Note: Descriptions are shown in the official language in which they were submitted.
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BACKGROUND OF THE INVENTION
- .: .
1. FIELD OF THE INVENTION
This invention relates to compositions, devices, and
methods for determihing the presence of a ligand in a liquid
medium based on the affinity of the ligand for a specific
binding partner thereof. In particular, this invention
relates to compositions, devices, and methods for use in
specific binding-assays which do not require a separation
step and which do not employ radioactive materials or modi-
~ fied enzymes as the labeling substance.
The desirability of a convenient, reliable, and non-
~ hazardous means for detecting the presence of low concen-
,d, trations of substances in liquids is self-evident. This is
particularly true in the field of clinical chemistry where
constituents of body fluids which may appear in concentra-
tlons às low as 10 11 molar are known to be of pathological
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significance. The difficulty of detecting such low concen-
trations is compounded in the field of clinical chemistry
where sample size is usually quite limited.
~!, 20 Classically, substances have been detected in liquids
`; based on a reaction scheme wherein the substance to be
i
~`; detected is a necessary reactant. The presence of unknown
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is indicated by the appearance of a reaction product or
the disappearance of a known reactant. In certain instances,
such an assay method may be quantitative, based on a measure-
ment of either the rate of appearance of product or dis-
appearance of reactant or measurement of the aggregate
amount of product produced or reactant consumed in attaining
- equilibrium. Each assay reaction system is necessarily
either limited to use in the detection of only a small group
of substances or is non-specific.
The search for assay systems which are highly specific
yet adaptable to the detection of a wide range of substances
has evolved the radioimmunoassay. In this system a known
.,,
amount of a radiolabeled form of the substance to be de-
tected is allowed to compete with the unknown for a limited
; 15 quantity of antibody specific for the unknown. The amount
of the labeled form that becomes bound to antibody varies
,;
; inversely with the level of unknown present. Inherent in
the radioimmunoassay technique is the need to separate the
labeled form of substance to be detected which becomes bound
;.:,
to antibody from that which does not become so bound. While
various ways of accomplishing the required separation have
, . .
' been developed, as exemplified in U.S. Patents Nos. 3,505,019;
3,555,143; 3,646,346; 3,720,760; and 3,793,445, all require
i at least one separate manipulative step, such as filtering,
`~ 25 centrifuging, or washing, to insure efficient separation of
l the bound-labeled form from the unbound-labeled form. The
,. j
elimination of the separation step would greatly simplify
; the assay and render it more useful to the clinical labora-
tory.
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The use of radioactive materials in immunoassays has
been eliminated to some degree by the use of enzyme
-tagged materials in place of radiolabels. As exemplified -
by U.S. Patents Nos. 3,654,090 and 3,791,932, the manipula-
tive steps necessary for carrying out the enzyme-tagged
immunoassays are for the most part the same as those re-
quired in radioimmunoassays and include the cumbersome
separation step. An additional disadvantage of using
enzyme-tagged materials is that each enzyme used as a tag
must be individually chemically modified for use in the
formation of the tagged conjugate. The use of other tagging
materials has been suggested, such as the use of coenzymes
or viruses, ~ature 219:186(1968) and the use of fluorescent-
, labels, French Patent No. 2,217,350.
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2 DESCRIPTION OF THE PRIOR ART
While the radiolabeled and enzyme-tagged immunoassays
;, may undergo future improvement in terms of expansion of the
range of substances detectable thereby or of simplification
of the procedure, by their nature they will always require
some type of separation step. Recently, a different approach
was disclosed which does not require a separation step and
~, therefore has been referred to as a homogenous system, in
contrast to a heterogenous system in which separation is
; essential. U.S. Patent No. 3,817,837 discloses a competi-
` 25 tive binding assay method involving the steps of combining
the liquid to be assayed with a soluble complex consisting
of an enzyme as a labeling substance covalently bound to the
ligand to be detected and with a soluble receptor, usually
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an antibody, for the ligand; and analyzing for the effect of
the liquid to be assayed on the enzymatic activity of the
enzyme in the complex.
While this method has the advantage of not requiring a
separation step because reaction between the enzyme-bound-
ligand complex and the receptor results in inhibition of the
enzymatic activity of the enzyme in the complex, the method
nonetheless ls severely restricted in its ability to be
adapted to widely varied assay requirements. For instance,
it is clearly essential that in the fabrication of the
enzyme-bound-ligand complex, the substance or ligand to be
detected must be coupled to the enzyme in a carefully con-
trolled manner so that the coupling site is close to the
enzymatically active site on the enzyme. This is required
in order that upon reaction between the complexed ligand and
,j the receptor, the enzymatically active site is blocked.
; Enzymes vary greatly in their size, ranging in molecular
weight from about 10,000 to 1,000,000. Thus, for a receptor
in the form of an antibody having a molecular weight of
i 20 between 150,000 and 300,000 to be capable of physically
:. blocking the active site on an average enzyme of 500,000
molecular weight or greater, the coupling site must be
: '
precisely controlled. Due to the complex chemical structure
of enzymes, precise control of such chemical linkage is
indeed difficult, and one would expect that even upon
screening a wide variety of enzymes only a small number
would be found to be of use in this homogenous assay system.
Moreover, it is critical for the purpose of obtaining
; quantitative test results to precisely control the ratio of
the number of enzymes to the number of ligands in each
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enzyme-bound-ligand complex. Here also, the complex peptide
structure of enzymes makes such control difficult. It would
again be expected that only a small number of enzymes would
have suitable molecular structure to ensure necessary
control of the ligand/enzyme ratio.
The prior art homogeneous assay method is stated to
involve an enzyme amplification and thus to be highly
sensitive. However, since the labeling substance, namely
the enzyme, is itself the limiting factor determining the
. 10 sensitivity of the prior art assay method, the versatility
. of the method is severely restricted. The sensitivity is
clearly limited to the catalytic activity of the particular
enzyme in the enzyme-bound-ligand conjugate. The versatil-
ity of the prior art method is therefore restricted not only
by the coupling requirements for formation of a useful
conjugate but also by the dependence of the sensitivity of
i'
the assay that employs such conjugate on the activity of the
particular conjugated enzyme.
~' An additional disadvantage of the prior art homogeneous
assay method arises in its application to the testing of
) .
~ biological fluids such as urine and serum. It is to be
.r~ expected that significant amounts of the enzyme species
comprised in the enzyme-bound-ligand conjugate may appear in
'';'! the fluid sample to be tested thereby creating an uncontrol-
lable background activity which would severely affect the
accuracy of the assay method. Therefore, in order to form
` an assay system that is useable in testing biological fluids
of humans or animals, exotic enzymes not endogenous to such
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fluids must be selected for use in forming the enzyme-bound-
ligand conjugate with the result that the versatility of the
assay method is even further restricted.
It is therefore an object of the present invention to
provide a novel test composition, device, and method for
detecting a ligand in a liquid which do not require a separa-
tion step and which do not employ inconvenient radioactive
materials or modified enzymes as the labeling substance.
Further, it is an object of the present invention to
provide a homogenous specific binding assay method and
system which are more versatile and convenient than those of
the prior art.
Another object of the present invention is to provide
a homogenous specific binding assay method and system which
lS employ a labeling substance which is capable of being
coupled to the ligand or to a specific binding partner
thereof more conveniently than can the enzyme of the prior
art method.
` A further object of the present invention is to provide
a homogenous specific binding assay method and system which
employ a conjugate comprising a labeling substance whose
activity is more readily affected by a specific binding
reaction than is the enzyme of the prior art method.
It is also an object of the present invention to pro-
vide a homogenous specific binding assay method and system
which employ a conjugate comprising a labeling substance any
change in the activity of which is more conveniently detectable
using a wide variety of sensitive reaction systems than is
any change in the activity of the enzyme in the prior art
method.
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It is a further object of the present invention to
provide a homogenous specific assay method and system which
are more readily applicable to the testing of biological
fluids than those of the prior art.
:
SUMMARY OF THE INVENTION
The present invention provides a highly convenient,
versatile, and sensitive homogenous specific binding assay
method and system based on the use of, as labeling substance,
a substance which exhibits given reactant activity as a con-
i 10 stituent of a predetermined reaction, such substance beingreferred to herein as the reactant. The method is based, in
; part, on the fact that the reaction between a ligand and aspecific binding partner thereof to one of which the reac-
, tant is coupled alters the activity of the reactant in the -
predetermined reaction, which reaction thus serves as means
for monitoring the specific binding reaction. In view of
this basic phenomenon, various manipulative schemes involv-
ing various test compositions and devices may be employed in
performing the method of the present invention. The preferred
fundamental manipulative schemes are the direct binding
technique and the competitive binding technique. -
In the direct binding technique, a liquid medium
suspected of containing the ligand to be detected is con-
tacted with a conjugate comprising the reactant coupled to a
specific binding partner of the ligand, and thereafter any
change in the activity of the reactant is assessed. In the
competitive binding technique, the liquid medium is contacted
with a specific binding partner of the ligand and with a
; conjugate comprising the reactant coupled to one or both of
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108257~7
the ligand or a specific binding analog thereof, and there-
after any change in the activity of the reactant is assessed.
In both techniques, the activity of the reactant is deter-
mined by contacting the liquid medium with at least one
reagent which forms, with the reactant, the predetermined
monitoring reaction. Qualitative determination of the ;
ligand in the liquid medium involves comparing a character-
istic, usually the rate, of the`resulting reaction to that
; of the monitoring reaction in a liquid medium devoid of the
ligand, any difference therebetween being an indication of a
change in activity of the reactant. Quantitative determina-
.
tion of the ligand in the liquid medium involves comparing a
characteristic of the resulting reaction to that of the
monitoring reaction in liquid media containing known amounts
lS of the ligand.
The monitoring reaction preferably is enzyme
-catalyzed. Usually, a monitoring reaction is selected
which is highly sensitive to the reactant in the conjugate.
Luminescent or fluorescent reaction systems are very useful
in this regard. Particularly preferred are cyclic reaction
systems, especially those in which the reactant is the
cycled material. Of the preferred cyclic reaction systems,
those which are enzyme-catalyzed are particularly advan-
tageous. The reactant in the conjugate is usually an enzy-
`` 25 matic reactant, such as an enzyme substrate or, as is particu-
larly preferred, a coenzyme, and preferably has a molecular
, weight of less than 9000.
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BRIEF DESCRIPTION OF THE DRA~ING
Figure 1 is a graphical representation of the effect of
various levels of a ligand on the aggregate reaction rate in
a direct binding-cycling assay technique.
Figures 2 and 3, respectively, are graphical representa-
tions of the effect of various levels of two different
ligands on the aggregate reaction rate in a competitive
binding-cycling assay technique.
Figures 4 and 5, respectively, are graphical representa-
tions of the effect of various levels of two different
ligands on the peak light intensity produced in a competitive
binding-bioluminescence assay technique.
Figure 6 is a graphical representation of the effect of
various levels of a ligand on the net rates of two different
reactions, one enzyme-catalyzed and the other not, in a direct
binding-fluorescent assay technique.
Figures 7 and 8, respectively, are graphical representa-
tions of the effect of various levels of a ligand on reaction
rates in two different competitive binding assay techniques,
- 20 one involving a fluorescent monitoring reactlon and the other
involving a spectrophotometric monitoring reaction.
Figures 9 and 10, respectively, are graphical representa-
:~ tions of the effect of various levels of two different ligands
on the relative intensity of luminescence produced in direct
and competitive binding-bioluminescent assay techniques.
Figures 11 and 12, respectively, are graphical representa-
tions of the effect of various levels of a ligand on the peak
light intensity produced in two different competitive
~` binding-chemiluminescent assay techniques.
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` DESCRIPTION OF THE PREFERRED EMBODIMENTS
In the context of this disclosure, the following terms
shall be defined as follows: ligand is the substance, or
group of substances, whose presence or the amount thereof in
a liquid medium is to be determined; specific binding partner
of the ligand is any substance, or group of substances,
which has a specific binding affinity for the ligand to the
exclusion of other substances; and specific binding analog
; of the ligand is any substance, or group of substances,
which behaves essentially the same as the ligand with respect
to the binding affinity of the specific binding partner for
the ligand.
In general, the components of the specific binding
reaction, i.e., the liquid medium suspected of containing
the ligand, the conjugate, and/or a specific binding partner
of the ligand, may be combined in any amount, manner, and
, sequence, provided that the activity of the reactant in the
,. .
conjugate is measurably altered when the liquid medium
; contains the ligand in an amount or concentration of signifi-
:' 20 cance to the purposes of the assay. Preferably, all of the
:
components of the specific binding reaction are soluble in
the liquid medium, thus providing a homogenous assay system.
However, a heterogenous assay system wherein the conjugate
or a specific binding partner of the ligand is insoluble may
be employed if desired.
Where a direct binding technique is used, the compon-
ents of the specific binding reaction are the liquid medium
- 1 1 -
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~08Z57~7
suspected of containing the ligand and a quantity of a
conjugate comprising the reactant coupled to a specific
binding partner of the ligand. The activity of the conju-
gated reactant on contact with the liquid medium varies
inversely with the extent of binding between the ligand in
the liquid medium and the specific binding partner in the
conjugate. Thus, as the amount of ligand in the liquid
medium increases, the activity of the conjugated reactant
decreases. To obtain quantitative results, the amount of
the specific binding partner contacted with the liquid
medium is usually in excess of that capable of binding with
all of the ligand thought to be present in the liquid medium
during the time that the conjugate ~nd the liquid medium are
in contact prior to completion of the assessment of any
change in activity of the conjugated reactant. In practice,
an amount of the specific binding partner is chosen according
;, to the above-mentioned criterion based on an estimation of
the largest amount of the ligand which is likely to be
present in the liquid medium. A direct binding technique is
particularly useful in detecting high molecular weight
: .
ligands which have specific binding partners that are smaller
than themselves.
Where a competitive binding technique is used, the
components of the specific binding reaction are the liquid
`, 25 medium suspected of containing the ligand, a quantity of a
,! conjugate comprising the reactant coupled to the ligand or a
specific binding analog of the ligand, and a quantity of a
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specific binding partner of the ligand. The speciEic binding ~
partner is contacted substantially simultaneously with both ~ -
the conjugate and the liquid medium. Since any ligand in
the liquid medium competes with the ligand or specific
binding analog thereof in the conjugate for binding with the
specific binding partner, the activity of the conjugated
reactant on contact with the liquid medium varies directly
with the extent of binding between the ligand in the liquid
medium and the specific binding partner. Thus, as the
amount of the ligand in the liquid medium increases, the
activity of the conjugated reactant increases. To obtain
quantitative results, the amount of the specific binding
partner contacted with the conjugate and the liquid medium
is usually less than that capable of binding with all of the
ligand thought to be present in the liquid medium and all of
the ligand or ligand analog in conjugated form in the time
that the specific binding partner, the conjugate~ and the
liquid medium are in contact prior to completion of the
` assessment of any change in activity of the conjugated
:q : . .
reactant. In practice, an amount of the specific binding
. partner is chosen according to the above-mentioned criterion
, based on an estimation of the largest amount of the ligand
i which is likely to be present in the liquid medium. Usually,
the amount of the ligand or ligand analog in conjugated form
which is contacted with the liquid medium does not exceed
,
the smallest amount of the ligand to be tested for in the
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liquid medium. A competitive binding technique is particu-
larly useful in detecting ligands which have specific bind-
ing partners that are larger than themselves.
A variation of the competitive binding technique is the
displacement binding technique wheTein the conjugate is
contacted first with the specific binding partner of the
ligand and thereafter with the liquid medium. Competition
for the specific binding partner then occurs. In such a
method, the amount of the conjugate contacted with the
specific binding partner is usually that which comprises the
ligand OT analog thereof in excess of that capable of bind-
ing with the amount of the specific binding partner present
during the time that the conjugate and the specific binding
partner are in contact prior to contact with the liquid
medium suspected of containing the ligand. This ordeT of
contact may be accomplished in either of two convenient
ways. In one method, the conjugate is contacted with the
specific binding partner in a liquid environment prior to
:;
contact with the liquid medium suspected of containing the
ligand. In the second method, the liquid medium suspected
of containing the ligand is contacted with a complex com-
prising the conjugate and the specific binding partner, the
specific binding substance in the conjugate and the specific
binding partner being reversibly bound to each other. The
amount of the conjugate that becomes bound to the specific
.,,
binding partner in the first method, as well as the amount
thereof which is in complexed form in the second method, is
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1082S7~7 :
usually in excess of that capable of being displaced by all
of the ligand in the liquid medium in the time that the
specific binding partner, or complex, and the medium are in
contact prior to the completion of the assessment of any
change in the activity of the conjugated reactant.
Another variation of the competitive binding technique
is the sequential saturation technique wherein the com-
ponents of the specific binding reaction are the same as
those used in the competitive binding technique, but the
order of addition or combination of the components and the
relative amounts thereof used are different. Following a
sequential saturation technique, the specific binding
partner of the ligand is contacted with the liquid medium
suspected of containing the ligand for a period of time
` 15 prior to the contact of said liquid medium with the conju- ;
; gate. The amount of the specific binding partner contacted
with the liquid medium is usually in excess of that capable
of binding with all of the ligand thought to be present in
the liquid medium in the time that the specific binding
partner and the liquid medium are in contact prior to the
time that the liquid medium is contacted with the conjugate.
Further, the amount of the ligand or ligand analog in con-
jugated form is usually in excess of that capable of binding
with the remaining unbound amount of the specific binding
partner during the time that the liquid medium and the
conjugate are in contact prior to the completion of the
assessment of any change in activity of the conjugated
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~0~25'7~7
reactant. In practice, the amounts of the specific binding
partner and of the ligand or ligand analog in conjuga~ed
form are chosen according to the above-mentioned criterion
by estimating the largest amount of the ligand likely to be
present in the liquid medium.
It is contemplated that manipulative schemes involving
other orders of addition and other relative amounts of the
specific binding reaction components may be devised for
carrying out a homogenous specific binding assay without
departing from the inventive concept embodied herein.
The step of assessing any change in activity of the
conjugated reactant as a constituent of the predetermined
~; monitoring reaction is conveniently accomplished by contact-
ing the specific binding reaction mixture with at least one
substance which forms with the conjugated reactant, the
monitoring reaction, and determining the effect of the
specific binding reaction on a characteristic of such reaction.
The monitoring reaction may comprise a single chemical
~'~'! transformation or a plurality or series of chemical trans-formations. Unless otherwise specified, the term "reaction
system" as used herein refers to the whole or a portion of
; the predetermined monitoring reaction.
` Where an enzyme-catalyzed reaction system is used, itincludes, in addition to the conjugated reactant, at least
- 25 one enzyme and may include one or more enzymatic reactants
such as substrates and coenzymes. Such enzyme-catalyzed
reaction system may comprise a single simple enzymatic
reaction or a complex series of enzymatic and non-enzymatic
reactions. For instance, the enzyme-catalyzed reaction
system may consist of a single enzyme-catalyzed degradation
- 16 -
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1082S7q
or dissociation reaction. In such a system, the conjugated
reactant is the enzyme substrate which undergoes degradation ~ -
or dissociation, and the only component of the reaction
system necessary to be contacted with the specific binding
reaction mixture is an enzyme which catalyzes the degrada-
tion or dissociation reaction. A more complex enzyme-
catalyzed reaction system may consist of a single enzymatic
reaction involving two or more reactants or may consist of a
series of reactions involving several reactants, at least
one of which reactions is enzyme-catalyzed. In such a
system, the conjugated reactant would be one of the enzymatic
reactants in the enzyme-catalyzed reaction and the specific
binding reaction mixture would be contacted with the appro-
priate enzyme and reactant constituents, other than that in
the conjugate, which are necessary to provide the selected
i,.J
. enzyme-catalyzed reaction system.
It is further contemplated that the enzyme-catalyzed
reaction system may comprise a biochemical system as complex
as the metabolic system of a biological cell such as a
; 20 microorganism. For example, a nutrient substance essential
to the growth of a particular microorganism may be selected
.~ as the reactant in the conjugate. Any change in the activity
of the reactant would cause a change in a growth characteris-
tic of the microorganism when such microorganism would be
~ 25 placed in an environment wherein the only source of the
S reactant nutrient substance is the conjugate. Thus, for
` example, a change in the rate of microorganism growth when
~; contacted with the specific binding reaction mixture would
indicate the presence of the ligand therein.
- 17 -
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The appropriate reaction constituents which form, to-
gether with the reactant in the conjugate, the monitoring
reaction may bè contacted with the specific binding reaction
mixture singularly or in any combination either prior to,
simultaneous with, or subsequent to initiation of the speci-
fic binding reaction. After initiation of the specific
binding reaction, the reaction mixture, which may include
any or all of the necessary components for the monitoring
reaction is usually incubated for a predetermined period of
time before assessing any change in the activity of the
reactant in the conjugate. After the incubation period, any
components which are necessary for the monitoring reaction
and which are not already present in sufficient quantities
in the reaction mixture are added thereto, and any effect on
the monitoring reaction is assessed as an indication of the
presence or amount of the ligand in the liquid medium.
In the situation where the ligand is absent from the
;~ liquid medium, or is present in an insignificantly small
` amount, the predetermined monitoring reaction exhibits a
relatively constant character. When the ligand is present
in the liquid medium, at least one characteristic or property
of the monitoring reaction is altered. Generally, the
activity of the conjugated reactant is defined as the extent
or rate at which the reactant is capable of participating in
the monitoring reaction. Thus, the character of the monitor-
ing reaction is altered by the presence of the ligand in
the liquid medium, usually with respect to either the
aggregate reaction rate thereof or the equilibrium quantity
; - 18 -
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of one or more reaction products produced thereby. In the
usual case, the ability of the conjugated reactant to partici-
pate in the monitoring reaction is decreased upon reaction
between the specific binding substance to which it is conju-
; 5 gated and a specific binding counterpart of such specific
binding substance, that is, the conjugate in its free state
is more active in the monitoring reaction than in its bound
state. The relative amounts of free and bound conjugate
present after the incubation of the specific binding reac-
tion are a function of the amount of ligand in the liquid
r
` medium and are determinative of the effect on the monitoring reaction.
When the change in the aggregate reaction rate of the -
monitoring reaction is the characteristic used to determine
the presence of the ligand, as is preferred, such rate is
s usually determined by measuring the rate of disappearance ofa reactant or the rate of appearance of a reaction product.
Such measurement can be accomplished by a wide variety of
methods including the conventional chromatographic, gravi- -
metric, potentiometric, spectrophotometric, fluorometric,
turbidimetric, and volumetric analysis techniques. Since
the present method is primarily designed for the detection
of low concentrations of ligands, highly sensitive reaction ;
; systems have been developed for use in conjunction with the
. 25 novel specific binding reaction system.
,~ One preferred form of the monitoring reaction includes
a luminescent reaction system, preferably enzyme-catalyzed,
such as a reaction exhibiting the phenomenon of biolumines-
cence or chemiluminescence. The reactant in the conjugate -
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may be a reactant in either the light-producing reaction or
a reaction which is preliminary to an enzymatic or non
-enzymatic luminescent reaction. Any change in the activity
of the conjugated reactant resulting from the specific
: 5 binding reaction causes a change in the rate of light produc-
tion or in the total amount, peak intensity, or character of
the light produced. Examples of luminescent reaction systems
~ are given in Table A in which the following abbreviations
; are used:
ATP adenosine triphosphate
AMP adenosine monophosphate
NAD nicotinamide adenine dinucleotide
NADH reduced nicotinamide adenine dinucleotide
FMN flavin mononucleotide
. 15 FMNH2 reduced flavin mononucleotide
hv electromagnetic radiation, usually in the
infrared, visible, or ultraviolet region
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+
O
O ~ ~i Z
+
:'. ¢ O ~
~, ~ ~ O
E-~ O +
~' ~l X E~
+ ~. ~ O ~d ~
A ~cJ l A ,~ ~:
. ~ I U~
, * ~ ~ i ~ ~ ~ a. ~ +
! v~.~ ul v~
u~ ~ ~) Xo ~ ~ ~ A
o o h X X , I ,
cd r ~ x o ~ o
,i ~ 0~ 5: o
,.,; ~ ::C + o~ +
+ o + o
~ ~I o
::' C) ~ ~ ~ ~ ~ o
., ~ O ~i O
rl
-`.! ~ ~rl 0 ~r~ O + +
i 13 ~ E~ h
' `, ~1 p~ _I p, ~o o ,1
O O h
h h h 5-1 ''~
` . '
.
~ ~ X ~ ~ ,
`: j
2 2 -
, i - ,
, . . :
, ........................................................................... .
. ~
. . .
i`` , . . . . . . . . . . . . .. . .... . . . . .
108Z57~7
Further détails and discussion concerning luminescent
reaction systems which may be used in the present method may
be found in the following references:
J. BioZ. Chem. 236: 48(1961).
J. Amer. C~em. Soa. ~9: 3944(1967).
Cornier et al., BioZumines¢en¢e in Progress~
ed. Johnson et al., Princeton University
Press (New Jersey, 1966) pp. 363-84.
Kries, P. Purifioation and P~opertie~ of ReniZZa
Lu¢iferase, doctoral thesis University of
Georgia (1967).
Am. J. PhysioZ. 41: 454~1916).
BioZ. BuZZ. 51: 89(1926).
J. BioZ. C~em. 243: 4714(1968).
. :
Another type of preferred, sensitive, monitoring
reaction involves the phenomenon of fluorescence and is
enzyme-catalyzed. In such a reaction system the reactant in
the conjugate is a substrate in an enzymatic reaction which
produces a product which has fluorescent properties that
differ from those of the conjugated substrate. Any change
in the activity of the conjugated enzymatic reactant result-
ing from the specific binding reaction causes a change in
the fluorescent properties of the reaction mi~ture. A
-~ general reaction scheme for such an enzyme-catalyzed reactionl 25 system is as follows:
't
reayctant -X-Z (enZyme)~ produc*
(substrate)
~ wherein X is an enzyme-cleavable bond or linking group, such
.- 30 as an ester or amido group, and Z is a specific binding sub- -
- 23 -
~.
. ~ . - : , .
:: :: ~ : .
108257n7
stance which, depending upon the specific binding reaction
technique used, is the ligand, a specific binding analog of
the ligand, or a specific binding partner of the ligand.
Specific conjugates which may be used in this type of reac-
tion system are ~arious enzyme-cleavable derivatives of
fluorescein, umbelliferone, 3-indole, ~-naphthol, 3-py~idol,
resorufin, rhodamine B, and so forth. Examples of possible
structural formulas of such derivatives are as follows:
Derivative . Formula
fluorescein Rl~~R2
o
: \/
O . ~
umbelliferone z
R3
3-indole 2
H
~-naphthol ~ R2
. :
!. ~ .
, - 24 -
;. ' : .:
i
. _~
.- :
., . . -........ ~ - . - . . . . . . . .
:: : . ~ . : . .: .
1082s7q -
,~ R2
3-pyridol N 'J
0~ o~ R2
resorufin
,, ,
,:' ' ' ~ '
.. .
wherein Rl is -OH or -X-Z (as defined above in this para-
graph), R2 is -X-Z, and R3 is -H or -CH3.
A reaction system which is particularly preferably for
use in monitoring the novel specific binding reaction
'I of the present invention is a cyclic or cycling reaction
,l system. Such a reaction system is one in which a product of
l a first reaction is a reactant in a second reaction, which
i~ 10 second reaction has as one of its products a substance that
:;1
is also a reactant in the first reaction.
,~ The following diagram illustrates a model of a cyclic
reaction system:
products A ~ cycled material reactants B
~?~ 15 ~ (form 1)
REACTION A ~ ~ REACTION B
reactants A cycled material ~products B
(form 2)
,i y
.:~
~' .
- 25 -
j,, .
. . .
... .
, .,
- ~ , . -
..
-; .: ' - . : . .. ,. ' ' : - . ,
.
ZS7~7
In the above model cyclic reaction system, a small amount of
cycled material, if provided with sufficient amounts of
reactants A and B, will generate large amounts of products
A and B. Since the rate and amount of product produced by
the reactions constituting the cyclic reaction system is
highly sensitive to the amount of cycled material present,
it is most preferred to use the cycled material as the reac-
tant in the conjugate of the present invention. Examples of
cycling reaction systems contemplated for use in conjunction
with the novel specific binding reaction system of the
present invention are given in Tables b,C, and ~.
.' .
.~' ., .
';~ :'' '
.~ .
, .,: '-
. - 26 - :
, 1 :
., . , i ; . , . : . ~ - - :
~0f~2S7~7
TABLE B
product A ~ NAD* V reactant B
enzyme enzyme
reactant A ~ NADH** ~ product B ~ :
reactant A reactant B
or or :
reaction product B enzyme product A
1 lactaldehyde alcohol de- propanediol
. hydrogenase
2 a-ketoglutar- glutamic de- glutamate
. ate ~ NH3 hydrogenase
3 oxaloacetate malic dehy- malate
.~ drogenase
4 acetaldehyde alcohol de- ethanol
hydrogenase
, 5 a-ketoglutar- isocitric isocitrate
ate ~ CO2 dehydrogen- :
~, ase
6 dihydroxyace- a-glycerol L-a-glycerol
. 20 tone phos- phosphate phosphate
phate dehydrogen-
; ase
.,` 7 pyruvate lactic dehy- lactate
l~ drogenase
`~ 8 1,3-di~hos- giyceralde- glyceraldehyde :
phogl~cerate hyde-3-phos- -3-phosphate
phate dehy- + phosphate
drogenase
t;
i
* nicotinamide adenine dinucleotide
** reduced NAD
'^'~'s
,~
;
- 27 -
~; , .
~' ~ ' .
: . : . : . ,.
, . . : , ,. . , . , ~
.. . . . . , : ~:
: .:
1~82S7q
TABLE C
product A ~ NADP* ~ reactant B
enzyme enzyme
reactant A ~ NADPH** ~ product B
5reactant A reactant B
or or
reaction ~roduct B enzyme product A
1 6-phospho- glucose-6 glucose-6
gluconate -phosphate -phosphate
dehydrogenase
2 o~idized glutathione reduced glu-
glutathione reductase tathione
3 p-benzoqui quinone hydroqui-
none reductase none
4 nitrate nitrate nitrite
reductase
~-ketoglu- glutamic glutamate
tarate + NH3 dehydrogenase
,. . .
* nicotinamide adenine dinucleotide phosphate :
~ ** reduced NADP
: ,,
.
.,
.
, .
~' '
.
'
. i . . . . . . . .
.... . . . . . . . . . . .
- . ~ :. . . , -
. ~ .. . - . . -
1082S7!7
It should be noted that the cyclic reaction systems
illustrated in Tables B and C comprise the combination of
any one of the reactions listed in the respective tables
with any other reaction listed therein. For example, reac-
tion 1 in Table B may be paired with any one of reactions 2-
8 to form a useful cyclic reaction system. Thus, Tables B
and C represent respectively 56 and 20 possible cyclic reac-
tion systems for use in the present invention.
In addition to the cyclic reaction systems represented
in Tables B and C, it is contemplated that one of the reac-
tions in the cyclic reaction system may involve the enzymatic
or non-enzymatic conversion of a spectrophotometric indicator,
preferably colorimetric. In such a system, any change in
the reaction or cycling rate would be reflected in a change
in the spectrophotometric properties of the indicator.
Using the preferred colorimetric indicators such change
would be a color change. An example of a cyclic reaction
system involving a conversion of an indicator is the system
produced by combining one of the reactant B - product B
` 20 reactions from Table B with a reaction comprising an oxidation
-reduction indicator and an electron transfer agent. As
electron transfer agent, phenazinemethosulfate may be used.
Useful indicators include the oxidized forms of nitrotetra-
æolium, thiazoyl blue, and dichlorophenolindophenol.
., .
~'-' . .
~ .
29
.,'','` ~ . . .
. '
-
.
. . .
,.,: ' ~
iO82S~7
tPage 1 of 4 pages)
TABLE D
reduced
cytochrome C ~ ~ FMNa NADPH
cytochrome C cytochrome C
reductase reductase
- oxidized ~ b ~
:~ cytochrome C FMNH2 NADP
.
: aflavin mononucleotide
~ 10breduced FMN ~:
:, ,
(
,~ H202 --~ ~ ~ FADC v ~ D-aminoacid -~.
D-aminoacid D-aminoacid
:l oxidase oxidase
~ 15 2 ~ ~ FADH2 ~ ~ a~keNHaCid
~i . .
flavin adenine dinucleotide
~: dreduced FAD :
: : :
,~ . .
o~-ketoglutarate ~ a-aminoacid ~ ~ 02 '':~
transamlnase L-amlnoacid
~ 20 A oxidase
J\ A
glutamate / \ N-ketoacid~ ~H22
'' .
;
,, .
: .
: - 30 -
:.',
:. .
:''
~08257~7
(page 2 of 4 pages)
TABLE D
phosphate _ ADPe phosphoenol
pyruvate
adenosine pyruvate
triphosphatase kinase
ATPf ~pyruvate
. eadenosine diphosphate
fadenosine triphosphate
1 -
~, succinate + GTPg coenzyme A a~ketoglu-
10~ ~ tarate + NAD ::
succinic a-ketoglutarate
thiokinase dehydrogenase
phosphate + GDPh ~ succinyl-
coenzyme A
15gguanosine triphosphate
, hguanosine diphosphate
;
oxidized
ascorbate ~ glutathione ~ NADPH
~'~ dehydroascorbate glutathione
', 20reductase reductase
~ dehydro- ,~ ~ reduced ~ _ NADP
;~i ascorbate glutathione
:,
- 31 -
..
.', ~ ., , ' ; .' ~ '
1082S7~
(page 3 of 4 pages)
TABLE D
oxidized
glutathione ~ ascorbate ~ 2
dehydroascorbate ascorbate
reductase oxidase
reduced ~ ~ dehydro- ~ ;;
glutathione ascorbate H20
.
. ,:
ADP ~ GTP ~ oxaloacetate
nucleoside phosphoenol
diphosphate pyruvate kinase
kinase A
J\ ' :,
' ATP ~~ ~ GDP ~~ ~ phosphoenol
,1 pyruvate
, ~ ,
H O oxidized NADPH
lS 2 ~ cytochrome C ~ ;
/~ cytochrome C cytochrome C
,, peroxkdase redActa9e
O ~ ~ reduced ~ ~ NADP
' cytochrome C
~~ .
! ',
.'
'.' i
.'
32 -
.~ j
. ~ .
.: ;. . . .
!~ . ~ . . . . . . . . :
-, ,, , ; : .. , : . . , :. . ..
., ., . . . . :. : .. : : . : -. .
: ~ `
:
1082S7'7
.
(page 4 of 4 pages)
~' .
~; TABLE D
sl, _
~`
NADPH~ ~ oxidized \ ,--H2
~ ferridoxin V
pyridine hydrogenase
nucleotideA reductase A
, . / \ / \
NADP ~ ~ reduced
ferridoxin
, - .
, ...................................................................... . .
In forming any of the cyclic reaction systems illus- -
trated in Tables B,C, and D, where a component in the reac-
.~ " .
tion system is in an ionic form, it may of course be added
in a salt or acid form which is ionizable upon contacting
the liquid medium. A water soluble salt or acid of such
component is usually preferred.
It is also contemplated that an exponential cyclic
reaction system may be included in the monitoring reac-
"
tion system. An example of an expo~ential cyclic reaction
system is as follows: -
. .
,,. 1 . ~ .
; AMP + A~P myokinase~_ 2 ADP
ADP + PEP pyruvate kinaSe~ATp + pyruvate
20 Such a cyclic reaction is autocatalytic in the sense that
~l~ during each cycle the amount of cycled material is doubled.
¦ The cycling rate therefore increases exponentially with time
and affords a high degree of sensitivity. Further details
and discussion relating to such cyclic reactions may be
25 found in J. Bio~. Çhem. 247 j3558-70~1972).
- 33 -
.;
;,
.i - - , . . -
. ...
;J'
108Z57t7
Where a cyclic reaction system is used as a means of
assessing any change in activity of the conjugated reactant,
the rate of disappearance of a reactant or rate of appear-
ance of a reaction product can be determined by conventional
techniques or by using one or more additional cycling sys-
tems followed by a conventional determination of the aggre-
gate reaction rate.
The use of a cyclic reaction system in conjunction with
the specific binding reaction system provides a high degree
of assay versatility as well as sensitivity. A single
reactant-specific binding substance conjugate may be used
with a multiplicity of reactions to form cyclic systems
which have sensitivities varying over a wide range and which
provide a wide variety of responses detectable by the senses
or artificial means. Such versatility is lacking in the
homogenous enzymatic assay system of the prior art.
While unnecessary in the preferred embodiment of the
present invention, it may be desirable to employ a hetero-
genous assay technique even where the presence of the ligand
in the liquid medium affects the activity of the conjugated
reactant. Such a situation may present itself where a
heterogenous system offers particular convenience. Certain
, heterogenous systems have the ability to increase the effec-
;i tive concentration of the ligand in the assay system, thus
increasing sensitlvity. An example of such a heterogenous
system is that which employs a column device containing an
insoluble matrix comprising either the conjugate of the
' present invention or a specific binding partner of the ligand, -
.
- 34 -
;'
~ ~ ~ ' , . : . .
. ~., , : .
.. , , , ... , : .
: .. . . . : . . : . .
10~257t7
depending on the particular manipulative format selected.
All other heterogenous assay methods employing radio-labeled
or enzyme-tagged materials as a labeling substance may also
be followed using the reactant of the present invention as
the labeling substance.
The present invention may be applied to the detection
of any ligand for which there is a specific binding partner.
The ligand usually is a peptide, protein, carbohydrate,
glycoprotein, steroid, or other organic molecule for which
; 10 a specific binding partner exists in biological systems or
can be synthesized. The ligand, in functional terms, is
usually selected from the group consisting of antigens and
antibodies thereto; haptens and antibodies thereto; and
hormones, vitamins, metabolites and pharmacological agents,
and their receptors and binding substances. Specific
examples of ligands which may be detected using the present
.~ .
invention are hormones such as insulin, chorionic gonado-
tropin, thyroxine, liothyronine, and estriol; antigens and
haptens such as ferritin, bradykinnin, prostaglandins, and
tumor specific antigens; vitamins such as biotin, vitamin
B12, folic acid, vitamin E, and ascorbic acid; metabolites
such as 3',5' adenosine monophosphate and 3',5' guanosine
monophosphate; pharmacological agents such as dilantin,
digoxin, morphine, digitoxin, and barbiturates; antibodies
such as microsomal antibody and antibodies to hepatitis and
allergens; and specific binding receptors such as thyroxine
binding globulin, avidin, intrinsic factor, and transcobal-
amine.
In general, it is preferred that the conjugate comprise
the reactant coupled to the smaller of the ligand and its
!~ - 35
., ~
"' ' :
lO~Z57q
selected specific binding partner. It is preferred to use
a direct binding technique to detect the ligand where the
molecular weight of the selected specific binding partner is
about one-tenth that of the ligand or less. Thus, where the
ligand to be detected is an antibody or a specific binding
receptor, it is preferred to follow a direct binding tech-
nique wherein the conjugate comprises an enzymatic reactant
coupled to an antigen or hapten to the antibody or a lower
molecular weight binding partner of the receptor. Where
the molecular weight of the selected binding partner is ten
or more times larger than that of the ligand to be detected,
; as when an antigen, hapten, hormone, vitamin, metabolite or
pharmacological agent is to be detected, it is particularly
advantageous to employ a competitive binding or sequential
saturation technique iIl which the conjugate comprises the
reactant coupled to the smaller ligand.
In the conjugate of the present invention, the reactant
is coupled or bound to a specific binding substance, which
is the ligand, a specific binding analog of the ligand, or
a specific binding partner of the ligand depending upon the
i assay scheme selected, such that a measurable amount of
activity of the reactant is retained. The bond between the
reactant and the specific binding substance is usually sub-
~ stantially irreversible under the conditions of the assay
`I 25 such as where the monitoring reaction in which the reac-
tant has activity is not designed to chemically destroy such
bond as in the above-mentioned luminescent and cyclic reac-
tion systems. However, in certain instances such bond is by
.~,
~, design destroyed or otherwise affected by the selected
monitoring reaction as a means for assessing the change
` in reactant activity. Such a case is the enzymatic fluores- -
- 36 -
.. .
', :'
1082S~7
cent substrate reaction systems referred to previously
herein.
The reactant may be directly coupled to the specific
binding substance so that the molecular weight of the conju-
gate is less than or equal to the aggregate molecular
weight of the reactant and the specific binding substance.
Usually, however, the reactant and the specific binding
substance are linked by a bridge group comprising between 1
and 50, and preferably between 1 and 10, carbon atoms or
heteroatoms such as nitrogen, oxygen, sulfur, phosphorus and
so forth~ Examples of a bridge group comprising a single
atom would be a methylelle group (one carbon atom) and an
amino group ~one heteroatom). The bridge group usually has
I a molecular weight not exceeding 1000 and preferably less
'3l 15 than 200. The bridge group comprises a chain of carbon
atoms or heteroatoms, or a combination of both, and is
joined to the reactant and the specific binding substance,
or active derivative thereof, by a connecting group usually
in the form of an ester, amido, ether, thioester, thioether,
acetal, methylene, or amino group.
The reactant in the conjugate of the present invention
, may be any substance which has given (i.e. fixed!or known)
~, reactant activity as a constituent of a predetermined moni-
toring reaction. More particularly, for the purposes of
,~ 25 this disclosure, the terms "reactant" and "substance having
i reactant activity" refer to any chemical substance whlch is
capable of undergoing a finite measurable chemical transforma-
tion which yields one or more products different from itself
and which results upon interaction of said reactant with reac-
tion-initiating means, such as a chemical substance ~i.e. an-
- 37 -
, .
/ - :
108ZS7~7
.
other reactant, a catalyst, or other type of material which
participates in such chemical transformation), electromagnetlc -
radiation, thermal energy, or sonic energy.l The class of sub-
stances defined herein as "reactants" therefore includes con-
ventional inorganic and organic reagents andvarious biochemical
materials, but excludes such materials as catalysts, including
enzymes, and radioactive isotopes which are not reactants in
the monitoring reaction. It will be recognized that while a
particular chemical substance may be classified in several dif-
ferent catagories because it is able to function in several ~ -
ways depending on its chemical environment, it is the activity
; of such substance with respect to the selected monitoring reac-
.,
tion referred to herein which shall govern which functional
identity such substance shall have in the context of this dis-
closure.
Preferably, the reactant is an enzymatic reactant such as
an enzyme substrate, a coenzyme, or an active modification or
derivative thereof. An enzyme substrate is a compound or
moeity capable of undergoing a chemical transformation that is
catalyzed by an enzyme. Where a substrate is employed as the
conjugated reactant, the preferred molecular weight thereof is
less than 9000 and preferably less than 5000. Substrates of
such size, because of their lack of molecular complexity, are
most convenient for use in the fabrication of the conjugate.
Moreover, the activity of such substrates when coupled to a
specific binding substance is readily affected by reaction of
the conjugate with a specific binding counterpart of such
specific binding substance. Examples of enzyme substrates
which are contemplated for use in the present invention in-
clude the enzyme-cleavable fluorescent substrates referred
.... .
~ - 38 -
,
., ,
. . ' ' .
, ... . ~ - . . . .
, . ' ' . ,'- . .':, ., ,.' : .. ' ~ , ' .- ' . '
1082S7~
to previously such as fluorescein and umbelliferone deriYa-
tives; pH indica*ors; and spectrophotometric indicator dyes,
particularly chromogenic types.
For the above reasons and for reasons of versatility and
adaptability, coenzymes are especially preferred for use as
the reactant in the conjugate. A coenzyme is a non-protein
molecule which migrates from one enzyme protein to another in
facilitating the efficient performance of the catalytic func-
tion of the enzyme. All known coenzymes have a molecular
weight of less than 9000, the preferred coenzymes having a
molecular weight of less than about 5000. Useful coenzymes
include the nucleotide coenzymes, particularly those com-
prising adenine groups, such as the adenosine phosphates
~i.e. the mono-, di-, and tri-phosphate forms), nicotinamide
adenine dinucleotide and its reduced forms, and nicotinamide
adenine dinucleotide phosphate and its reduced forms. Other
useful coenzymes include the guanosine phosphates, flavin
mononucleotide and its reduced forms, flavin adenine dinu-
cleotide and its reduced forms, coenzyme A and its thio-
esters including succinyl-coenzyme A, 3',5' adenosine
diphosphate, and adenosine-3'-phosphate-5'-phosphosulfate.
Useful coenzyme-active conjugates comprise nucleotide
coenzymes having an adenine group to which the specific
binding substance, i.e., a ligand, a specific binding analog
.,
of a ligand, or a specific binding partner of a ligand, is
... .
coupled through a direct bond or a bridge group as referred
to hereinbefore. Such coenzyme-active conjugates which
comprise an adenosine phosphate, nicotinamide adenine di-
nucleotide or its reduced form, or nicotinamide adenine
dinucleotide phosphate or its reduced form, have the follow-
ing general formula:
- 39 -
.~ .
.
- , .
. j .
1082577 ~ ~
Rl-CH ~ R3
1 2
OH R
: ' : , '
wherein Rl is
o~ o'3 o~i'
5- -O-P-O -O-P-O-~-O .
.,
0~ 0~3 0~ o~' o9
-O-~-O-P-O-P-O~) or , -O-ll-O-P-O-C~ O R4
. ~_1/ ............
~I 10 1H 1H
o~ :
wherein R2 is - OH or -O-P-O~; -
,
. ;~ wherein R3 is
NH2 R5 NH2 NHR5
Nl ~ ~ , Nl ~N ~ N
.. ~ N,H2 R5 NH2 NH2
N ~ N J ' ~N ~ ~ RS
~., wherein R is
i ~ - N
. ~ NH 2 CONH 2
. - 40 -
~OI~ZS7~7
wherein R5 is -Y-Z; wherein Y is a bond or a bridge group;
and wherein Z is a ligand, a specific binding analog of a -
ligand, or a specific binding partner of a ligand. The
above formula represents the ionized forms of the coenzyme-
active conjugate, however, the protonized or acid forms are
equally useful. The extent of protonization depends on the
pH of the environment. Also, the salts of such conjugates
may also be used where appropriate.
~"
:
~",
:'
,.. .
i~'
" ,
. . .
, .,
i l
..,
,.,
,i .
. .
,~....
.
. . .
,
. .
" ~.
.,
'~:
.. - 41 -
,
. .
.,
, .
,
. .
. . .
, . ~ -
1~257~7
Synthesis of such compounds may be accomplished in a
variety of wa~-s. It is contemplated that the synthesis
routes which are schematically illustrated below are ad-
vantageously followed in the preparation of the useful
compounds. In the illustrated syntheses, the positions on
the adenine ring structure are referred to according to the
following: NH2
\NJ\~N3J
Also, the following abbreviations are used;
Rib refers to the ~ ~
ribose moiety, i.e., H ~ H
OH OH
, 15 Rib' refers to the phosphated - CH2
ribose moiety, i.e., / O ~
h I I H
; :
20 Ph refers to a phosphate group;
.
AP derivatives refers to derivatives of adenosine
-5'-phosph2te, i.e., the mono- (AMP), di- (ADP),
or tri- (ATP) phosphate form;
NAD derivati~-e refers to a derivative of either
nicotinamide adenine dinucleotide or a reduced
form thereof;
NADP derivative refers to a derivative of either
nicotinamide adenine dinucleotide phosphate or a
reduced fo~m thereof;
R refers to the specific binding substance or a
`: modification thereof; and
X refers to a leaving group, usually a halogen.
- 42 -
: '
' 5a~
.........
.,
:.' , :
.~ .
;,;'~ ', . . .. :
'' ' . '
108257~7
.,
Z ~ Z
U ~ U ,~
Q~ ¢ zQ~ ~ ~
., o ~ ~
!r ,
.
' ,
X 5
... ~
.
. ~ .,, ~ , o
... , .. ~ ,
\
O
Z~
:~ 3 ~ cq
a ~
` - 43 -
'`''~
. ~
" ' '
; . . . .
.. . ...
., ' , . . : . ~ .
-
lO.~ZS7q
'~ :
0
z~
z ~
O ~
:
. ~ r~
A Z-~ "
i~ ~ ¢ ~d
,. ~
,~ ~ ¢ ~ :
h 3:~ , , ~
N
v
- 4 4
.....
.. .. . . . .
~08257'7
o
X .,
o .c
_,
_,
.~ , ~ o U~
:C
c~ ~
,, ~ Z~Z~
,
U G A
P ~
.. o \_ , ~ ~ ~
~ ~ ~ C
,
- 45 -
.. . . ~ . ,,` ,~ , :....... . - :
1~257~
Z
~ Z~ , .
Z~O~z
` ~z/O z ~
t~ ~, ~
, ~ ~ ~_, ~, , .
~ _, o
~ ~, ~
., ~ ~ .
,, .,, .,, ~
zQz~
; ~ , ~ ~ :~ I X
O O ~
y~ IZ o ~
.` zo~ ~ ;~V
~i" ~ --<Oj~; / h ~ o ~: ~1 . .
O ~ h `--O ~ t~
Q z ~ o~ ~ o ~ ~ ~ ~ h
.; ,_, ~ ~ 0 ~ o
:, C u~ ~z_ ~ ,~ h ~, O z ~`I 1
11 ~ , ~ ~ 'r ~0., ~0~0~
_I ~_ ~--~ c. td h ~ o to C~
I ~ u~ ~_ /
.;i ~ O ~_ ~_
: .~ . IP........... ~ / ~_ h ~ ~ ,~
~;, . I ~, ~C 1 i~
. I ~ y Z
lo~ ~ ~ ~ z--< ~ ~ ~ o
~, I ~ ~0~~_ O X ~ ~ U h h ^
,:'1 . I ~ ~ O r_ u~ h S: ~ ~ 0
. I I ~ :~ZOZ C~ o ~0--I
~i 11 ~ ~ ~ ~ '~a~E~
o ~,~ o ~ ~
~ .
4 6
'r.
:~'
~0~257~7
Z~O/ ~ ~
Z ~ ¢
Z ~ ¢Z ~
~ \ ~ ~ ~ ,
. ~ ~-~ ~1 ~
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10~2S7 7
In addition to the compounds mentioned above, useful
coenzyme-active conjugates include the adenosine phosphates
to which are coupled the specific binding substance through
the phosphate grouping. Such compounds have the following
general formula: NH2
~ - ~ JN
; 1 N'- N
~ R -C ~ O ~
;' I .
OH OH
O~ OQ O~
;~ wherein Rl is O p-O-R2 o p o-P-O-R2 or
O , O O
~) ~.,-
-O-P-O-P-O-P-O-R
1 5 : ~:
, .
-~ wherein R2 is -Y-Z; wherqin Y is a bond or a bridge group;
; and wherein Z is a ligand, a specific binding analog of a
ligand, or a specific binding partner of a ligand. Also,
the protonized or acid forms, as well as the salt forms
where appropriate, may be used.
Synthesis of such compounds may be accomplished in a
variety of ways. It is contemplated that the synthesis
.
routes which are schematically illustrated below are advan-
: tageously followed in the preparation of the useful com-
~ 25 pounds. The abbreviations used hereinbefore also apply to
,~, the illustration to follow.
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1t)82S7~7
In one form of the present invention, the components of
the specific binding reaction which are to be combined with
the liquid medium suspected of containing the ligand are in
a liquid or solid form. In the preferred homogenous assay
system, the components are usually in solution or in a solid
form capable of ready dissolution in the liquid medium.
Since the liquid medium to be tested is normally aqueous in
character, the components are generally in a water soluble
form, that is, either in aqueous solution or in a water
soluble solid form such as a powder or resin. The assay
method may be carried out in a standard laboratory vessel
such as a test tube with the specific binding reaction
components and the components of the reaction system being
added thereto in solid or liquid form.
; 15 It is also contemplated that one or more of the speci-
..~
~: fic binding reaction components and/or one or more of the
components of the monitoring reaction may be incorporated
with a carrier. In one aspect, the carrier may be a liquid-
holding vessel such as a test tube or capsule containing
such component or components in an interior portion thereof,
for instance, in the ~orm of a liquid or loose solid or a
coating on an interior surface of the vessel. In another
i aspect, the carrier may be in the form of matrix which is
,, .
insoluble and porous, and preferably absorbent, relative to
the liquid medium to be tested. Such matrix may be in the
form of bibulous papers; polymeric films, membranes, fleeces,
or blocks; gels; and so forth In such a form, the device
- 60 -
:`:
. . .
~:.
~082S7~
would provide a convenient means for contacting the liquid
medium to be tested, for carrying out the specific binding
reaction and/or the monitoring reaction, and for observing
the resulting response.
The liquid medium to be tested may be a naturally
occurring or artificially formed liquid suspected of con-
taining the ligand, and usually is a biological fluid or a
liquid resulting from a dilution or other treatment thereof.
; Biological fluids which may be assayed following the present
method include serum, plasma, urine, and amniotic, cerebral,
and spinal fluids. Other materials such as solid matter,
for example tissue, or gases may be assayed by reducing them
to a liquid form such as by dissolution of the solid or gas
in a liquid or by liquid extraction of the solid.
In contrast to the prior art homogenous assay system,
biological fluids containing substances which have reactant
activity similar or identical to that of the conjugated
labeling substance may be assayed for the ligand without
background interference. Endogenous background reactant
activity can be readily eliminated in several manners. The
biological fluid can be treated to selectively destroy the
endogenous reactant activity. Such treatment may involve
the action of a clearing agent which chemically destroys the
endogenous activity followed by treatment to inactivate the
destructive action of such clearing agent.
For instance, reactant-degrading enzymes often appear
naturally in biological fluids, particularly if the reactant
is a coenzyme such as NAD, NADP, or ATP. There are many
. . .
~ - 61 -
;,' ,.
1~82S7t7
inhibitors of s~ch coenzyme-degrading enzymes, for example,
chelating agents which operate to deprive the enzymes of
essential metal -on activators. As a specific example, NAD-
degrading enzyr s are found in normal serum and have suf-
S ficient enzymatic activity to remove essentially all endogen-
ous NAD activit~- from isolated serum within a few hours.
The degrading ac.ivity of such enzymes may be effectively
inhibited by adc-tion of a chelating agent such as ethylene-
diamine tetraace~ic acid. Elimination of the degrading
activity may alc~ be accomplished by adding a specific
enzyme inhibito~. For example, ATP-degrading enzymes may be
inhibited by addition of ~y methylene ATP or ~ methylene
ATP.
The present invention will now be illustrated, but is
not intended to ~e limited, by the following Examples,
"
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EXAMPLE 1
Preparation of nicotinamide 6-(2-aminoethylamino)
purine dinucleotide.
Two (2) grams of nicotinamide adenine dinucleotide
(NAD) were dissolved in 10 ml of water and 0.6 ml of ethyl-
eneimine was added dropwise, the pH being maintained below 7
by the addition of 1 M perchloric acid. When addition of
ethyleneimine was complete, the pH was adjusted to 4.5 and -
the reaction was incubated at 20-25C. At 24 hour intervals
0.6 ml of ethyleneimine was added and the pH readjusted to
4.5. After 96 hours, the solution was poured into 10 volumes
of acetone at -10C. The oil which formed was collected,
;i washed with ether, and dissolved in approximately 50 ml of
water in a flask.
The resulting solution was adjusted to pH 7.0-7.5 with
1 N sodium hydroxide, and 1 gram of sodium bicarbonate was
.. . .
added. Nitrogen was bubbled through the solution for from 4
to 5 minutes and 1 gram of sodium hydrosulfite was added.
`l The flask was sealed tightly and allowed to stand at room
' 20 temperature for 45 minutes. The solution was then oxygenated
,1 for 15 minutes and adjusted to pH 11.3 with sodium hydroxide.
The solution was heated at 75C for 1 hour. Then the reaction
mixture was cooled to room temperature and 0.6 grams of
:i
tris-(hydroxymethyl)-aminomethane was added, followed by 5 N
~` 25 hydrochloric acid to adjust the pH to 7.5. To the resulting
`; solution was added 1000 International units of alcohol de-
hydrogenase and 1 ml of acetaldehyde. The decreasing
optical density of the reaction mixture was monitored at 340
` nm and when no further decrease was observed, the pH was
.. .
- 63 -
: '.' "
. . .
, .
~ .
. . . . ..
~08257~7
adjusted to 3.5. The solution was poured into 10 volumes of
acetone at -10C. The oil which formed was separated and
washed ~ith ether, after which it was dissolved in 10 to 15
ml of water.
The resulting solution was introduced into a 2.5x90 cm
column of Sep~adex G-10, available from Pharmacia AB,
Uppsala, Sweden, equilabrated with water. Fractions of 12
ml volume were collected. The wavelength of maximum optical
absorption in the ultraviolet region and the optical density
at such wavelength were determined for each fraction. Also,
the optical density at 340 nm of each fraction after reduc-
tion with alcohol dehydrogenase was determined. The frac-
; tions which had an optical absorption maximum at 264 nm and
had a ratio of optical density at 340 nm to that at 264 nm
~ - .
; 15 greater than 0.05 were pooled. The pooled material was
concentTated to from 15 to 20 ml on a rotary evaporator and
.*
passed through a 2.5x28 cm column of Dowex l-X8, available
from Bio-Rad Laboratories, Richmond, California, equilabrated
with water. Additional water was added to wash the pooled
,~ .
material through the column, and 10 ml fractions were
collected. The fractions which had an optical absorption
maximum at 264 nm and had a ratio of optical density at 340
nm to that at 264 nm greater than 0.1 were p'ooled.
The pooled material was passed through a 5x45 cm
; 25 column of ~owex 50-X2, available from Bio-Rad Laboratories,
Richmond, California, equilibrated with water. Additional
,:,
~ater was added to wash the poole~d material through the
column and 20 ml fractions were collected. The fractions
,, .
- 64 -
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C
~ ~ * Trade Mark
.: :
~, ' ' .: , ." ';:
~08ZS7~7
- which had an optical absorption maximum at 264 nm and had a
ratio of optical density at 340 nm to that at 264 nm greater
than 0.18 were pooled. The pooled material was concentrated
to from 4 to 5 ml and purified by electrophoresis as follows.
The concentrated material was applied to a sheet of
.,, :
Whatman 3MM paper, available from Reeve Angel, Clifton, New
Jersey, in a 1 to 2 cm wide strip perpendicular to the
aj direction of current flow. The paper was then wetted with -
0.02 M sodium phosphate at pH 6Ø Electrophoresis was
; 10 conducted according to the Durrum hanging paper method, as
described in Science 121:829(1955), for 4-7 hours with a
! potential gradient of about 8.5 volts/cm. The location of
. . . .the desired pyridine nucleotide derivative was determined by
fluorescence developed after spraying a test strip of the ;
paper with 0.5 M sodium cyanide according to the procedure
~¦ described in J. Bio~. Chem. 191:447(1951). The area contain-
ing the desired derivative was cut out of the paper and
extracted with three (3) 50 ml volumes of water. The result-
~ ing extracts containing nicotinamide 6-(2-aminoethylamino)
l 20 purine dinucleotide were pooled, concentrated to from 3 to 4
` ml, and stnred at -20C.
,., :
, EXAMPLE 2
Preparation of nicotinamide adenine dinucleotide
-biotin conjugate.
A 16 mg quantity of biotin was suspended in 1 ml of
water containing 22 mg of nicotinamide 6-(2-aminoethylamino)
,~ .
purine dinucleotide prepared as in Example 1. A few drops
. - 65 -
~, .
,. ' .
. .
. . . -
' ' ' ' ' : ; ' ' ': ~
lO~:S7~7
of 0.1 N sodium hydroxide was added to aid dissolution of
the biotin. A 240 mg quantity of 1-cyclohexyl-3-(2-morpho-
linoethyl)-carbodiimide metho-p-tolulene sulfonate was added
to the resulting solution and brought into solution by
dropwise addition of 0.1 N hydrochloric acid. The reaction
mixture was allowed to incubate at room temperature for 5
hours and was then poured into 10 ml of acetone at -10C.
The oil which formed was separated, washed twice with from 5
to 10 ml of ether and dissolved in from 1 to 2 ml of water.
The resulting material was purified by electrophoresis on
.I paper as in Example 1. Two fluorescent bands appeared after
spraying with sodium cyanide, one having migrated toward the
cathode and the other toward the anode. The latter band,
which contained the NAD-biotin conjugate, was eluted with
, 15 water and stored at -20C.
,
EXAMPLE 3
~: Preparation of nicotinamide adenine dinucleotide
-2,4 dinitrophenyl conjugate.
.,
A 26 mg quantity of sodium bicarbonate was dissolved in
1.5 ml of water containing 23 mg of nicotinamide 6-~2
-aminoethylamino) purine dinucleotide prepared as in Example
; 1. To the resulting solution was added 3 ml of ethanol
containing 17 ~1 of 2,4 dinitrofluorobenzene. The reaction
. ,1
mixture was stirred at room temperature in the dark for 5
hours after which 45 ml of acetone at -10C was added there-
, to. The precipitate which formed was separated, washed
. twice with 10 ml of acetone, and stirred with 5 ml of
water. The yellow soluble material which separated was
- 66 -
..
, ,
~082S7~7
purified by electrophoresis on paper as in Example 1 for 5
hours. The band which migrated toward the anode, and which
contained the NAD-2,4 dinitrophenyl conjugate, was eluted
with water, concentrated to from 3 to 5 ml, and stored at
-20C.
'" " '
EXAMPLE 4
; Preparation of biotin-umbelliferone conjugate.
A reaction mixture was formed by dissolving in 10 ml of
dimethylformamide 100 mg of umbelliferone, 167 mg of biotin,
i 10 and 141 mg of dicyclohexyl carbodiimide. The reaction
I mixture was incubated at -18C for about 4 hours, then
- overnight at 7C and allowed to stand at room temperature
.i, ,
for from 3 to 4 hours. An additional 141 mg of dicyclohexyl-
carbodiimide was added and the reaction mixture was stirred
.:~ -, .
at 7C for from 3 to 4 hours and allowed to stand at room ;
temperature overnight. The resulting precipitate was fil-
tered off and discarded. To the filtrate was added 75 ml of
ice water, and the resulting mixture was incubated at 0C
for 1 hour. The precipitate which resulted was filtered off
and discarded. The filtrate was evaporated to dryness and
the residue dissolved in from 3 to 4 ml of methylene chloride.
i, I
To the resulting solution was added 5 ml of diethylether.
The resulting precipitate which comprised the biotin-
umbelligerone conjugate was filtered off, dried, and stored
at room temperature.
.
...
- 67 -
''
.
.
~0~32S7~7
.'
EXAMPLE 5
Effect of avidin and biotin on the enzymatic cycling
rate of NAD and NAD-biotin conjugates.
The cycling reaction system used in this Example was
based on the following reactions:
,i
a) NAD-ligand + lactate dehydrog
NADH-ligand + pyruvate
i (b) NADH-ligand + thiazolyl blue (oxidized) diaphoraseJ_
~ 10 NAD-ligand + thiazolyl blue (reduced)
.
Eight specific binding reaction mixtures were prepared, each
having a total volume of 0.5 ml and containing 0.12 M N,N
bis-2-hydroxyethylglycine hydrochloride buffer at pH 7.8 and
respectively containing the concentrations and activities
indicated in Table 1 of NQD, NAD-biotin conjugate prepared
as in Example 2, biotin, and avidin, which latter has an
affinity for binding with biotin. One (1) unit of avidin
activity is that quantity of avidin capable of binding 1 yg
....
. of biotin. The reaction mixtures were incubated at room
temperature for from 2 to 3 hours. Each reaction mixture
was contacted with an aqueous enzyme/substrate mixture by
.;
~1 the addition of 0.1 ml of 1 M lithium lactate, 0.05 ml of 10
;~l mM thiazolyl blue in its oxidized form, and a sufficient
....
~I quantity of 0.12 M N,N bis-2-hydroxyethylglycine hydrochloride
i,
; 25 buffer at pH 7.8 containing 0.38 International units of
bovine heart lactic dehydrogenase, and 1.5 International
'!''~ units of porcine heart diaphorase to give a total reaction
.:.,
- 68 -
`~ .
~: , . . , , . -
.. . .. ~ : . . .: .
1082S~
volume of 1 ml. The relative rate of production of the
reduced form of thiazolyl blue was then determined in each
of the reaction mixtures by measuring the total change in
the optical density in each thereof at 570 nm during a 24
minute period within the first hour after the addition of
the enzyme/substrate mixture. The entire procedure was
performed in duplicate and the averaged results appear in
Table 1.
. `
,,
; - 69 -
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. . . . .
,. . . . . . .
.
~rl
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h C) R
R o o o ~ o o ~ sD o
~ o ,1 o o ~ o o o
Cd U~
a~ oooooooo
.,
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.~ ~.rl ~ I I I . . .
: R o o o o
." ~
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h-~ I I I ~D
R O t
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.sl R R
~ 3 U ~
Y' td R~
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R o ~ ~ t~
R, l
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.~ U ¢
`:i Z
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h
a
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... , ~Z
, ! g
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.
.. , .. . .. ` . , .. , .: .. , .. , ,, . ,. ` ., . , . ~: .
10~
Reactions 1~ 4, and 8 were controls and show that in
the absence of NAD and the NAD-biotin conjugate essentially
no cycling occurred. The results of reactions 2 and 3
demonstrate that the NAD-biotin conjugate has a significant
S amount of coenzyme activity relativ0 to native NAD. It can
be seen from the results of reactions 3 and 6 that the
presence of avidin in the reaction mixture inhibits the for- '
mation of thiazolyl blue (reduced form) where the NAD present
is conjugated with biotin. By comparing the results of
reactions 6 and 7 it can be seen that the presence of free
biotin reduces the amowlt of inhibition of thiazolyl blue
(reduced form) formation in proportion to the concentration
of biotin in the reaction mixture.
It was thus demonstrated in this Example that the
activity of the NAD in the NAD-biotin conjugate relative to
; the cycling reaction system was decreased in the presence of
,.~
avidin and that the magnitude of such decrease in activity
was reducet by the additional presence of biotin.
, . . .
;, , EXAMPLE 6
Direct binding-cycling assay for avidin; effect of
' varying levels of avidin on the cycling rate.
,~ The cycling reaction system used in this Example was
'i the same as that diagrammed in Example 5. Seven specific
.~i binding reaction mixtures were prepared, each having a total
,
volume of 0.6 ml and each containing 0.12 M N,N bis-2-
hydroxyethylglycine hydrochloride buffer at pH 7.8 and
. ~ .
,, 250 nM NAD-biotin conjugate prepared as in Example 2. Six
of the reaction mixtures also contained avidin in the
!~ amounts indicated in Table 2,
~ - 71 -
:.
,:
... .
. .
.
"- ~,0~2S7q
The reaction mixtures were incubated at room tempera-
ture for from 2 to 3 hours. Each reaction mixture was
contacted with an aqueous enzyme/substrate mixture by the
addition of 0.1 ml of 1 M lithium lactate, 0.05 ml of 10 mM
thiazolyl blue in its oxidized form, and a sufficient quan-
tity of 0.12 M N,N bis-2-hydroxyethylglycine hydrochloride
buffer at pH 7.8 containing 0.38 International units of
bovine heart lactic dehydrogenase and 1.5 International
units of porcine heart diaphorase to give a total reaction
volume of 1 ml. The relative rate of production of the
reduced form of thiazolyl blue was then determined in each
of the reaction mixtures by measuring the total change in
the optical density in each thereof at 570 nm during a 24
minute period within the first hour after the addition of
the enzyme/substrate mixture. The ratio, expressed as
percent, of the change in optical density in each reaction
mixture containing avidin to that in the reaction mixture
not containing avidin was calculated and is referred to in
j Table 2 and Figure 1 as the relative cycling rate. The
; 20 results appear in Table 2 and in graphical form in Figure 1
of the drawing.
TABLE 2
amount of relative
reaction avidin added cycling
mixture (units) rate (~)
,! 1 0 0 0 0 1 0 0
2 0.005 96
3 0.010 93
4 0.045 84
' 30 5 o.090 68
6 0.120 51
7 0.180 8
- 72 -
. . . . .
108257~7
It was demonstrated in this Example that the relative
cycling rate of the cycling reaction system, and thus the
activity of the NAD in the NAD-biotin conjugate, was an
inverse function of the amount of avidin present in the
specific binding reaction mixture. The present invention
therefore provides a test composition and method for quanti-
tatively determining the presence of the ligand avidin in a
liquid medium using a direct binding-cycling assay technique.
EXAMPLE 7
Competitive binding-cycling assay for biotin; effect of
varying levels of biotin on the cycling rate.
." ~
The cycling reaction system used in this Example was
' the same as that diagrammed in Example 5. Seven specific
binding reaction mixtures were prepared, each having a total
volume of 0.45 ml and each containing 0.12 M N,N bis-2-
;j~ hydroxyethylglycine hydrochloride buffer at pH 7.8 and 180
nM NAD-biotin conjugate prepared as in Example 2. Six of
( the reaction mixtures, i.e. nos. 1 through 6 in Table 3,
s~ additionally contained 0.11 units of avidin. Also, biotin,
~i~ 20 at the concentrations indicated in Table 3, was included in
five of the six reaction mixtures containing avidin, i.e.
.
mixtures 2 through 6 in Table 3.
The reaction mixtures were incubated at room tempera-
ture for from 2 to 3 hours. Each reaction mixture was
contacted with an aqueous enzyme/substrate mixture by the
addition of 0.1 ml of 1 M lithium lactate, 0.05 ml of 10 mM
thiazolyl blue in its oxidized form and a sufficient quan-
tity of 0.12 M N,N bis-2-hydroxyethylglycine hydrochloride
. . .
- 73 -
.
.
'
. ,: '
' " , , ~ ~
:, , .
-
~OBZ57 7
buffer at pH 7.8 containing 0.38 International units of
bovine heart lactic dehydrogenase and 1.5 International Units
of porcine heart diaphorase to give a total reaction volume of
1 ml. The relative rate of production of the reduced form of
thiazolyl blue was then determined in each of the reaction
mixtures by measuring the total change in the optical density
in each thereof at 570 nm during a 24 minute period within
the first hour after the addition of the enzyme/substrate
mixture. The ratio, expressed as percent, of such change in
optical density in each reaction mixture containing biotin to
that in the reaction mixture not containing either biotin or
avidin was calculated and is referred to in Table 3 and
Figure 2 as the relative cycling rate. The results appear
in Table 3 and in graphical form in Figure 2 of the drawing.
; TABLE 3
; reactioni concentration relative
mixture of biotin cycling
(nM) rate (%)
1 0 8
2 80 22
3 160 35
4 320 63
400 80
6 800 92
It was demonstrated in this Example that the relative
cycling rate of the cycling reaction system, and thus the
activity of the NAD in the NAD-biotin conjugate, was a direct ~-
function of the amount of biotin present in the specific -
; binding reaction mixture. The present invention
- 74 -
', r~
- . : . ,: ;~ ~,
~.' :. ' -: ,: - :
-"` 108Z57~
therefore provides a test composition and method for quanti-
tatively determining the presence of the ligand biotin in a
liquid medium using a competitive binding-cycling assay
technique.
EXAMPLE 8
; Direct binding-cycling assay for antibody to 2,4
- dinitrophenyl and derivatives thereof.
The cycling reaction system used in this Example was
the same as that described in Example 5. Eight 0.6 ml
specific binding reaction mixtures were prepared, each
containing 0.12 M N,N bis-hydroxyethylglycine hydrochloride
buffer at pH 7.8 and respectively containing the amounts and
concentrations indicated in Table 4 of NAD, NAD-2,4 dinitro-
phenyl conjugate prepared as in Example 3, antiserum to 2,4
' 15 dinitrophenyl, and nicotinamide mononucleotide (NMN). The
reaction mixtures were incubated at room temperature for
from 3 to 4 hours. Each reaction mixture was contacted with
an aqueous enzyme/substrate mixture by the addition of 0.1
. .
ml of lM lithium lactate, 0.05 ml of 10 mM thiazolyl blue in
its oxidized form, and a sufficient quantity of 0.12 M N,N
bis-hydroxyethylglycine hydrochloride buffer at pH 7.8
containing 0.38 International units of bovine heart lactic
dehydrogenase and 1.5 International units of porcine heart
diaphorase to give a total reaction volume of 1 ml. The
relative rate of production of the reduced form of thiazolyl
,~ blue was determined in each of the reaction mixtures by
;i measuring the total change in the optical density in each
.,;
~ - 75 -
,
..
;.~
..,
. .
.; .
.. ~., . - -
': . .!i.. ` .
.~:,. : .
~()82S7~7
thereof at 570 nm during a 24 minute period within the first
hour after the addition of the enzyme/substrate mixture.
The entire procedure was performed in duplicate and the
averaged results appear in Table 4.
'; ,:
'
- "
.~ ''
:,
. . ~:
. ' .
: -
.,
~,~
?,~
~ ...................................................................... .
:,~' .
.;
;., :
~ 76 -
.. .
.:," ~ '.
~`: .
~D, ' ' . , ~ ' ' .
10~3Z57~7
rl
Ul ,,
V~
R
R o o ~ o cnr~ ~t ~ ~
.~ _"~ o ~ ~ ~ o o o
a~u~ oooooooo
~d
s~ P~
~ o
o ~ :~ '
~ ,_
,; ~ ~ ,~ o o o o
I I ~ o o o o
o ~ . .
" ~ . .
~ .
,: o
'`i o~ :~
,~ t'
`i h ~ , , , o , o , o
?, a~
.-
,~ o
.:~ V
, . .
~1 ¢~ ..
,` ~1 Z
.
"': ¢ ~o ~ '.
o o o
O ~`-- I ~ I I I Io. o~. .
.. o ~ ~ '~
"~ o : .
R
U
U ~
~:,', o
., ~
`'' .,1 ,_
~r~ h `-- ~ I~ t`
¢
Z
.',
~`'`' U
.:
r.;~ O
~rl
u ~1 (~ ~ ~ In ~ t~ oo
h
~i;. - 77
'
`:
~'
,. . . :
.. , . . . ~
,.:
,: :.
~.osz57q
Reaction 1 was a control and shows that in the absence
of NAD and the NAD-2,4 dinitrophenyl conjugate essentially
no cycling occurred. From the results of reaction 2 it is
demonstrated that the NAD-2,4 dinitrophenyl conjugate is -~
active in the enzymatic cycling system. The results of
reactions 3 and 5 indicate that the presence of antibody to
2,4 dinitrophenyl inhibits the cycling of NAD. As shown by
the results of reaction 6, such inhibition is reversed by
addition of NMN. From the results of reactions 3 and 4 it
is seen that the cycling rate in the presence of NMN is ~-
; about 15% greater than in its absence. This result is
probably due to contamination by extraneous NAD because
other measurements have shown that NMN does not influence
the cycling rate in the absence of antibody. Nevertheless,
the antiserum contains some activity with respect to NAD
itself which activity is inhibited by the presence of NMN.
- It was thus demonstrated in this Example that the
activity of the NAD in the NAD-2,4 dinitrophenyl conjugate
relative to the cycling reaction system was decreased in the
presence of antibody to 2,4 dinitrophenyl. The present
invention therefore provides a test composition and method
; for determining the presence of the ligand antibody to 2,4
dinitrophenyl in a liquid medium using a direct binding - `-
' cycling assay technique.
::
EXAMPLE 9
Competitive binding-cycling assay for 2,4-dinitrobenzene
- and derivatives thereof; effect of various levels of N (2,4
,,.;
dinitrophenyl)-6-aminocaproate on the cycling rate.
- 78 -
:;:
~,.,
: .
: , .
- `
108Z57~
The cycling reaction system used in this Example was
the same as that diagrammed in Example 5. Seven specific
binding reaction mixtures were prepared, each having a total
volume of 0.6 ml and each containing 0.12 M N,N
. 5 bis-hydroxyethylglycine hydrochloride buffer at pH 7.8, 300
nM NAD-dinitrophenyl conjugate prepared as in Example 3, and
50 ~M nicotinamide mononucleotide. Six of the seven reac-
tion mixtures, i~eO nos. 1 through 6 in Table 5, also
contained an amount of antibody to 2,4 dinitrophenyl suffi-
cient to inhibit the cycling rate of the other reaction
mixture by 85 percentO N (2,4 dinitrophenyl)-6-aminocaproate,
a derivative of 2,4 dinitrobenzene prepared by the method
described in Biochem. J. ~2: 287~1948), was also included in
;~ five of the six antibody-containing reaction mixtures, i.e.
nos. 2 through 6 in Table 5, at the concentrations indicated
! in said Table.
The reaction mixtures were incubated at room tempera-
ture for about 4 hoursO Each reaction mixture was contacted
l,!
with an aqueous enzyme/substrate mixture by the addition of
Ool ml of 1 M lithium lactate, 0.05 ml of 10 mM thiazolyl
i .,
blue in its oxidized form, and a sufficient quantity of 0.12
. :
M N,N bis-hydroxyethylglycine hydrochloride buffer at pH 7.8
. .,
containing 0.38 International Wlits of bovine heart lactic
dehydrogenase and 1.5 International units of porcine heart
diaphorase to give a total reaction volume of 1 ml. The
, ~ .
;~` relative rate of production of the reduced form of thiazolyl
i blue was determined in each of the reaction mixtures by
measuring the total change in the optical density in each
~t thereof at 570 nm during a 24 minute period within the first
30 ! hour after the addition of the enzyme/substrate mixture.
.,
- 79 -
`~:
. .
:"' ' ' ' '' ~ - ' '
.. ~ . .
~O~}ZS7~7
.
The ratio, expressed as percent, of such change in optical - -
density in each reaction mixture containing N (2,4
dinitrophenyl)-6-aminocaproate to that in the reaction
mixture containing neither N (2,4 dinitrophenyl)-6-amino-
caproate nor antibody to 2,4 dinitrophenyl was calculated
and is referred to in Table 5 and Figure 3 as the relative
cycling rateO The results appear in Table 5 and in graphi-
cal form in Figure 3 of the drawing.
TABLE 5
concentration of relative
reactionN (2,4 dinitrophenyl) cycling
mixture-6-aminocaproate (nM) rate (%)
1 0 16
2 17 19
;15 3 42 30
; 4 83 35
166 41
6 415 76
It was thus demonstrated in this Example that the
relative cycling rate of the cycling reaction system, and
thus the activity of the NAD in the NAD-dinitrophenyl con-
jugate, was a direct function of the amount of N (2,4
dinitrophenyl)-6-aminocaproate present in the specific bind-
ing reaction mixture. The present invention therefore
provides a test composition and method for quantitatively
determining the presence of the ligand N (2,4 dinitrophenyl)
, -6-aminocaproate in a liquid medium using a competitive
binding-cycling assay technique.
- 80 -
, :
:.'; -, .
10~257~7
EXAMPLE 10
Direct binding-bioluminesence assay for avidin; effect
of presence of biotin on the peak light intensity produced.
The bioluminescence reaction system used in this Example
was based on the following reactions:
(c) NAD ligand ethanol dehydrogenase
NADH-ligand + acetaldehyde
(d) NADH-ligand + FMN* + H~ dehydrAODgHenaSe~-
NAD-ligand + FMNH2
(e) FMNH2 + long-chain aldehyde + 2 luciferase~_ -
FMN + long-chain acid + H20 + hv
,,
*flavin mononucleotide
~, .
A light-generating solution for carrying out reactions
(d) and (e) was prepared as follows. A reagent mixture was
prepared containing 0.13 M phosphate buffer at pH 7.0, 0.67
wt% bovine serum albumin, 15.7 ~M flavin mononucleotide
;; (FMN), and 13.3 mM sodium acetate, and this mixture was
stored in the dark at -20C. An emulsion of 5 ~1 of dode-
canal in 5 ml of water was prepared the day the light-
generating solution was to be used. Lypohilized luciferase
extracted from Photoba¢terium f%sheri ~enzyme available from
Worthington Biochemical Corp., Freehold, New Jersey) was
added to 0.013 M phosphate buffer at pH 7.3 to a concentra-
tion of 20 mg/ml. After 30 minutes the resulting suspension
- 81 -
',~
lO~Z57~
was centrifuged at 1500xg for 10 minutes and the pellet was
discarded. The light-generating solution was then prepared
within 5 minutes of use by combining 75 ~1 of the reagent
mixture, 5 ~1 of the dodecanal emulsion, and 20 ~1 of the
luciferase solution.
To detect the light produced by reaction (e) a photo-
meter was constructed consisting of a photodetector and a 6
x 50 mm cuvette mounted within a light integrating sphere
such that light generated in the cuvette was reflected onto
the photodetector. The electronic signal produced by the
photodetector was passed to a strip chart recorder. The
peak light intensity, as the term is used herein, was mea-
sured from the recorder trace and assigned arbitrary units
; based on the chart paper divisions.
Nine specific binding reaction mixtures were prepared,
~' each having a total volume of 0.2 ml and each containing
' 0.1 M tris-(hydroxymethyl)-aminomethane hydrochloride
buffer at pH 8.0, 0.01 M semicarbazide hydrochloride, and
respectively the amounts or concentrations indicated in
Table 6 of ethanol, NAD, NAD-biotin conjugate prepared as in
Example 2, biotin, and avidin. The reaction mixtures were
incubated at room temperature for 10 minutes. Then, 0.025
International units of alcohol dehydrogenase was added to
each reaction mixture to initiate a reduction reaction.
Semicarbazide combines with the acetaldehyde produced in
reaction (c) to form a semicarbazone and thus to drive
reaction (c) in the desired direction.
. '
. .
- 82 -
'; '
1()1!3Z57~7
The reaction mixtures were incubated at room tempera-
: ture for about 30 minutes. A 10 ~1 volume of each reaction
mixture was then injected into a separate cuvette mounted in
~ the photometer previously described and containing 100 ~1 of
S the previously prepared light-generating solution which had
been pre-incubated at 28C for from 2 to 3 minutes. The
results appear in Table 6.
,
:i ,
.
.
,1~
.1
-
'.,
.,
.
,, /
..
;
~ .
. ~ .
j - 83 -
..~
~:
.
-,' " ' ,: - ' ' ' `
1082s7~
~ ,~ ~ ~ o ~ ~ .,., L~
~ U~ ~ ~ U~
q~ ~
.,,
~ ~t d d ' '
., ~ ~.,, , , o , , o o o
o o o o
., ~d
.;
o o
o
r~H, .,."
.. ,.~ , .
ol . .
'1
, b O
.; ~ o ~' I I ~ el ~ e~
a
~ ~ `D t~ I
ib~ w o d
, .. 1 0 ~_ 1~ U~
h ~ ~ ~
.,.. , ~ .. ,
~j" ~
.'~, .~
o o o o o o
:: :
.. o
~ o
g ~ ~
- 84 -
. :'` .
'.`"`~ .
...
....
.: .; . ' . '' .
:.
.. . . , , :~: ,
. ,
~0~:57~
Reactions 1, 4, and 7 were controls and show that in
the absence of ethanol essentially no reaction occurredO
The result from reaction 5 demonstrates that the NAD-biotin
conjugate is active in the bioluminescence reaction system.
It can be seen from the results of reactions 5 and 6 that
the presence of avidin in the reaction mixture inhibits the
amount of light produced. From a comparison of the results
of reactions 6 and 8 it is seen that the presence of free
~ biotin reduces the amount of inhibition of light production- 10 as the concentration of biotin increases in the reaction
mixture. Reactions 2 and 3 demonstrate that avidin does not
inhibit the activity of free NAD and reactions 5 and 9 show
that the presence of biotin alone does not affect the
activity of the NAD-biotin conjugate.
It was thus demonstrated in this Example that the
activity of the NAD in the NAD-biotin conjugate relative to
the bioluminescense reaction system was decreased in the
presence of avidin and that the magnitude of such decrease
in activity was reduced by the additional presence of biotin.
EXAMpLE 11
Competitive binding-bioluminescence assay for biotin;
effect of varying levels of biotin on the peak light inten-
sity produced.
The biolumlnescence reaction system used in this Example
was the same as that diagrammed in Example 10. Seven specific
binding reaction mixtures were prepared, each having a total
. volume of 0.2 ml and each contain~ng 0.1 M tris-~hydroxy-
- methyl)-aminomethane hydrochloride bufer at pH 8.0, 0.6 M
- 85 -
.
' ' ' ' :
, ,. ~
: `
1()8Z57~7
ethanol, 0.01 M semicarbazide hydrochloride, 343 nM NAD
-biotin conjugate prepared as in Example 2, 0.025 Inter-
national units of alcohol dehydrogenase, and 0.055 units of
avidin. Biotin was added to six of the seven reaction
mixtures, i.e. nos. 2 through 7 in Table 7, in the concen- -
trations indicated in said Table. The order and manner of
j addition was the same as in Example 10.
.. The reaction mixtures were incubated at room tempera-
; ture for about 30 minutes. A 10 ~1 volume of each reaction
mixture was injected into;a separate cuvette mounted in the
photometer described in Example lQ and containing 100 ~1 of
a light-yielding solution prepared in the manner described
in Example 10 and pre-incubated at 28C for from 2 to 3
minutes. The entire procedure was run in duplicate, and the
averaged results appear in Table 7 and in graphical form in
, ~ ,
Figure 4 of the drawing.
.", :
-~ TABLE 7
average
reaction concentration of peak light
mixture biotin (nM) intensity
1 0 36
- 2 25 44
;.i
. . .
3 50 57
s 4 100 79
. 25 5 150 go
~ ,
6 200 97
7 300 104
.~ ' .
- 86 -
... . .
,',' ~ .
,': :
r,'.
'','" ' ' ~ . ,
. ' ' ' ' .
92$7~
It was thus demonstrated in this Example that the
rnagnitude of the peak light intensity produced by the bio-
:Luminescence reaction system, and thus the activity of the
NAD in the NAD-biotin conjugate, was a direct function of
the amount of biotin present in the specific binding reaction
mixture. The present invention therefore provides a test
composition and method for quantitatively determining the
presence of the ligand biotin in a liquid medium using a
competitive binding-bioluminescence assay technique.
EXAMPLE 12
Competitive binding-bioluminescence assay for
2,4 dinitrobenzene and derivatives thereof; effect of
various levels of N (2,4 dinitrophenyl)-6-aminocaproate on
the peak light intensity produced.
The bioluminescence reaction system used in this Example
was the same as that diagrammed in Example 10. Seven speci-
fic binding reaction mixtures were prepared, each having a
total volume of 0.1 ml and each containing 0.1 M tris-
; (hydroxymethyl)-aminomethane hydrochloride buffer at pH 8.0,0.01 M semicarbazide hydrochloride, 0.6 M ethanol, 35 ~M
nicotinamide mononucleotide, and 367 nM NAD-dinitrophenyl
conjugate prepared as in Example 3. N (2,4 dinitrophenyl)-
; 6-aminocaproate was added to six of the seven reaction
mixtures, i.e. nos. 2 through 7 in Table 8, at the concen-
. ~
~25 trations indicated in said Table, and to each of said six
: reaction mixtures was also added an amount of antibody to
2,4 dinitrophenyl sufficient to reduce the peak light-
intensity produced to 39% of that produced in the absence of
N (2,4 dinitrophenyl)-6-aminocaproate and antibody to 2,4
dinitrophenyl.
- 87 -
:- , -
, - . . : -
~08~S7~7
The reaction mixtures were incubated at room rempera-
ture for 3 hours. Then, 0.025 International units of alco-
hol dehydrogenase was added to each reaction mixture to
initiate a reduction reaction. The reaction mixtures were
then incubated at room temperature for about 30 minutes. A
10 ~1 volume of each reaction mixture was injected into a
separate cuvette mounted in the photometer described in
Example 10 and containing 100 ~1 of a light-generating
solution prepared in the manner described in Example 10 and
pre-incubated at 28C for from 2 to 3 minutes. The entire
procedure was run in duplicate, and the averaged results
appear in Table 8 and in graphical form in Figure 5 of the
drawing.
.
TABLE 8
concentration of average
reaction N (2,4 dinitrophenyl) peak light
mixture -6-aminocaproate ~M) intensity -
0.00 14
2 0.125 17
3 0.25 20
4 0.50 22
0.75 24
6 1.00 27
7 1.50 28
. . .
It was thus demonstrated in this Example that the
magnitude of the peak light intensity produced by the bio-
; luminescence reaction system, and thus the activity of the
NAD in the NAD-2,4 dinitrophenyl conjugate, was a direct
- 88 -
,.;
,
'
.~ ., ,: ............................ .
!
'
' ,
' I . '
10~257q
function of the amount of N (2,4 dinitrophenyl)-6-aminocaproate
present in the specific binding reaction. The present inven-
tion therefore provides a test composition and method for
quantitatively determining the presence of the ligand N
' 5 (2,4 dinitrophenyl)-6-aminocaproate in a liquid-medium using
i a competitive binding-bioluminescence assay technique.
EXAMPLE 13
Specific binding assays for biotin and avidin employing
an enzyme substrate as labeling substance.
- Th-e specific binding assay system used in this Example
was based on the following reaction:
~., O
' J~
~ 0--C--(CH2)4~ esterase
umbelliferone-biotin conjugate
(maximum fluorescence at 378 nm)
~ l H~ I biotin
(maximum fluorescence at 448 nm)
,, .
, Ten specific binding reaction mixtures were prepared,
each having a total volume of 0.3:ml and each containing 0.1
M tris-(hydroxymethyl3-aminomethane hydrochloride buffer at
p~ 8.0 and the respective amounts or-concentrations of
~ 25 umbelliferone-biotin conjugate prepared as in Example 4,
'1 biotin, and avidin indicated in Table 9. The-reaction
':,.`,',
, . ~ .
;,' .
,. . .
- ~08257t7
mixtures were incubated at room temperature for from 1 to 3
minutes. Reaction mixtures nos. 2 through 10 in Table 9
each also contained 0.26 International units of bovine liver
carboxylate hydrolase (esterase). The relative reaction
rate in each of the reaction mixtures was then determined by
monitoring the fluorescence produced by each thereof at 448
nm with a Model 111 Turner fluorometer (available from G.K.
Turner Assoc., 2524 Pulgas Street, Palo Alto, California)
set for excitation at 364 nm. The electronic signal produced
by the fluorometer was passed to a strip chart recorder, and
the amount of fluorescence praduced per minute was measured :~
from the recorder trace and assigned arbitrary units based
on the chart paper divisions. The results appear in Table 9.
, . .
'1 . ~
. .
., .
' .
.
:,.
~:
.~ i
.,,
,',; .
- 90 -
~r,~,.,
....
,'
. ......................................................................... .
,
' 1
108Z57q
a ~
a~ ~
R ~ o ~ U~ ~ oo o 1~ ~ ~ Ln
o ~ ~ o u~ o ~ t~
a) o o ~1 ~1 0 0 0 O` 0 ~1
h
o oooooooooo
4~
~, Ul
.- .,1
R ~ ~ u) t~
I I I o ~
~ ~ o o o o o o o
: ~ o o o o o o o
C~ ~
.' ~
,
i 4~
.,` o
~,_
"~ ~
C~o~ ' ' ' ' ' I ' ' o
~,D
:~ ~ .
¢ (~ o
.,., .~ ,_
o ~ o U~ o o o o o o o o
h h ~ ~ ~ ~ ~ ~ t~
o a) o
~} ~
.^.,
'`I a~
.`~ ~,_
~ ~ O `D ~ ~O
'~ h 3 ~ t~
OOOOOOOOO
` ! V) `--
. .
d ~ ~
h
:j - 91 -
, .' 1
~,
,
.;~ . .
:. . ` . : . :,. . .` ., , ` :
108Z57t7
Reaction 1 was a control and shows that in the absence
of esterase no reaction occurs. The results of reactions 2
and 3 demonstrate that the umbelliferone-biotin conjugate
was active in the enzymatic reaction, and comparing such
S results to those of reactions 4 through 8 demonstrates that
the presence of avidin inhibits the reaction rate in propor-
tion to the amount of avidin in the reaction mixture.
.~ Comparing the results of reactions 8, 9, and 10 shows that
the amount of inhibition of the reaction rate by avidin is a
direct function of the amount of biotin present in the
reaction mixture.
It was thus demonstrated in this Example that the rate
of fluorescence produced by the esterase reaction, and thus
the substrate activity of the umbelliferone-biotin conjugate,
was decreased by the presence of avidin and that the magni-
tude of such decrease in activity was reduced by the presence
~` of biotin. The present invention therefore provides a test
composition and method ~or determining the presence of the
~ ligands biotin and avidin in a liquid medium using a speci-
,. 20 fic binding assay technique employing an enzyme substrate as
r, the labeling substance.
.
,
,~.
, . .
~ j .
., . ~
. ~, .
,~ .
~ - 92 -
..
.:
!
';`'' '
,' :. :
. ,
' ' : - : , : ,
. , , , . :
: ' ~. . : ' . '' :', :. ' " ' '
o~æs77
EXAMPLE 14
Preparation of 2,4-dinitrophenyl-fluorescein conjugate.
Fluorescein-3'-[6-(2,4-dinitroanilino)hexanoate].
The synthesis basically involved the reaction of the
acid chloride of 6-(2,4-dinitroanilino)hexanoic acid with
the disodium salt of fluorescein. 6-(2,4-dinitroanilino)
hexanoic acid was prepared by the method described in
B~och~m. J. 42:287-94(1948).
A solution of 1.5 g (5 mmol) of 6-(2,4-dinitroanilino)
hexanoic acid was converted to the acid chloride by reaction
with 10 ml of warm thionyl chloride for 15 min followed by
cooling and dilution with 20 ml of hexane. The solid acid
chloride which formed was collected by filtration and after
thorough drying was added to 600 mg of the disodium salt of
fluorescein in 10 ml of dry acetone. After 5 hr at reflux,
the reaction was quenched by the addition of 2 ml water and
5 ml acetone. After 30 min at 25C, the mixture was concen-
trated to dryness and the residue partitioned between ethyl
acetate and aqueous sodium bicarbonate solution. The
organic phase was separated and washed with 1% aqueous
- sulfuric acid, dried over anhydrous magnesium sulfate and
evaporated. The red oil was chromatographed on 60 g of
.. ..
silica gel 60, available from E. Merck, Darmstadt, Germany,
with 20% (v/v) acetone in carbon tetrachloride as eluant.
The 1.2 g of impure bis-ester was rechromatographed on 60 g
of silica gel using 10~ (v/v) acetone in carbon tetrachloride.
Appropriate fractions were combined and evaporated to yield
180 mg of a yellow, glassy solid.
'~'' '
.~ .
~ - 93 - ~
` ` ~V~ 57~7
Calculated for C44H38N615
Found: C,60.92; H,4.35; N,6.65.
The infrared spectrum displayed the expected ester
carbonyl stretching absorption at 1765 cm 1.
.
EXAMPLE 15
Specific binding assays for derivatives of
2,4-dinitrophenyl and antibody thereto employing an enzyme
substrate (modified fluorescein) as labeling substance.
The specific binding assay systems used in this Example
was based on the reaction shown in Diagram 1.
A. Direct binding-fluorescent assay for antibody to
'r 2,4-dinitrophenyl; effect of various levels of antibody on
the reaction rate.
Seven specific binding reaction mixtures were prepared
and analyzed. For each reaction mixture, 20 ~1 of 1 ~M
2,4-dinitrophenyl-fluorescein conjugate (prepared according
to Example 14) in dimethylsulfoxide was combined with a
."
volume of antiserum to 2,4-dinitrophenyl as indicated in
Table 10 and with a sufficient volume of 0.1 M
bis-hydroxyethylglycine hydrochloride buffer at pH 7.0 to
`~ make a total volume of 2.0 ml. The background rate of
;` hydrolysis of the ester linkage in the conjugate was deter-
;' mined for three minutes for each reaction mixture by deter-
mining the rate of increase of fluorescence intensity at 510 nm
,.,~
;-~ 25 using the general technique described in Example 13 with the
''., :
:,,
,;........ :
, ~ .
, ,
~ 94
. .
.. ~ .
.
~` ~0~32S7~7
DIAGRAM 1
;
/ esterase
H20, pH 7 . 0 7
\l
`"~ O ::
2,4-dinitrophenyl--luorescein conjugate
,,
' . ~ - ~o~30 ' ~
.~ / + other products . -
.~.. ~ /
.,i ~, . . .
:. /
., O ,:
,~
(maximum fluorescence at 510 nm) :
. .
,.; .
' : ~
' ... :
;'` . :.
~,..................................................................... ....
R = - (CH2)5 - NH f ~ ~2 :
O N'
;.': ~
; :
-` ~08Z57~
fluorometer set for excitation at 470 nm. A 10 ~1 volume of
0~1 M bis-hydroxyethylglycine hydrochloride buffer at pH
7.0 containing 0.54 units of Type I esterase (E.C. No.
3.1.1.1, obtained from Sigma Chemical Co., St. Louis, Missouri)
was then added to each reaction mixture. The resulting
overall reaction rate was measured in the same manner as the
background hydrolysis rate. The results appear in Table 10.
The background hydrolysis rate was substrated from the
overall reaction rate to obtain the net reaction rate attribu-
table to the esterase-catalyzed reaction. The relationships
between both the net enzyme~catalyzed reaction rate and the
background alkaline hydrolysis reaction rate and the amount
of antiserum present in the reaction mixture is shown in
graphical form in Figure 6 of the drawing.
', .
1; TABLE 10
.1 :
` amount of background overall
'~3~ reaction antiserum hydrolysis reaction
~l mixture (~1) rate rate
. .
~i 20 1 0 0.02 2.68
2 5 0,07 2.48
~` 3 10 0,11 1.91
,~i 4 20 0.17 1.08
~ 5 30 0.21 0.63
;,il25 6 40 0.22 0.45
~; 7 60 0.26 0.30
. ' ' .
.
- 96 -
,; .
"' ~ ' . ,
. . . .
2S7~
.
It was demonstrated in this part of the Example that
the net reaction rate of the hydrolysis reaction was an
inverse function of the amount of antibody to the ligand,
2,4-dinitrophenyl, present in the specific binding reaction
mixture. It was likewise demonstrated that the reaction
rate of the background hydrolysis reaction was a direct
function of the amount of antibody present in the specific
binding reaction mixture. The present invention therefore
provides a test composition and method for determining the
presence of the ligand antibody to 2,4-dinitrophenyl in a
liquid medium using a direct binding-fluorescent assay
technique.
. .. .
B. Competitive binding-fluorescent assay for derivatives
i~ of 2,4-dinitrophenyl; effect of various levels of 2,4-
dinitrophenyl-~-alanine on the reaction rate.
Ten specific binding reaction mixtures were prepared,
~, .
each having a total volume of 2.0 ml and each containing
0.1 M bis-hydroxyethylglycine hydrochloride buffer at pH 7.0 -
and 2,4-dinitrophenyl-~-alanine, prepared according to the
method described in J. Amer. Chem. Soc. 76:1328~1954), at
~ the concentrations indicated in Table 11. To nine of the
g ten reaction mixtures, i.e. nos. 2 through 10 in Table 11,
, j .
5 ' was added an amount of antiserum to 2,4-dinitrophenyl suf-
, . . . .
ficient to inhibit the rate of the esterase-catalyzed reac-
~j 25 tion in the other mixture, i.e. no. 1, by 60 percent. After
mixing, 20 ~1 of 1 ~M 2,4~dinitrophenyl-fluorescein conjugate
(prepared as in Example 14) in dimethylsulfoxide was added
''' . `
- 97 -
~,
''`` , .
: .
., :
i~
~08:ZS7~7
to each reaction mixture. A 10 ~1 volume of 0.1 M
bis-hydroxyethylglycine hydrochloride buffer at pH 7.0
containing 0.54 units of Type I esterase (E.C. No. 3.1.1.1,
obtained from Sigma Chemical Co., St. Louis, Missouri) was
then added to each reaction mixture. The resulting reaction
rate was measured as in Part A of this Example. The percen-
; tage value of the rate of reactions nos. 2 through 10 to
that of reaction no. 1 (no antibody present) was calculated.
The results appear in Table 11 and in graphical form in
Figure 7 of the drawing.
., .
TABLE 11
concentration of percent of
reaction 2,4-dinitrophenyl reaction rate of reaction
mixture-~-alanine (nM) rate no. 1
t, 1 0 2.78
.~', 2 0 1.04 37
3 S 1.01 36
;'~ 4 10 1.04 37
; s 20 1.24 45
,' 20 6 30 1.51 54
,~:
7 50 1.54 56
8 75 1.80 65
9 100 1.85 67
150 2.33 84
`' .
,. :
!~ - 98
... .
::,
. .
: . , , .:
,.:
.
~o8z~7~7
It was demonstrated in this part of the Example that
the reaction rate of the hydrolysis reaction was a direct
function of the amount of 2,4-dinitrophenyl-~-alanine in the
reaction mixture. The present invention therefore provides
a test composition and method for determining the presence
of ligands such as derivatives of 2,4-dinitrophenyl in a
liquid medium using a competitive binding-fluorescent assay
technique.
C. Competitive binding-spectrophotometric assay for
derivatives of 2,4-dinitrophenyl; effect of various levels
of 2,4-dinitrophenyl-~-alanine on the reaction rate.
Eight specific binding reaction mixtures were prepared,
each having a total volume of 1.0 ml and each containing 0.1 M
; tris-(hydroxymethyl)-aminomethane hydrochloride buffer at
pH 7.0 and 2,4-dinitrophenyl-~-alanine at the concentrations
indicated in Table 12. To seven of the eight reaction ~ ;
mixtures, i.e. nos 2 through 8 in Table 12, was added an
amount of antiserum to 2,4-dinitrophenyl sufficient to
inhibit the rate of the esterase-catalyzed reaction in the
other mixture, i.e. no. 1, by 82 percent. After mixing, 10 yl
of 0.1 mM 2,4-dinitrophenyl-fluorescein conjugate ~prepared
as in Example 14) in dimethylsulfoxide was added to each
reaction mixture. A 20 ~1 volume of 0.1 M tris-~hydroxymethyl)
-aminomethane hydrochloride buffer at pH 7.0 containing
; 25 2.16 International units of Type I esterase ~E.C. No. 3.1.1.1,
obtained from Sigma Chemical Co., St. Louis, Missouri) was
then added to each reaction mixture. The change in absorbance
. . .
99
..~
~8ZS~7
, ,-
of each reaction mixture at 489 nm per minute was recorded
with a Gilford 2000 spectrophotometer. The results appear
in Table 12 and in graphical form in Figure 8 of the drawing.
. .
TABLE 12
. 5 concentration of
reaction2,4-dinitrophenyl rate of change
mixture-~-alanine (yM) of absorbance
i 1 0 0.0261
; 2 0 0.0047
3 1.25 0.0118
. ,~
v 4 2.5 0.0131
,; , .
5.0 0.0185
. 6 7.5 0.0202
7 10.0 0.0192
8 12.5 0.0223
~`' 'I ,.
~, It was demonstrated in this part of the Example that
.~ the reaction rate was a direct function of the amount of
2,4-dinitrophenyl-~-alanine in the reaction mixture. The
~, present invention therefore provides a test composition and
method for determining the presence of ligands such as
derivatives of 2l4-dinitrophenyl in a liquid medium using a
~, competitive binding-spectrophotometric assay technique.
~`,
,: ~
!, ~.`. .
". ~ - 100
,'~', .
`,':
;", "
'.~, .
~.`;'1' ' ~ `.`: ' `. `
o~257~7 1
D. Competitive binding-fluorescent assay for deriva-
tives of 2,4-dinitrophenyl; use of non-enzymatic monitoring
reaction.
The specific binding assay system used in this part was
the same as shown in Diagram 1 except that no esterase was
used to catalyze the hydrolysis of the ester linkage in
the conjugate.
Eight specific binding reaction mixtures were prepared,
each having a total volume of 2 ml and each containing 0.1 M
tris-(hydroxymethyl)-aminomethane hydrochloride buffer at pH
7.5 and 2,4-dinitrophenyl-~-alanine at the concentrations
indicated in Table 13. To each reaction mixture was added
50 ~1 of antiserum to 2,4-dinitrophenyl. After mixing, 20 ~1
of 2 ~M 2,4-dinitrophenyl-fluorescein conjugate ~prepared
as in Example 14) in dimethylsulfoxide was added to each ;~
reaction mixture and the resulting reaction rate was measured
as in Part A of this Example. The results appear in Table
13.
TABLE 13
concentration of
reaction2,4-dinitrophenyl reaction
- mixture -~-alanine ~nM) rate
. . .
1 0 0.96
: 2 12.5 0 94
3 31.2 0.84
4 62.5 0.78
94.0 0.70
6 125 0.59
7 187 0.57
8 250 0.53
- 101 -
.~'' ~ .
' lO~Z57q
It was demonstrated in this part of the Example that
the background hydrolysis rate, in the absence of esterase,
was an inverse function of the amount of 2,4-dinitrophenyl
-~-alanine in the reaction mixture. The present invention
therefore provides a test composition and method for deter-
mining the presence of ligands such as derivatives of
2,4-dinitrophenyl in a liquid medium using a competitive
binding-fluorescent technique wherein the binding partner, upon
becoming bound to the ligand in the conjugate, participates in
the monitoring reaction.
,,
EXAMPLE 16
Preparation of cortisol-umbelliferone conjugate.
Cortisol-21-hemisuccinate-umbelliferone.
A. Cortisol-21-hemisuccinate.
lS Succinic anhydride (0.5 g) was added to a solution of
0.5 g cortisol in 10 ml dry pyridine and stirred overnight at
room temperature. Water ~100 ml) was added and the mixture
extracted with 100 ml ethyl acetate. The organic phase was
,, .
:; washed once with water and extracted with 100 ml saturated
sodium bicarbonate solution. The aqueous phase was separated
and acidified to pH 4 with 10% hydrochloric acid. The precipi-
tate which formed was collected by filtration, dried, and re-
crystallized from a hexane-acetone mixture to yield the desired
intermediate (meeting point=171-2C).
.
~,
- 102 -
.,
~.
, . .
~.~)8Z57~7
B. Cortisol-umbelliferone conjugate. ~
; Carbodiimide (50 mg) was added to a solution of 100 mg ~ I
of the intermediate from Part A of this Example in 3 ml dry
dimethylformamide and stirred for 30 minutes. A solution of
50 mg 7-hydroxycoumarin in 2 ml dimethylformamide was added
and the reaction mixture was stirred overnight at room tem-
perature~ The precipitate which formed was filtered and
discarded. Water was added to the filtrate and the mixture
was extracted with ethyl acetate. The organic phase was
washed once with water, separated, dried with anhydrous
sodium sulfate, filtered, and evaporated to dryness under
vacuum. The residue was crystallized from an acetone-hexane
mixture yielding the desired conjugate ~melting point=126C).
EXAMPLE 17
Specific binding assay for cortisol employing an
enzyme substrate (modified umbelliferone) as labeling 1
substance.
The specific binding assay system used in this
, Example was based on the following reaction:
, ~ .
cortisol-umbelliferone H5OerpaHe8 5~ umbelliferone
conJugate 2
Eight specific binding reaction mixtures were prepared, each
~ having a total rolume of 2 ml and each containing 0.1 M bis-
`q hydroxyethylglycine hydrochloride buffer at pH 8.5 and
25cortisol at the concentrations indicated in Table 14. To
seven of the eight reaction mixtures, i.e. nos. 2 through 8
- 103 -
. . .
, ,
: .
: . . . .
r~
108257q
in Table 14, was added an amount of antiserum to cortisol
sufficient to inhibit the rate of the esterase-catalyzed
reaction in the other reaction mixture, i.e. no. 1, by 70
percent. After mixing, 20 ~1 of 2 yM cortisol-umbelliferone
S conjugate (prepared as in Example 16) in 0.1 M
bis-hydroxyethylglycine hydrochloride buffer at pH 8.5 was
added to each reaction mixture. After mixing again, 15 yl
of porcine esterase (0.81 units/ml) were added to each reac-
tion mixture. The resulting reaction rate was measured in
each reaction mixture in a manner similar to that described
in Example 13. The results appear in Table 14.
.
TABLE 14
.
reactionconcentration of reaction
, mixture cortisol (nM) rate -~
1 0 0.0838 -~
2 0 0.0256
~; 3 2.5 0.0296
4 10 0.0306
0.0381
~-' 20 6 30 0.0408
7 50 0.0654
' 8 100 0.0603
!
It was demonstrated in this Example that the reaction
rate was a direct function of the amount of cortisol in the
~`
-~-25 reaction mixture. The present invention therefore provides
a test composition and method for determining the presence
of cortisol in a liquid medium using a competitive binding
-fluorescent assay technique.
- 104 -
,~
:
'~ ' ' '
~,-- ' 10
EXA~PLE 18
Preparation of 2,4-dinitrophenyl-ATP conjugate (6-
position derivative).
N6-[2-(2,4-dinitrophenyl)aminoethyl] adenosine-5'
-triphosphate.
A. N6-(2-aminoethyl) adenosine-5'-monophosphate.
Two g (7 m~ol) 6-chloropurine riboside (available from
Sigma Chemical Co., St. Louis, Missouri) ~as stirred with ~-
17 ml triethylphosphate and was reacted with phosphoryl
chloride in the presence of rater as described in Chem. Scrip
19:165-70 (1972). After hydrolysis of the phosphodichloridate, -~
9.5 ml ethylenediamine (140 mmol) was added and allowed to react
at room temperature for 3 hr. The reaction mixture was
diluted to 4 litérs with water and ad~usted to pH 12 with
;' lS sodium hydToxide. This solution was passed through a 5x30 cm
column of Do-rex lx8 ~available from Bio-Rad Laboratories,
.,,; .`,~ Richmond, California) in the acetate form. Then the column
~' was ~ashed with 3 liters 0.01 M ammonium chloride and the` chromatogram was developed with a linear gradient generated -~;
with 3 liters water and 3 liters 1 M acetic acid. An
isolated peak of uv absorbing material eluted bet-reen 1800 ml
3 and 2050 ml of the gradient was concentrated to about 25 ml
undeT ~acuum. l~'hile this solution stood at 7C overnight,
rhite crystals formed and these were collected and dried to
... . - : .
`~ 25 give 65 percent yield of the product. A sample was recrystal-
lized from hof water for analysis. Calculated for
- C12 H19~16O7P.2H2O: C,33.8; H,5.45; N,19.7. Found:~C,34.3;
H,5.22; N,19.7. Separate thin-layer chromatograms developed
' . '';
. .
- 105 -
* Trade Mark
' ,
~ i()8~57q
with two solvent systems, the first consisting of 4 parts
0.5 M ammonium acetate to 1 part ethanol and the second con-
sisting of 3 parts isobutyric acid to 5 parts 1 M ammonium
hydroxide, each showed one component which quenched fluores-
cence and reacted with ninhydrin. The compound in 0.1 N
hydrochloric acid had an absorption maximum at 264 nm and the
millimolar extinction coefficient was 17.7 which spectral
properties are characteristic of I~6-alkylated adenosine
derivatives.
' ~ .
B. N6-[2-(2,4-dinitrophenyl)aminoethyl] adenosine-5
-monophosphate.
Two hundred fifty mg N6-(2-aminoethyl)adenosine-5'
; -monophosphate tO.65 mmol) from Part A of this Example was
, dissolved in 20 ml water at pH 8. Then 168 mg sodium bicar-
bonate was added, followed by 0.2 ml 1-fluoro-2,4-dinitrobenzene
~1~58 mmol dissolved in 2 ml ethanol). The reaction mixture
;.',
was stirred in the dark at room temperature for 4 hr and
then an additional 0.1 ml 1-fluoro-2,4-dinitrobenezene in
- 1 ml ethanol was added. After the reaction mixture had stirred
overnight, it was adjusted to pH 2.0 with hydrochloric acid
,( and was poured into 200 ml ethanol. The precipitate which
,;:,1
;:;; formed was dissolved in 200 ml water and this solution was
.:,
~' adjusted to pH ~.0 with sodium hydroxide and chromatographed
. on a 2~5x30 cm column of DEAE-cellulose in the bicarbonate
:'-
form (available from Reeve Angel, Clifton, New Jersey). The
chromatogram was developed with a linear gradient generated
with 1.5 liters water and 1.5 liters 0.7 M ammonium bicarbonateO
A major peak of yellow material which absorbed uv light was
eluted between 1200 and 1500 ml of the gradient. Ammonium
` 30 bicarbonate was removed by repeated evaporation to dryness to
. .
....
~ - 106 -
.:
.. . : .
_~` i()~Z~;7~
. ~:
give 40 percent yield of the desired product. This product ~ ;
migrated as one yellow spot on thin-layer chromatograms
developed with the same two solvents mentioned in Part A of
this Example and on epichlorohydrintriethanolamine anion-
S exchange paper developed with 0 25 M sodium acetate-acetic
acid buffer, pH 5Ø In 0.02 N hydrochloric acid the product
had optical absorption maxima of 264 nm and 363 nm with -
millimolar extinction coefficients of 21.8 and 14.2, respect-
ively. - -
C. 2,4-dinitrophenyl-ATP conjugate.
N6-[2-(2,4-dinitrophenyl)aminoethyl] adenosine-5'-
monophosphate (from Part B of this Example) ~0.3 mmole) was
converted to the pyridinium salt by chromatography on a
l.5x20 cm column of ~Dwex 50x2 in the pyridinium form (avail-
able from Bio-Rad Laboratories, Richmond, California). The
yellow effluent was concentrated to dryness and lS ml dimethyl-
formamide and 0.3 mmol tri-n-butylamine were added. This ~ -
mixture was evaporated to dryness and the residue was dried
~ further by repeated evaporation. The monophosphate inter- -
- 20 mediate was then converted to the triphosphate form using
the method disclosed in J. Amer. Chem. Soc. 8~:1785-8(1965).
The reaction products which were soluble in dimethylformamide
were added to 250 ml water which was then adjusted to pH
8Ø This solution was passed into a 2.5x58 cm column of
~DEAE-cellulose in the bicarbonate form and the chromatogram
was developed with a linear gradient generated with 3 liters
ater and 3 liters 0.5 M ammonium carbonate. The first
eluted peak of yellow material was identified as the diphos-
phate derivative. A second peak of yellow material, which
- 107 -
'
* Trade Mark
'. '' `
-~ ~0~2S7q
-
eluted between 4.15 and 4.4 liters of the gradient, ~ as
evaporated to dryness to give 20 percent yield of the desired
conjugate which, by analysis, was found to contain 3.0
residues of phosphate per ribose residue. ' '
,.~`. .
EXAMPLE 19
Preparation of 2,4-dinitrophenyl-ATP conjugate (8-
' position derivative).
8-[2-(2,4-dinitrophenyl)aminoethyl] aminoadenosine-5'
'' -triphosphate.
A. 8-(2-aminoethyl)aminoadenosine-5'-monophosphate.
. . .
' A reaction mixture consisting of 2.2 mmol 8-bromoadenosine
, -5'-monophosphate, prepared according to the method described
in Arch. Biochem. Biophys . 163: 561-9(1974), 66 mmol ethyl-
... .
- enediamine and 25 ml water was heated in an oil bath at 140D
~! 15 for 2 hr. The cooled mixture was adjusted to pH 11.5 with
'~ sodium hydroxide and passed into a 2.5x55 cm column of Dowex
lx8 (200-400 mesh, bicarbonate form). The column was
'' - washed with 300 ml ~1ater and then with a linear gradient
generated with 3 liters wateT and 3 liters 0.5 M ammonium
bicarbonate. The absorbance of the effluent at 254 nm was
~'~ monitored and a major pealc of absorbing mateTial eluted be-
~- tween 4.6 and 5.8 l'iters of the gradient.
Ammonium bicarbonate was removed by repeated evapoTation
'' (five times, 20 to 30 ml wateT each time) to dryness under
,,:
"'' 25 vacuum and the final residue was dissolved in 20 ml ~ater
' by additlon of ammonium hydroxide to pH 8;0. The solution
:',: ., .
'~' - 108 -
...... .
:
. -
'' * Trade Mark
,,~. ,~r^
'''' ' _
. , ~ ' '
1~;Z57~7
was filtered, adjusted to pH 5.0 with formic acid and allowed
to stand at 5C for one day. Crystals which formed were
collected, dissolved at pH 8.0 and recrystallized at pH 5.0O -
The yield of the desired intermediate was 27 percent. On -
examination by thin layer chromatography in a solvent con-
sisting of 4 parts 0.5 M ammonium acetate to 1 part ethanol,
the product migrated as one ninhydrin positive spot which
quenched fluorescence. The optical absorption maxiumum in ~-
- 0.02 N hydrochloric acid was 275 nm and the millimolar
` 10 extinction coefficient was 17.5, which spectral properties
are characteristic of alkylated 8-aminoadenosine derivatives.
Calculated for Cl2H20N7o7p H2O C~3 ;
Found: C,34.1; H,5.28; N,23.9.
B. 8-[2-(2,4-dinitrophenyl)aminoethyl]aminoadenosine
-5'-monophosphate.
8-(2-aminoethyl)aminoadenosine-5'-monophosphate from
Part A of this Example (0.64 mmol) was dissolved in 20 ml
water by addition of sodium hydroxide to pH 8Ø Then
168 mg sodium bicarbonate was added, followed by 0.2 ml
. 20 1-fluoro-4-dinitrobenzene (1.58 mmol dissolved in 2 ml
ethanol). The reaction was stirred for 18 hr at room tem-
perature and then 0.1 ml 1-fluoro-2,4-dinitrobenzene in
1 ml ethanol was added. After stirring for an additional
4 hrs, the mixture was adjusted to pH 2.0 with hydrochloric
acid and poured into 200 ml cold acetone (-10C). The yellow
precipitate which formed was collected by filtration, dis-
solved in 200 ml water and passed into a 2.5x45 cm column
of DEAE-cellulose in the bicarbonate form. The chromatogram
:, - 109 -
'' ~ !
-: . . .
: - . ' ~ ; ' .
:
,r~
2577
was developed with a linear salt gradient generated with
2 liters water and 2 liters 0.7 M ammonium carbonate. A
peak of yellow material with an absorption maximum at 275 nm
eluted between 2 and 3 liters of the gradient. Ammonium bi-
carbonate was removed from this material by evaporation
under vacuum and the yield of the desired intermediate was
; 37 percent. Optical absorption maxima measured in 0.02 N
hydrochloric acid occurred at 275 and 363 nm and the mill-
;
imolar extinction coefficients were 21.8 and 15.5, respec-
tively. Further analyses showed 1.07 phosphate residue per
ribose residue.
C. 2,4-dinitrophenyl-ATP conjugate.
The monophosphate intermediate (0.5 mmol) was converted
. to the tri-n-butylammonium salt by addition of 0.8 mmol tri-
n-butylamine. The mixture was dried by repeated evaporation
; from dry dimethylformamide (four times, 10-15 ml each). The
final reslidue, dissolved in 1 ml dimethylformamide, was
. .
mixed with 2.0 mmol carbonyldiimidazole also in 1 ml dimethyl-
~.~
formamide and was allowed to react at room temperature for
4 hr. The excess carbonyldiimidazole was destroyed by reac-
tion with 15 ~1 methanol for 30 min. Finally, 3 mmol tri-
` n-butylammonium pyrophosphate in 4 ml dimethylformamide was
added and allowed to react for 20 hr. The solid residue
. .
'~ which formed was separated by centrifugation and washed
twice with 5 ml portions of dimethylformamide. The combined
supernatants were added to 200 ml water, which was then
~ . ~
,:
., .
- 110 -
., ' , '
`` io~257q
'' adjusted to pH 8 and chromatographed on a 2.5x25 cm column
of DEAE-cellulose in the bicarbonate form. The chromatogram
was developed with a linear gradient generated with 2 liters
water and 2 liters 0.5 M ammonium bicarbonate. A peak of
ye,llow material with an optical absorption maxima at 275 and
363 nm was eluted between 2.0 and 2.9 liters of the gradient.
' The ammonium bicarbonate was removed by evaporation to give ' '
,' a 22 percent yield of the desired conjugate. Results ofanalyses indicated that this product contained 3.2 residues
~ 10 of phosphate per residue of ribose.
,...
~'
' EXAMPLE 20
:' Preparation of 2,4-dinitrophenyl-ATP conjugate (terminal
phosphate derivative).,
Pl{2-[N-(2,4-dinitrophenyl)amino]ethyl}P4-tS'-adenosine)
tetraphosphate. ''
'' : - ' ' ~
. ., - .
',~ A. 2-[N-(2,4-dinitrophenyl)amino]ethylphosphate.
', A solution containing 20 mmol ethanolamine phosphate, '
0.4 mol sodium bicarbonate and 0.2 g benzyltriethylammonium
' chloride in 20 ml water was stirred while 20 mmol l-fluoro-
,. .2, 4-dinitrobenzene was added dropwise. The resulting two- ',
' phase mixture was stirred at room temperature for 3 days.
Then 600 ml ethanol was a~ded and a yellow solid formed at
'`~ 0C overnight. The solid was dissolved in 50 ml water and
,J'' the solution was adjusted to pH 1.5 with 3 N hydrochloric
'',` 25 acid. The precipitate which formed was collected by filtra-
,~ . .
tion and triturated at 0 with 200 ml anhydrous ethanol.
The solid residue was dried in a vacuum at room temperature
'
- 111 -
.,':
,
:~,
.,
. . . .
.~ .
- ~()8ZS7~7
to yield 4 gm of yellow product (65 percent of theoretical).
This material melted at 200-202 and migrated as one yellow
spot on thin-layer chromatograms developed with a solvent
consisting of 7 parts ethanol to 3 parts triethylammonium
S bicarbonate (pH 7.5). The optical absorption spectrum
measured in 0.02 N hydrohcloric acid had maxima at 359 and
264 nm and the millimolar e~tinction coefficients were 17.0
and 9.2, respectively. The neutralization equivalent was
- 325 which is the value calculated for the monohydrate.
; 10 Calculated for C8HloN3O8P H2O
Found: C,29.38; H,2.94; N,12.81.
B. 2,4-dinitrophenyl-ATP conjugate.
The intermediate prepared in Part A of this Example was
reacted with dipheny~phosphorochloridate by the method
described in Eur. J. Biochem. 28:492-6(1972) to produce the
activated pyrophosphate ~hich was reacted with ATP. One g
2-[N-(2,4-dinitrophenol)amino]ethylphosphate (3.25 mmol) was
converted to the pyridinium salt by chromatography on a
2.5x25 cm column of Dowex 50x2 in the pyridinium form. The
yellow effluent was concentrated to dryness and the residue
was suspended in 50 ml methanol to which 1.4 ml tri-n-
octylamine (3.25 mmol) was added. The mixture was stirred
until the solid dissolved and then the methanol was removed
under vacuum. The residue was taken up in pyridine (20 to
25 ml) and evaporated to dryness (repeated twice). Then the
` residue was dissolved in 30 ml dry dimethylformamide and
evaporated to dryness (repeated three times). The~dried
. . .
- 112 -
.
C * Trade Mark
_ ~ . ..
- :
~ .
10~2S77
residue ~as dissolved in 30 ml dimethylformamide and 0.97 ml
diphenylphosphorochloridate (4.9 mmol) was added, followed
by 1.6 ml tri-n-butylamine (,.25 mmol). The reaction mixture
was stirred for 2 hr at room temperature and then evaporated
to dryness. The residue was shaken with 70 ml dry diethyl
ether for 2 min and then 150 ml petroleum ether was added.
After 1 hr the yellow supernatant was decanted, leaving a
yellow oil which was dissolved in 30 ml dry dimethylformamide
; and evaporated to dryness. The oil which remained was dis-
solved in 100 ml pyridine:dimethyl formamide (1:1 v/v) and
one-half of this solution was reacted with the tri-n-octyl
, ammonium salt of ATP.
ATP ~0.7 mmol) was converted to the pyridinium salt by
,~ . .
, chromatography on a 2.5x25 cm column of Dowex 50x2 in the
pyridinium form. The aqueous solution of this salt was '
evaporated to dryness and 15 ml methanol and 1.4 ml tri-n
-octylamine (1.4 mmol) were added. The mixture was stirred :~
;' until the ATP dissolved and the solvent was removed under
vacuum. 'The residue was dried by repeated evaporation from
pyridine and then dTy dimethylformamide. Finally, the, oily '
residue was combined with the activated 2-~N-(2,4-dinitrophenyl)
,' amino]ethylphosphate and the reaction mixture was stirred at
,. room temperature overnight. Then the solvent was removed by
',; evaporation under vacu,um and the residue was stirTed,wlth
'', 25 100 ml water for 1 hr, while the pH was maintained at 6.5 to
i - -
7.5 by addition of sodium hydroxide. The soluble mateTial
as applied to a 3x45 cm column of Sephadex A-50 (DEAE) in
i, - 113 -
'~ * Trade Mark
.,,,,," ~ , . .
,,; :
. i,, . . . . -
.~ ~ ' . ' - ~. -
i ............................. . .
'. , ~ . . ' ~, ' :
iO~2S7~7
the bicarbonate form (available from Pharmacia Fine Chemicals,
Piscataway, New Hersey. The chromatogram was developed with
a linear gradient generated with 2 liters each 0.1 M and
0.5 M ammonium carbonate. Material which eluted between 0.18
and 0.26 M ammonium carbonate was concentrated by evapora-
tion under vacuum and the ammonium carbonate was removed by
- repeated evaporation to dryness from water. Further purifi-
cation was carried out by thick-layer chromatography on
silica gel with a solvent consisting of 7 parts ethanol to
3 parts 1 M triethylammonium carbonate ~pH 7.5). Two major
yellow bands separated and were each scraped from the plate.
The silica gel was stirred for 1 hr with methanol-water
~1:1 v/v) and the soluble materials from each band were passed
separately into 2.5x20 cm columns of DEAE-cellulose in the
bicarbonate form~ The columns were washed with water and
then 0.5 M ammonium carbonate. The yellow materials eluted
by the salt were concentrated to dryness under vacuum. The
compound which migrated faster on silica gel was identified
as unreacted 2-[N-(2,4-dinitrophenyl)amino]ethylphosphate.
The second band, Rf = 0.64, from the silica gel plate
had optical absorption maxima at 257 and 259 nm, and the
millimolar extinction coefficients of a solution in 0.02 N
hydrochloric acid were 21.1 and 16,2, respectively. This
product, the desired conjugate, was found to contain 4.3
residues of phosphate per residue of ribose.
`"
'
. - 114 -
,.,
' :
~' :
.,.~
, . , : . , , ,~ , . ~ " , :
~ lUR2S7~7
EXAMPLE 21
Direct binding-bioluminescent assay for antibody to
2,4-dinitrophenyl; use of ATP as labeling substance.
The bioluminescence reaction system used in this Example
was based on the following reaction:
ATP-ligand (enzyme) ~_ ATP + modified llgand
ATP + reduced luciferin luciferase ~_
AMP + oxidized luciferin + hv
" '
A light generating solution was prepared containing ~
10 mM morpholinopropane sulfonate buffer at pH 7.4, 10 mM -
magnesium sulfate, 0.7 mM luciferin and 0.15% (w/v) bovine
serum albumin.
Nine specific binding reaction mixtures were prepared,
each having a total volume of 10 ~1 and each containing
20 mM tris-(hydroxymethyl)-aminomethane hydrochloride buffer
at pH 7.4, 10 mM ethylenediaminetetraacetic acid, 45 mM
` 2,4-dinitrophenyl-ATP conjugate (terminal phosphate deriva-
tive, prepared according to Example 20), and antiserum to
` 2,4-dinitrophenyl in the amounts indicated in Table 15. After
incubation at 25C for 1.5 hour, duplicate 10 ~1 aliquots of
, each reaction mixture were assayed by injection into a
~f
;1~ 0.1 ml volume of the above-described light generating solution
~ previously incubated at 25C for at least 2 minutes and
y~ contained in a test tube mounted in a Dupont Model 760
25 Bioluminescence Photometer (E.I. duPont de Nemours, Willming-
;~ ton, Delaware).
- 115 -
rj~, i
X'
~,.......................................................................... .
~,' .
~8257q
The peak light intensity was read from the photometer. The
average peak light intensity for each reaction mixture was
calculated as well as the relative intensity (100% times the
ratio of average peak intensity for the sample to that in
the absence of antiserum). The results appear in Table 15
and in graphical form in Figure 9 of the drawing.
'
TABLE 15
~- antiserum average relative
reaction to 2,4-dinitrophenyl peak light intensity
mixture (~1) intensity ~rcent)
:
1 0 373 100
2 0.2 351 94
' 3 0.4 324 85
4 0.8 254 68
1.0 211 57
6 2.0 91 24
7 4.0 40 11
8 6.8 40 11
9 8.0 37 10
'',;
It was demonstrated in this Example that the activity of
` the ATP in the 2,4-dinitrophenyl-ATP conjugate relative to the
bioluminescent reaction system was an inverse function of the
amount of antiserum to 2,4-dinitrophenyl present in the reaction
mixture. The present invention therefore provides a test
. .
.
- 116 -
.' .
.- ,' , .
., ~.
257~
composition and method for determining the ligand antibody
to 2,4-dinitrophenyl in a liquid medium using a direct bind-
ing-bioluminescent assay technique.
EXAMPLE 22
Competitive binding-bioluminescent assay for derivatiYes
of 2,4-dinitrophenyl; use of ATP as labeling substance.
The bioluminescence reaction system used in this Example
was the same as that described in Example 21.
Thirteen specific binding reaction mixtures were prepared,
each having a total volume of 100 ~1 and each containing 20 mM
tris-(hydroxymethyl)-aminomethane hydrochloride buffer at
pH 7.4, 10 mM ethylenediamine tetraacetic acid, 45 mM
2,4-dinitrophenyl-ATP conjugate (terminal phosphate derivatiYe,
prepared according to Example 20), and 2,4-dinitrophenyl
` 15 -~-alanine at the concentrations indicated in Table 16. To
twelve of the thirteen reaction mixtures (i.e. nos 2 through
~; 13) was added an amount of antiserum to 2,4-dinitrophenyl
sufficient to inhibit the peak light intensity produced by
the bioluminescence reaction in the other reaction mixture
~! 20 (i.e. no. 1) by 75 percent. After incubation at 25C for
`~ 2 hours, duplicate 10 ~1 aliquots of each reaction mixture
were assayed as in Example 21 and the average light intensity
~i and relative intensity were calculated for each. The results
appear in Table 16 and in graphical form in Figure 10 of the
drawing.
, . .
.: :
~ - 117 -
, -
.. . ' .
.
, :.
,
: - - . . .. .
S7~7
TABLE 16
concentration of average relative
reaction 2,4-dinitrophenyl peak light intensity
mixture -~-alanine (~M) intensity (percent)
1 0.00 377 100
2 0.00 89 24
3 0.01 97 26
4 0 04 130 35
0.06 143 38
l 6 0.10 170 45
7 0.20 210 56
8 0.30 257 68
9 0.60 314 83
0.80 334 89
11 1.0 344 91
12 2.0 363 96
13 4.0 385 102
.
~ It was demonstrated in this Example that the relative
,i ,
intensity produced was a direct function of the amount of
2,4-dinitrophenyl-~-alanine present in the reaction mixture.
The present invention therefore provides a test composition
and method for determining ligands such as derivatives of
2,4-dinitrophenyl in a liquid medium using a competitive
binding-bioluminescent assay technique.
:c~ :
,~ ~
EXAMPLE 23
Competitive binding-bioluminescent assay for derivatives
of 2,4-dinitrophenyl; use of ATP as labeling substance.
. ~ . - 118 -
. ' '
, ~
~, :
-- lOR257~7 ;
.:
The bioluminescence reaction system used in this Example
WclS based on the following reaction:
; ,'
ATP-ligand + reduced luciferin luciferase
AMP-ligand + oxidized luciferin + hv
. .
A. Assay using 6-position derivative of ATP.
Three specific binding reaction mixtures were prepared,
each having a total volume of 100 ~1 and each containing
20 mM tris-(hydroxymethyl)-aminomethane hydrochloride buffer
at pH 7.4, 10 mM ethylenediaminetetraacetic acid, 20 ~1 anti-
. 10 serum to 2,4-dinitrophenyl, 512 nM 2,4-dinitrophenyl-ATP
......
~ conjugate (6-position derivative, prepared according to
i, `
, Example 18), and 2,4-dinitrophenyl-~-alanine at the concen-
trations indicated in Table 17. After incubation at 25C for
2 hours, duplicate 10 ~1 aliquots of each reaction mixture
were assayed as in Example 21 and the average peak light inten-
,~",
~ sity was calculated for each. The results appear in Table 17.
,.
TABLE 17
concentration of average
reaction2,4-dinitrophenyl peak light
mixture -~-alanine (~M) intensity
1 0 7
"
-l 2 25 14
3 2500 185
" ~ ~ .
~'';';J :
- 11 9
,.
.,
~, .
!;`
,, .
- . ' `;' ;' ' ;' ' "
;~
S7~7
B. Assay using 8-position derivative of ATP.
Four specific binding reaction mixtures were prepared,
each having a total volume of 100 ~1 and each containing 20 mM
tris-(hydroxymethyl)-aminomethane hydrochloride buffer at pH
7.4, 10 mM ethylenediaminetetraacetic acid, 20 ~1 antiserum to
2,4-dinitrophenyl, 594 nM 2,4-dinitrophenyl-ATP conjugate
(8-position derivative, prepared according to Example 19),
and 2,4-dinitrophenyl-~-alanine at the concentrations indicated
in Table 18. After incubation at 25C for 2 hours, duplicate
10 ~1 aliquots of each reaction mixture were assayed as in
Example 21 and the average peak light intensity was calculated
for each. The results appear in Table 18.
TABLE 18
concentration of average
reaction 2,4-dinitrophenyl peak light
mixture -~-alanine (~M) intensity
l 0 18
2 25 51
3 250 191
4 2500 190
-
;` The results of this Example and those of Example 22
demonstrate that the labeling substance, ATP, may be deriva-
, . .
`I tized at various positions about its structure in the prep- -
aration of a useful conjugate for use in the specific binding
assay method of the present invention. ~-
~ :
.. . .
- 120 -
.. . . ~ : . . . . - .
57q
EXAMPLE 24
Preparation of biotin-isoluminol conjugate.
6-(3-Biotinoylamido-2-hydroxypropylamine)-2,3
-dihydrophthalazine-1,4-dione.
A. 4-(3-chloro-2-hydroxypropylamino)-N-methylphthalimide.
; Twenty-five grams (0.142 mole) 4-amino-N-methylphthalamide,
prepared according to the method described in J. Chem. Soc. 2~:
(1937), and 20.7 g (0.21 mole) 1-chloro-2,3-epoxypropane were
added to 150 ml 2,2,2-trifluoroethanol and the reaction mix-
ture was heated to reflux with stirring for 48 hrs. Seventy
to eighty ml of 2,2,2-trifluoroethanol was removed by distilla-
tion and a heavy yellow precipitate formed when the remaining
solution cooled to room temperature. This precipitate was
triturated with ethyl acetate, collected by filtration and
"
~ 15 dried to give 29.5 g (77% yield) of the desired intermediate
:;
.~ m.p. 136-138.5C. Calculated for C12H13ClN2O3: C,53.64; H,4.88;
N,10.45. Found: C,53.87; H,4.85; N,10.81.
B. 4-[3-(N-Phthalmido)-2-hydroxypropylamino]-N -
methylphthalimide.
The intermediate prepared in Part A (13.5 g,
.,; ~ . .
0.05 mole) and 15.7 g (0.085 mole) potassium phthalimide
were heated to reflux with stirring in 150 ml dimethyl-
formamide for 24 hrs. The dimethylformamide was removed
~;~ t
-` and the residue was washed with water and filtered. The
,:-
~; 25 yellow filter cake was recrystallized from acetic acid-water
to give 12.8 g (67% yield) of product, m.p. 247-248.5C.
Calculated for C20H17N35 C~63-32; H~4- ; N~
Found: C,63.16; H,4.38; N,10.93.
' - 121 -
,
, :.
. ,.
1082S77
C. 6-[3-Amino-2-hydroxypropylamino]-2,3
-dihydrophalazine-1,4-dione.
The intermediate from Part B (5.0 g, 13.2 mmole), 90 ml
absolute ethanol and 35 ml 95% hydrazine were refluxed with
- 5 stirring for 4 hrs. The solvent was removed under a vacuum
and the resulting solid was dried for 24 hrs under vacuum at
120C. This material was stirred for 1 hr with 70 ml of
0.1 N hydrochloric acid. The insoluble 2,3-dihydroxyphthala-
zine-1,4-dione was removed by filtration and the filtrate was
adjusted to pH 6.5 with saturated sodium bicarbonate. The
white precipitate which formed was collected by filtration and
dried to give 2.2 g of the product (67% yield). After re-
crystallization from water, the compound decomposed at 273C.
Calculated for CllH14N2O3: C,52.79; H,5.64; N,22.39. Found: -
C,52.73; H,5.72; N,22.54.
D. Biotin-isoluminol conjugate.
Biotin (0.29 g, 1.2 mmole) and 0.17 ml triethylamine
were dissolved in 20 ml dry dimethylformamide under anhydrous
conditions and cooled to -10C. A solution of 0.141 ml
ethylchloroformate in 2.86 ml ether was added slowly and the
reaction was stirred for 30 min. A precipitate which formed
was separated by filtration. A suspension consisting of
600 mg (2.4 mmole) of the intermediate from Part C, 20 ml
dry dimethylformamide and 1 ml dry pyridine was added to the
filtrate quickly, This mixture was stirred at -10C for
30 min and then at room temperature overnight. During this
- 122 -
!~
082S71'7
period a solution was obtained. The dimethylformamide was
removed by distillation at 60C and 0.10 mm Hg pressure. The
oily residue was stirred with 50 ml 0.1 N hydrochloric acid
for 1 hr. A white solid which formed was filtered and washed
with 0.1 N hydrochloric acid and then water. After drying
under a vacuum at room temperature overnight, 0.55 g (97%
yield) of the product was obtained, m.p. 170-3C. Calculated
for C21H28N6O5S: C,52.92; H,5.92; N,17.64. Found: C,51.69;
. H,5.90; N,17.63.
-
j 10 EXAMPLE 25
Specific binding-chemiluminescent assays; effect of
avidin and biotin on activity of a biotin-isoluminol conjugateO
I The chemiluminescent reaction system used in this
t example was based on the following reaction:
~1
biotin-isoluminol + H2O2 lactoperoxidase ~ _
,,
.,
i biotin-aminophthalate + N2 + hv
,i .
, Nine specific binding reaction mixtures were prepared,
each having a total volume of 140 ~1 and each containing
0.1 M tris-(hydroxymethyl)-aminomethane hydrochloride buffer
at pH 7.4 and biotin, biotin-isoluminol conjugate ~prepared
according to Example 24), and avidin ~added last) in the con-
centrations indicated in Table 19. After 5 minute incubation
, '
~.
~ - 123 -
:' ~
'
,- .. ,. , : ' .,~ ~ ~
l~ZS7t7
at 25C, lO ~1 0.1 M tris-~hydroxymethyl) aminomethane
hydrochloride buffer at pH 7.4 containing 20 units/ml lacto-
peroxidase ~available fTom Sigma Chemical Co., St. Louis,
Missouri; assayed as described in MethodR ~n EnzymoZogy
S XVIIA,~1970)p.653-Assay 2) was added to each reaction mixture.
After incubation at 25C for 2 additional minutes, lO ~1
0.95 mM hydrogen peroxide in 10 mM tris-(hydroxymethyl)
-aminomethane hydrochloride buffer at pH 7.4 was injected into
each reaction mixture and the peak light intensity produced in
. 10 each was measured using a Dupont Model 760 Bioluminescence
; Photometer ~E.I. duPont de Nemours, Willmington, Delaware).
The results appear in Table 19.
. .. .
.. " ~ .
.-
`.' . ~
...
:`.'1
.,, .: .
,`''' . ~ :
.
. ~ .
:
.',~ ' :'
. ' ':
. ~ :
.~...................................................................... .
''~,. ' .
,
~ - 124 -
.;.
.,.~. .1
iO8257'7
.,..,.
,~ ~ o o ~ o ~ o ~ o
~d ~ ~ _I
a~ ~
.,,
o
o ~
. ~ ~ o o o o o : .
8 . ~;
, ;
,. .
,.. .
o .
. o ~
,~ o~ ..
,, .,, ~
,, o~ o ~
~., f~.,, ~
.. 1 P~ ~ ~ I I ~ ~ I ~ I
.~ ¢ ~ o ~
i.i o ~ o
.,
.~ ~o . -
.,, ~
~ .,,
~ ~ o : .
i~
~,
.'.'A
1 O
, t h ~
- 125 -
'"
., .
:~,, - ` - , ,.. . ,- - `.
-- 1013~S7~7
Reactions 1,2,5 and 7 were controls and show that in
the absence of biotin-isoluminol conjugate, only a low background
amount of light was measured. The result of reactions 3 and
6 indicate that the biotin-isoluminol conjugate was active in
the chemilumescent reaction and that the presence of free ;
biotin had no significant effect on such activity. The re-
sult of reaction 4 shows that in the presence of avidin, a
binder for biotin, the activity of the biotin-isoluminol conju-
gate increased. This result is rather unexpected since one
would anticipate that binding of avidin to the conjugate
should limit the availability of the isoluminol moeity for the
chemiluminescent reaction. The reason for the observed
enhancement of light-production is not understood. A com-
parison of the results of reactions 4,8, and 9 demonstrate
that the enhancement of light production is decreased inversely
with the amount of free biotin present.
This Example demonstrates that the ligands avidin and
biotin can be determined using specific binding-chemilumescent
assay techniques and that according to the present invention
the effect of binding between the labeling substance in the
conjugate and a corresponding binding partner may be an en- -
..,
' hancement, rather than inhibition, of the activity of the
labeling substance.
,......................................................................... .
~,:
''
. . .
~ - 126 -
. . .
, :
, . .
: - -. . , . , - - . . - - ,. .. .
., , .- , . ~ . , ~ .
i - . ~ ... . - ~- .
.
lO~ZS77
EXAMPLE 26
Competitive binding-chemiluminescent assay for biotin;
effect of various levels of biotin on the peak light intensity
produced.
The chemiluminescent reaction system used in this Example
was the same as that described in Example 25.
Six specific binding reaction mixtures were prepared,
each having a total volume of 140 ~1 and each contalning
O o l M tris-(hydroxymethyl)-aminomethane hydrochloride buffer
at pH 7.4, 84 nM biotin-luminol conjugate (prepared as in
Example 24), biotin at the concentrations indicated in Table
20, and 0.035 units/ml avidin ~added last). After a 5 minute
incubation at 25C, 10 ~1 of lactoperoxidase ~20 units/ml)
were added to each reaction mixture.
After an additional 2 minute incubation, 10 ~1 0.95 mM
hydrogen peroxide in 10 mM tris-(hydroxymethyl)-aminomethane
hydrochloride buffer at pH 7.4 was injected into each reaction
mixture and the peak light intensity produced in each was
measured as in Example 25. The results appear in Table 20
`; and in graphical form in Figure 11 of the drawing.
,. TABLE 20
,
. .
reactionconcentration ofpeak light
mixture biotin (nM) intensity
, 1 0 23.5
2 67 21.1
! 3 134 15.5
, 4 200 12.6
268 12.3
6 400 8.1
.~
- 127 -
.. . .
. . .
;~
:-- - lOl~Z57~7
It was thus demonstrated that the magnitude of the peak
light intensity produced by the chemiluminescent reaction
system was an inverse function of the amount of biotin present
in the specific binding reaction mixture. The present inven-
tion therefore provides a test composition and method for
determining the presence of the ligand biotin in a liquid
medium using a competitive binding-chemiluminescent assay
technique.
.
EXAMPLE 27
Competitive binding-chemiluminescent assay for biotin.
The chemiluminescent reaction system used in this Example
was based on the following reaction:
biotin-isoluminol + K02 ~
biotin-aminophthalate + N2 + hv
Sixteen specific binding reaction mixtures were prepared,
each having a total volume of 150 ~1 and each containing 0.1 M
tris-~hydroxymethyl)-aminomethane hydrochloride at pH 8.0,
42 nM biotin-luminol conjugate ~prepared as in Example 24),
biotin at the concentrations indicated in Table 21, and 0.12
units/ml avidin (added last). After incubation at 25C for
5 minutes, 10 ~1 of dimethylformamide containing 0.15 M
potassium superoxide (K02) (available from Alpha Products,
Beverly, Massachusetts) and 0.10 M 1,4,7,10,13,16
-hexaoxyacylcooctadecane (available from Aldrich Chemical
Co., Milwaukee, Wisconsin) were injected into each reaction
: .
- 128 -
~ ~ . .
- 10~257~ :
mixture and the peak light intensity produced in each was
measured as in Example 25. The results appear in Table 21
and in graphical form in Figure 12 of the drawing.
:.
TABLE 21
reaction concentration of peak light
mixture biotin (nM) intensity
1 0 38.5
2 13 38.5
3 27 34.3
. 4 40 36.1
53 35.2
6 67 36.2 ~-'
7 101 34.0
: 15 8 133 31.7
: 9 166 29.1
200 24.2
. 11 267 22.8
, 12 333 20.5
:: 20 13 400 13.4
-~ 14 534 8.6
.`-, 15 667 8.3
' 16 800 7.0
, .
.,, ' .
~ It was demonstrated that the magnitude of the peak light
. ,.
~ 25 intensity produced by the chemiluminescent reaction system
`~. was an inverse function of the amount of biotin present in the
,. .
~. ..
~ 129 -
;, ~
i,
,:."
;; . \
". .
:,
`~;
~ ,, .
108257q
specific binding reaction mixture. The present invention there-
fore provides a test compositlon and method for determining
the presence of the ligand biotin in a liquid medium using a
competitive binding-chemiluminescent assay technique which
does not employ an enzyme-catalyzed monitoring reaction.
, ~ .
`.:
.
.,
. .
.
- 130 -
....
.'', .
