Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
9L8~80
AN IMPROVED SPECIFIC BINDING ASSAY EMPLOY-
ING AN ENZYME-CLEAVABLE SUBSTRATE AS LABEL
:
BACKGROUND OF THE INVENTION
1. FIELD OF THE INVENTION
This invention relates to assay methods, and re-
agent means for use therein, of the homogeneous and hetero-
geneous specific binding type for determining qualitatively
or quantitatively a ligand in a liquid medium. In particu-
lar, the invention relates to an improved nonradioisotopic
binding assay employing a novel enzyme substrate label.
'
-'j 2. DESCRIPTION OF THE PRIOR ART
.. . .
-` In German Offenlegungschriften Nos. 2,618,419 and
2,618,511, corresponding respectively to Canadian Patents
~ - .
- Nos. 1,078,712 and 1,082,577, both assigned to the present
assignee, there are described homogeneous and heterogeneous
.
specific binding assays employing an enzyme-cleavable sub~
strate label. In exemplified embodiments there are dis- -
closed the use of fluorogenic-labeled conjugates comprising
umbelliferone or fluorescein coupled via an ester group to
.
a ligand under assay or to a binding partner therefor. The
amount of labeled conjugate in the bound-species and/or
~- free species resulting from the binding reaction system em-
" ployed is determined by addition of an esterase which cleaves
~ ; ,, ~ . .
the ester group linking the um~elliferone or fluorescein
25 residue to the ligand or binding partner to release the free
; fluorescent products, umbelliferone and fluorescein,
' .
- ` .
. ~
` ~ 2 ~ '
c~
., ~
, . . ..
~,', .
. ;.- . .
1~4~3~80
respectively. The rate of fluorescence production, which
follows the rate of release of the fluorescent product, is a
function of the amount of ligand in the liquid medium tested.
Performance of this assay depends upon the ability to
determine the amount of labeled conjugate which results in
either the bound-species or the free-species relative to the
`; amount initially introduced. Where the measured character
of the labeled conjugate in the bound-species is essentially
~ indistinguishable from that in the free-species, the two
species must be physically separated in order to complete the
; assay. This type of binding assay follows what is conven-
tionally known as a "heterogeneous" format. On the other
hand, where the measured character of the labeled conjugate
in the two species is distinguishable, a "homogeneous" for-
15 mat may be followed if desired and the separation step
avoided.
While the above described binding assays employing an
` enzyme-cleavable substrate label offer a generic, novel
,. ~
approach to the pertinent art, the application of the assays
to the detection of ligands in certain types of liquid media
using the ester linked labele~ conjugate is restricted. For
example, the ester based assay has been found to be incon-
venient for the detection of ligands appearing in the milli-
~,,
gram per liter concentration range in physiological fluids
such as serum and plasma. It has been found in this situa-
` tion that the fluid under assay may contain a high endogen-
ous es~terase activity and, independently, the ester linked
conjugate may exhibit a significant instability as the re-
~ sult of background hydrolysis under the conditions of the
; 30 assay, which are usually alkaline.
~ - 3 -
;''
,`': -
. ,
8G180
SUMMARY OF THE INVENTION
It has now been found that the specific binding assay
`; employing an enzyme-cleavable substrate label is greatlyimproved by the use of the novel label component described
herein in formation of the labeled conjugate. According to
~`~ the previously described assay method, the liquid medium under
assay for a particular ligand is combined with reagent means,
including a conjugate having a label component and a binding
component, to form a binding reaction system having a bound
-species and a free-species of such labeled conjugate, the
label component of the conjugate comprising an enzyme-substrate
~' active portion and an indicator portion, whereby the conjugate
~`; is cleavable by an enzyme to produce a detectable indicator
product. The resulting bound-species and/or the free-species
~`i.3 15 is contacted with the cleaving enzyme and the resulting indi-
.'';s
cator product measured as a function of the presence or
~^~ amount of the ligand to be determined in the liquid medium
~ :. . . . .
`~ assayed.
, The present improvement comprises employing as the label
~- 20 component of the conjugate, a residue of the formula:
. ,~, .
~ .
... .
G-D-R
. ............ ..
v wherein G is a glycone, D is a dye indicator moiety, and R is
a linking group through which the dye indicator moiety is
covalently bound to the binding component of the conjugate.
,.
The cleaving enzyme employed to monitor the la~el in the
bound-species or free-species accordingly is one capable of -
cleaving the glycosidic linkage between the glycone and the
- 4 -
'~ .
., ~ . ~ .
"':
..
... . .
,''~`' '- - ' . ' , . ' ~ :
:.".~ ~ . ' , .
, ............ .
~48~80
.
dye indicator moiety. The most preferred glycone and dye
indicator moiety for the labeled conjugate are, respectively,
a ~-galactosyl group and an umbelliferone residue. The assay
is adaptable to the detection of any specifically bindable
S ligand and is particularly useful in the detection of haptens
- such as drugs, particularly the aminoglycoside antibiotlcs.
~' The presently improved assay method and means feature
the advantages of involving a cleaving enzyme for which
negligible, if any, endogenous activity is found in physio-
logical fluids such as serum and plasma, and of employing a
labeled conjugate wherein the cleavable linkage is very stable
under assay conditions in the absence of enzyme. For these
; reasons the present invention offers a significantly more
,.;
~`- accurate and-reproducible assay than that previously known in
the art. Further, antibody-induced hydrolysis of the cleavable
, ,.~ .
linkage, which hydrolysis is sometimes found using the
ester-linked labeled conjugates, is absent using the present
glycosidic-linked conjugates. Even further, the reagents
necessary for performing the assay generally exhibit greater
;~ 20 stability, particularly the labeled conjugate, than prior
reagents. The glycosidase enzymes involved in the present
. :.
invention generally are stable over long storage periods and
in dilute solutions.
A
:
, . .
- .
..
.. ~ , . .
.. .
.. ~ . .
. . .
. .
- 5 -
..... .
:-;
,~,~.
:;. .
: .
...
i ..
.',
;~' .
.. . . .
:- BRIEF DESCRIPTION OF THE DRAWINGS
,
` Fig. 1 is a schematic representation of the basic
;~ principles of a specific binding assay employing an enzyme
., .-cleavable substrate label as applied to the immunoassay
determination of a drug wherein the cleaved product is
-, fluorescent.
, Fig. 2 is a graphical representation of the effect of in-
,`~ creasing antibody concentration on the rate of release of
cleaved product from a labeled conjugate for use in an assay
for gentamicin as described in Example 1.
Fig. 3 is a graphical representation of the relation ~e-
~-~ tween gentamicin concentration and reaction rate as determined
using standards as described in Example 1 for use as a stand-
~.. .
~ ard curve in a rate assay for gentamicin.
,~ . .
Fig. 4 is a graphical representation of the relation be-
~` tween gentamicin concentration and fluorescence intensity as
. .
,'`,jt'`~' determined using standards as described in Example 1 for use
~ .,, - . .
as a standard curve in a fixed-time assay for gentamicin.
Figs. 5 through 9 are graphical representations of the
relations between the concentration of various aminoglycoside
:,~ .. . ..
antibiotics and reaction rates as determined using standards
as described in Examples 2 through 6, respectively.
Fig. 10 is a graphical representation of the relation
between the concentration of diphenylhydantoin and fluores-
cence intensity as determined using standards as described in
~j Example 7 for use as a standard curve in a fixed-time assay
-~ for diphenylhydantoin.
....
.. .
.~"'"- .
,~. ,~ .
.
,..... . .
. .
~,;,
, .... .
.,` ~ ,
"'~'; ` ` , ' '
~8~80
DESCRIPTION OF THE PREFERRED EMBODI~ENTS
In the context of this disclosure, the following terms
shall be defined ~s follows: "ligand~ is the substance, or
class of related substances, whose presence or the amount
thereof in a liquid medium is to be determined; "specific hind-
ing partner of the ligand~ is any substance, or class of sub-
stances, which has a specific binding affinity for the ligand
to the exclusion of other substances; "specific binding analog
of the ligand" is any substance, or class of substances, which
behaves essentially the same as the ligand with respect to
the binding affinity of the specific binding partner for the
ligand; "monitoring reaction" is the reaction in which the
f~
glycosidic linkage in the labeled conjugate is cleaved enzymati-
cally to release a detectable indicator product; "lower alkyl"
I5 is an alkyl group comprising from l to 6 carbon atoms, inclusive,
such as methyl, ethyl, isopropyl, and hexyl.
,: i
. . 3
` LABEL RESIDUE
",i ................................... . .
In the novel label residue of the present invention, the
~; glycone may be any group which constitutes the carbohydrate
.. ~ .
portion of a glycoside. In general, therefore, the glycone
is a sugar residue bound through an acetal linkage to the dye
i indicator moiety in the labeled conjugate. The sugar resi-
due may be selected from residues of monosaccharides, in-
` ~ cluding the aldo-, keto-, deoxy-, and~derivatized forms of
e~: ~
the trioses, tetroses, pentoses, hexoses and heptoses in
~` their D- or L-stereoisomeric forms; oligosaccharides, such
.~................................................... . . .
.. , . ~ .
s- - 7 -
.;.~,
~ '' .
... .
` ~4~3~80
as disaccharides and trisaccharides; and polysaccharides.
Where the acetal iinkage to the dye indicator moiety is
adjacent to an anomeric carbon in the glycone, both the a- and
~-stereocon~igurations may be used. It is preferred that the
glycone be a monosaccharide such as a pentose, e.g., ribose,
arabinose, xylose, and lyxose, with hexoses being particularly
`i preferred, e.g., galactose, glucose, mannose, and gulose.
Derivatized monosaccharide residues which may be used include,
` without limitation, amino-substituted sugars, e.g., glucosa-
mine and galactosamine, O-acyl and O-methyl derivatives, and
; glucuronides. It is contemplated that oligo- and poly-
saccharides and their derivatives may be used as well, e.g.,
the disaccharide cellobiose.
The most preferred group from which the glycone is
~` 15 selected consists of galactosyl, particularly a- and
~-D-galactosyl; glucosyl, particularly a- and ~-D-glucosyl;
N-acetyl-galactosaminyl, particularly N-acetyl-a- or
~` N-acetyl-~-D-galactosaminyl; N-acetyl-glucosaminyl, particularly
N-acetyl-a- and N-acetyl-~-glucosaminyl; glucuronyl, particu-
larly ~-D-glucuronyl; arabinosyl, particularly a-L-arabinosyl;
fucosyl, particularly ~-L-fucosyl; mannosyl, particularly
a-D-mannosyl; and xylosyl, particularly B-D-xylosyl~ The
,~
most preferred glycone is a ~-galactosyl group.
.',,
. ` DYE INDICATOR NOIETY
.
~- 25 With regard to the dye indicator moiety in the novel
label residue of the present invention, this moiety-may com-
.. ~ prise any constitutent, usually one containing an organic
. ~ .
` - 8 -
8~;80
>
nucleus especially of aromatic- character, couplable to the
glycone through a glycosidic linkage and to the binding component
of the labeled conjugate through a suitable linking group, such
` that upon cleavage of such glycoside linkage by an enzyme
appropriate for the glycone, there results a detectable dye
.~
.~ product distinguishable from the intact labeled conjugate.
Preferably the dye indicator moiety is of a type such that
the detectable dye product of the enzymatic cleavage is fluoro-
metrically or colorimetrically active. The desired distinctive
indicator property of the cleaved product is obtained, in
general, by linking the glycone and the dye indicator moiety
;~ . .
~ at a site on the nucleus of the latter such that the fluoro-
.. . .
genic or chromogenic character of the cleave.d dye product is
. distinct from that of the intact labeled conjugate. For
example, the fluorogenic and chromogenic characters of many
. ,~
'`1 known aromatic dyes can be altered by modifying.an aryl
~ . , .
~....... hydroxyl group. Such a group provides an available site for
~, ..... . . .
: linkage to the glycone through a glycosidic linkage which upon
.` enzymatic cleavage results in release of a dye product hav-
ing a fluorogenic or chromogenic character similar to that
of the aromatic dye before formation of the labeled conjugate.
. . .
. Usually the fluorescence spectrum of the dye indicator moiety
.~' in the labeled conjugate will be shifted from that.of the
,;.. ,~ .
.", ~ .
t
~,............................................................. .
,.,,; . .
,
,; , g
.:,
~,.j.
":
.~ ~
,
8~
.
aromatic dye itself. The cleavage reaction is shown schemati-
cally below wherein
j:`
O OH
would represent the sugar precursor of the glycone with only
- 5 the anomeric hydroxyl group specifically shown, A is an aryl
, nucleus,, R is the linking group and L is the binding component
' of the labeled conjugate: -
- ~ ~o ~lycosidase , HO-A-R-L + ~ O OH
~ \A-R-L H2 ' ~detectable , ~
, .................................................................... .
'~ Examples of dyes useful for incorporation into the
''' labeled conjugate of the present invention as the dye indica-
;' tor moiety are umbelliferone, fluorescein, naphtholj indole,
.~ pyridol and resorufin, and active derivatives thereof. Follow-
ing in Table 1 are representative labeled conjugates compris-
ing residues of such dyes which are contemplated for use in
',' the present invention. G~O}- represents the glycone terminat-
~; .
' ing in a bridging oxygen atom which forms a part of the acetal
linkage with the dye indicator moiety and -R-L repre~ents a
,, 20 linking group and the linked binding component for the
,,' conjugate.
.: .
,~,.
.
, . .~;
, - 1 0
.~
~48~80
TA BLE
dye residue . structural formula
~ .
umbelliferone
wherein one of Rl G (0~0~0
and R2 is -R-' and
the other is hydro- l2
~.. ..
" gen or methyl]
, fluorescein G(O ~ O ~ R3
.. [wherein R3 is
hydroxyl or ~ \
-{O)G] L-R
",,,, , `I(
~ o
.. . .
.
`' 3-indole - I -
~i R-L
~ -, . . .
,.. .
::, 15
R-L
naphthol ~J
,; j
"~.~ ,
~. pyridol ~ R-L
,.;, -
vt 20 resorufin ~NO~-L
~. - 11 -
.;~ ' .
..
i . ~
.
`
. . .
~48~8~
Other variations of labeled conjugates based on the above
listed dye residues are clearly evident. Various derivatives,
particularly in the nature of aryl side chain derivativcs,
; which retain sufficient ability to be coupled to the glycone
and binding component and to exhibit appropriate fluorogenic
or chromogenic character in the cleaved indicator product may
be used in preparing labeled conjugates. Labeled conjugates
which are prepared using such a substituted dye as starting
material will possess substantially the same properties as the
conjugates prepared from the above-listed dyes. Such conjugates
will be recognized as equivalents and are exemplified by
; addition of one, two or more simple substituents to an avail-
able aromatic ring site, such substituents including without
limitation lower alkyl, e.g., methyl, ethyl and butyl; halo,~ 15 e.g., chloro and bromo; nitro; carboxyl; carbo lower alkoxy,
e g,, carbomethoxy and carbethoxy; amino; mono- and di-
lower alkylamino, e.g., methylamino, dimethylamino and
methylethylamino; amido; hydroxyl; lower alkoxy, e.g.,
methoxy and ethoxy; and so forth.
..,;, , .
,; .
LIN~ING CROUP
~`'?~
.,
It will be recognized that there are many methods,
available for linking the binding component of the labeled
, , .
; conjugate, e.g., the ligand to be detected, a binding analog
.~ ,
; thereof, or a binding partner thereof, to the dye indicator -
moiety, The particular chemical character of the iinking
group will depend upon the nature of the respec~ive available
~; linking sites on the binding component and the dye indicator
~,~ , ' .
':
- 12 -
~' '
~'
8~
. `
moiety. The important considerations in selecting the linking
sites are (1) preservation of the ability of the linked bind-
ing component to participate effectively in the selected bind-
ing assay system and (2) preservation of the ability of the
linked dye indicator molety upon enzymatic cleavage to yield an
~-` effectively detectable product, in both cases, to the extent
that a useful assay will result for the particular ligand under
. . .
assay and for the particular concentrations or amounts in
which such ligand is to be detected. Usually the linking
` 10 group will comprise a chemical bond, usually a single, but
~;. sometimes a double bond, or a chain containing between 1 to 10,
more commonly 1 to 6, carbon atoms and 0 to 5, more commonly
i 1 to 3, heteroatoms selected from nitrogen, oxygen, and sulfur.
~` Both the dye indicator moiety and the binding component,
~"~ 15 of course, will offer a great diversity of available function-
'"!''''` alities for attachment of the linking group. Commonly the
! .,~ , .
functionalities that can be expected to be available to the
linking group are amino, usually primary amino; hydroxyl; halo,
. usually chloro or bromo; carboxylic acid; aldehyde; keto;
isothiocyanate; isocyanate; and so forth, Accordingly, the
chemical structure of the linking group itself will vary
;;~ widely with its terminal groups depending on the functionalities
available on the dye indicator moiety and the binding component
and its overall leng*h being a matter of choice witXin the
basic constraint of maintaining the essential enzymatic sub-
strate and binding component characters of the resulting con-
jugate. With regard to the length of the linking group in
preparing a conjugate for use in a homogeneous assay format,
it is usually desirable to use as short a group as possible
., , ~
- - 13 -
1~8~80
without causing the resulting binding component in the con-
jugate to interfer significantly with the substrate activity
. ~
~ of the conjugate. Where the binding component is of low
i
~`~ molecular weight (e.g., a hapten of molecular weight between
~- 5 100 and 1000), the linking group is preferably a chemical
,
~;~ bond or a 1 to 3 atom chain such as carbonyl, amido, and the
like. In other circumstances, such as where the binding com-
. . ,~. . .
ponent ln the con~ugate is of relatively high molecular
weight, such as a polypeptide or protein (e.g., an antibody),
a longer linking group is usually desirable to prevent steric
hindrance of the substrate-active site of the conjugate. In
these cases, the linking group will comprise usually 4 to 10
.''.'
carbon atoms and 0 to 5 heteroatoms as previously discussed.
Chains of any significantly greater length will tend to re-
sult in conjugates in which the binding component will tend
to fold-back into the substrate-active site. With these con-
siderations in mind, examples of linking groups are shown in
Table 2. Particular examples of linking groups will be seen
hereinafter and further variations will be readily recognized
i~, 20 as being state-of-the-art.
s
~ "~,
'ti ~l
`,~.!.,,
~;'
3,~; ~
~, !i
~ . .
~`~
''''~'
s` - 14 -
-
,.~ ,
.
,
~8~;8V
TABlE 2
linking group
_ _
-R4-C-R5-
X
-R4-l-X-R5-
-R4-X- 11 -R5-
X'
label - indicator_ -R4-X-C X R5 -binding component
component moiety .
C R4 11
-R4-X-R5-
-X-R4-
-R4-X-
-X-R -X- .
wherein X is imino, sulfur or, preferably, oxygen; and R4
and R5 are, independently, a bond or lower alkylene such as
methylene, ethylene, butylene or hexylene.
- 15 -
i80
The preferred dye indicator moiety is an umbelliferone
residue which is bound directly to the glycone by an acetal
linkage at the 7-position and bound to the binding component
through a linking group at the 3 or 4-position, preferably
the former. Especially useful are labeled conjugates com-
prising such an umbelliferone residue coupled to a ~-galac-
tosyl group as the glycone at the 7-position and to the bind-
ing component through the 3-position. Such conjugates are
represented as:
CH2H
`?~ ~ ~R-L
wherein R is a linking group and L iS the binding component
such as a hapten of molecular weight between 100 and 1000,
or an analog thereof, particularly a drug or drug analog.
Such conjugates find application in the detection of anti-
convulsants such as diphenylhydantoin, phenobarbital, primi-
done, carbamazepine, ethosuximide, and sodium valproate; and
particularly for detecting aminoglycoside antibiotics such
as gentamicin, tobramycin, amikacin, kanamycin, sisomicin,
and netilmicin; as well as others as described hereinafter.
The linking group R is usually a single bond or a chain con-
taining 1 to 10 carbon atoms and 0 to 5 heteroatoms selected
from nitrogen, oxygen and sulfur. Where the binding compo-
nent has an available primary amino group, the linking group
may be carboxyl, forming an amide bond between the umbelli-
ferone residue and such binding component.
~ - 16 -
~8~80
`~ The present assay 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, glyco-
protein, steroid, or other organic molecule for which 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 gonadotropin, thyroxine, liothyro-
nine, and estriol; antigens and haptens such as ferritin,
bradykinin, prostaglandins, and tumor specific antigens;
vitamins such as biotin, vitamin B12, folic acid, vitamin E,
vitamin A, and ascorbic acid; metabolites such as 3',5' adenosine
monophosphate and 3',5' guanosine monophosphate; pharmacological
agents or drugs, particularly those described below; anti-
bodies such as microsomal antibody and antibodies to hepatitis
and allergens; and specific binding receptors such as thyroxine
binding globulin, avidin, intrinsic factor, and transcobalamin.
The present assay is particularly useful for the detection of
haptens, and analogs thereof, of molecular weight between 100
and 1000, particularly drugs and their analogs, including the
aminoglycoside antibiotics such as streptomycin, neomycin, and
especially gentamicin, tobramycin, amikacin, kanamycin,
sisomicin, and netilmicin; anticonvulsants such as diphenyl-
hydantoin, phenobarbital, primidone, carbamazepine, ethosuxi-
mide, and sodium valproate; bronchodialators such as theophyl-
line; cardiovascular agents such as quinidine and procainamide;
- 17 -
~1~8~80
drugs of abuse such as morphine, barbiturates and amphetamines;
and tranquilizers such as valium and librium.
As stated previously, the present assay method may
follow, in appropriate circumstances, either a homogeneous
or a heterogeneous scheme.
HOMOGENEOUS SCHEMES
A homogeneous scheme, i.e., one which does not require a
physical separation of the- bound-species and the free-species,
is available where reaction between the binding component of
the labeled conjugate and a corresponding binding partner
causes a measurable change, either in a positive or a negative
sense, in the ability of the label component of the labeled
conjugate to participate in the monitoring reaction, i.e., in
the ability of the labeled conjugate to be cleaved-enzymatically
to release the detectable product. In such a case, the distri-
bution of the label component between the bound-species and
the free-species can be determined by adding the enzyme
directly to the binding reaction mixture and measuring
therein the activity of the substrate-active label component,
i.e., the rate or total amount of detectable product that
results, which preferably comprises measuring the rate of
fluorescence or color production or the total amount thereof
produced. Several manipulative schemes are available for
carrying out a homogeneous assay with preference being given
to the direct binding and competitive binding techniques.
Briefly, in the direct binding technique, a liquid medium
suspected of containing the ligand to be detected, usually a
compound of high molecular weight (e.g., an antibody) relative
to a selected binding partner (e.g., an antigen or hapten), is
~8~130
\
contacted with the present labeled conjugate in which the
binding component is the selected specific binding partner
of the ligand, and thereafter any change in the substrate
activity of the label component is assessed. In the competi-
tive binding technique, primarily useful for the detection
of a compound of low molecular weight (e.g., an antigen or
hapten) relative to a selected binding partner (e.g., an
antibody), the liquid medium is contacted with the selected
specific binding partner of the ligand and with the present
labeled conjugate in which the binding component is one of
the ligand or a specific binding analog thereof, and thereafter
any change in the substrate activity of the label component is
assessed. In both techniques, the substrate activity of the
label component is determined by contacting the liquid medium
with an enzyme which can cleave the glycosidic linkage in the
label component of the free-species form of the labeled conju-
gate and then measuring the rate or amount of detectable
product which results. Qualitative determination of the ligand
in the liquid medium involves comparing a characteristic,
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 the presenGe
of such ligand in the liquid tested. Quantitative determina-
tion of the ligand in the liquid medium involves comparing a
characteristlc of the resulting reaction to that of the moni-
toring reaction in liquid media containing various known
amounts of the ligand, e.g., a comparison to a standard curve.
A schematic representation of the principles of a competi-
tive binding type of homogeneous immunoassay for a drug is
shown in Figure 1 of the drawing. As shown, the free labeled
- 19 -
8~80
drug is acted upon by the enzyme to release a fluorescent
product. However, upon addition of antibody to the drug, the
action of the enzyme on the resulting labeled drug~antibody
complex is inhibited, probably by steric hindrance. In the
competitive binding reaction then, the ability of the enzyme
to release the fluorescent product is dependent upon the
ratio of labeled drug remaining free to that bound to anti~
body. Thus, the reaction rate of production of fluorescence
is proportional to the amount of drug to be assayed w~ich
competes with labeled drug for antibody binding.
In general, when following a homogeneous assay
scheme, the components of the specific binding reaction, i.e.,
the liquid medium suspected of containing the ligand, the
labeled conjugatej and, in some systems, a specific binding
partner of the ligand, may be combined in any amount, manner,
and sequence, provided that the activity of the label com-
ponent of the labeled conjugate is measurably altered when
the liquid medium contains the ligand in an amount or con-
centration of significance to the purposes of the assay.
Preferably, all of the components of the specific binding
reaction are soluble in ~the liquid medium.
Known variations of the above briefly described
homogeneous methods and further details concerning the
speci-fic techniques discussed are readily available in the
literature, e.g., German OLS No. 2,618,511, corresponding
to Canadian Patent No. 1,082,577, assigned to the present
assignee.
. 20
~48~8(3
HE~E~OGENEOVS SCHEMES
The use of the present novel substrate-active labels
can also be applied to the conventional heterogeneous type
assay schemes wherein thè bound- and free-species of the
labeled conjugate are separated and the quantity of label in
one or the other is determined. The reagent means for per-
forming such a heterogeneous assay may take on many different
forms. In general, such means comprises three basic consti-
tuents, which are (1) the ligand to be detected, ~2) a
specific binding partner of the ligand, and (3) the labeled
conjugate. The binding reaction constituents are combined
simultaneously or in a series of additions, and with an
appropriate incubation period or periods, the labeled con-
jugate becomes bound to its corresponding binding partners
lS such that the extent of binding, i e., the ratio of the
amount of labeled conjugate bound to a binding partner tthe
"bound-species") to that unbound tthe "free-species"~, is a
function of the amount of ligand present. The bound- and
free-species are physically separated and the amount of label
present in one thereof is compared to a negative control or
standard results, e.g., a standard curve.
Various means of performing the separation step and of
forming the binding reaction systems are available in the art.
Separation may involve such conventional techniques as those
- 21 -
380
involving what is commonly known as a solid-phase antibody
or antigen, a second antibody, or a solid phase second anti-
body, as well as the use of immune complex precipitatin~
agents and adsorbents, and so forth. Binding reaction sy;-
stems that can be followed include the so-called competi-
tive binding techni~ue, the sequential saturation technique,
the "sandwich" technique, and so forth. Further details
concerning- the various known heterogeneous systems are readi~
ly/available in the literature, e.g., German OLS No.
2,618,419, corresponding to Canadian Patent No. 1,078,712,
assigned to the present assignee.
It should be recognized that manipulative schemes
involving other orders of addition and other binding reac-
tion formats may be devised for carrying out homogeneous
and heterogeneous specific binding assays without departing
from the inventive concept embodied herein.
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, saliva, and amniotic~
cerebral, and spinal fluids. Other materials such as solid
matter, for example tissue, or gases may be assayed by re~
ducing them to a liquid form such as by dissolution of the
solid or gas in a liquid or by liquid extraction of the
solid.
" ~486~80
In general, in those instances where for purposes of a
selected binding assay system the binding component in the
labeled conjugate is the ligand or an analog thereof, the
present labeled conjugate may be termed a glycone-dye-labeled
ligand and may be represented by the formula:
G-D-R-L
wherein G, D and R have their meanings as hereinabove and L
is the ligand or analog thereof. Particularly useful conjugates
for use in assays for haptens, especially those of molecular
weight between 100 and 1000, are the ~-galactosyl-umbelliferone
-hapten conjugates of the formula:
C~ OH
~ R-L
lS As stated hereinabove, the present invention finds
particular application.to the detection of aminoglycoside
antibiotics, in particular, gentamicin, sisomicin, tobramycin,
amikacin, kanamycin, and netilmicin. Particularly useful
corresponding ~-galactosyl-umbelliferone-ligand conjugates
are represented by the formula below wher.ein the linking
group between the umbelliferone residue and the aminoglycoside
antibiotic is attached to the latter via a primary amino
group of the isolated antibiotic`. Since there are several
available primary amino groups in each of the various anti-
biotics listed, one, two or more ~-galactosyl-umbelliferone
residues may be associated with one labeled antibiotic.
- 23 -
.
.
.
~48Q180
t R ~ L
5 wherein R is a linking group as described hereinbefore
terminating in an amino-linking group, preferably carbonyl;
L is an aminoglycoside antibiotic selected from the group con-
sisting of gentamicin, tobramycin, amikacin, kanamycin, siso-
micin, and netilmicin, coupled by a covalent bond to the
linking group R through a primary amino group therein; and n
equals 1 to the total number of primary amino groups in the
selected antibiotic, inclusive.
In preparing the above ~-galactosyl-umbelliferone-amino-
glycoside antibiotic conjugates wherein the linking group R
is carbonyl, one first obtains the intermediate of the formula:
~j:H20H
NO~o
wherein Z is hydrogen or a suitable salt cation such as
potassium or sodium, by reaction of 3-carboethyoxyumbelliferone
and tetraacetyl-~-D-galactosyl bromide according to the method
of Leaback, C~in. Chem. Acta 12 :647 (1965) . Conjugation with
the selected antibiotic then proceeds under suitable amide-bond
producing conditions such as in the presence of acid and
carbodiimide (cf. Table 3 below).
- 24 -
~8~80
To perform an assay for an aminoglycoside antibiotic
according to the present invention there may be used a labeled
conjugate wherein the binding component is said antibiotic
under assay or a binding analog thereof. Where an antibody
is used as binding partner in the assay, such as in a homo-
geneous or heterogeneous competitive binding assay, it has
been found that other aminoglycoside antibiotics may
cross-react with the antibody for the antibiotic under assay.
Thus such other antibiotics qualify as binding analogs and
could be used to form the labeled conjugate. Further, the
antibody qualifies as reagent for use in assays for the
cross-reacting antibiotic. For example, in an assay for
gentamicin it has been found that with appropriate antiserum
the binding component in the labeled conjugate may be
gentamicin itself or sisomicin which cross-reacts. Thus,
gentamicin antiserum and a labeled sisomicin conjugate could
be used in an assay for gentamicin. Specificity problems are
not encountered in clinical situations because it would be
known what antibiotic was administered and only one amino-
glycoside antibiotic is administered at a time.
The present invention will now be illustrated, but is
,~ . .
not intended to be limited, by the following examples.
- 25 -
` 11~8~80
EXAMPLE 1
Gentamicin Assays
A. Preparation of glycone-dye-drug conjugate
The reaction sequence for the preparation of the gly-
cone-dye-drug conjugate is given in Table 3. 3-carboethoxy-
7-hydroxycoumarin (II J was prepared by a Knoevenagel conden-
sation of 2,4-dihydroxybenzaldehyde (Aldrich Chemical Co.,
Milwaukee, Wisconsin, U.S.A.) with diethylmalonate in acetic
acid, benzene, and piperidine as described in J. Am. Chem.
Soc. 63:3452(1971). The potassium salt of ~-[7-(3-carboxy-
coumarinoxy)3-D-galactoside (III) was prepared by the reac-
tion of 3-carboethoxy-7-hydroxycoumarin (II) and 2,3,4,6-
tetraacetyl-a-D-galactosyl bromide (I, Sigma Chemical Co.,
St. Louis, Missouri, U.S.A.) as described by Leaback for the
preparation of methylumbelliferyl-~-D-galactoside in C~in.
Chim. A~ta 12:647(1965). The potassium salt of this compound
was purified by chromatography on silica gel-60 (E. Merck,
St. Louis, Missouri, U.S.A.) with a gradient of n-butanol/-
methanol/water (4/2/1 by volume) and methanol/water (1/6).
After recrystallization from acetone-water, the corrected
melting point of the product was 258-263C (decomp.). Ana-
lysis: calculated for C16H15OloK: C 47.28%, H 3.73%,
K 9.62%; found: C 47.30%, H 3.74%, K 9.34%. Optical rota-
tion [a]D = -77.40 (lg.H2O). NMR ( H2O), ~8.2(s, lH), 7.6
(m,lH), 7.0 (m,2H), 5.1 (s,lH), and 4.0 (m,6H). Infrared
analysis (KBr) indicated a carbon-oxygen double bond and a
carbon-carbon double bond (1705 and 1620 cm 1).
",
~'''`!
- 26 -
~1~8~380
TA ~LE 3
CH20R6
~Br ~oOCH2CN3
(I ) (II )
R6 =-CCH3
~ NaOH, acetone, 5C
KOH, methanol
CH20H
OH ~c_o~D
(III )
H20, pH 4.0, 5C.
. carbodii-ide, sisomicin
CH20H ,,,
~NH-SISOMICIN)
~IV)
- 27 -
' ` ' ;, ' ' ',
~B~80
~ -Galactosyl-umbelliferone-sisomicin ~IVJ was prepared
by mixing 50 milligrams (mg) (117 ~mol) of the potassium salt
of ~-[7-(3-carboxycoumarinoxy)]-D-galactoside ~III) with 171
mg of sisomicin sulfate (223 ~mol of sisomicin free base,
Schering Corp., Bloomfield, N.J., U~S.A.) in 2 ml of water.
The pH was adjusted to 3.8 by dropwise addition of 1 molar
hydrochloric acid. The solution was cooled in an ice bath
and 30 mg (150 ~mol) of 1-ethyl-3-(3-dimethylaminopropyl)-car-
bodiimide hydrochloride (Pierce Chemical Co., Rockford,
Illinois, U.S.A.) was added. After 2 hours the mixture was
chromatographed at 25C on a 2.5 x 50 centimeter (cm) column
of CM-Sephadex C-25 (Pharmacia Laboratories, Inc., Piscataway,
N.J., U.S.A.) 5.8 ml fractions were collected, and their ab-
sorbance was monitored at 345 nanometers (nm). The column
was washed with 200 ml of 50 mmol/liter ammonium formate to
elute unreacted 3-[7-(3-carboxycoumarinoxy)3-D-galactoside
(III). A linear gradient, formed with 400 ml of 50 mmol/-
liter and 400 ml of 1.8 mol/liter ammonium formate, was
applied to the column. A peak of material absorbing at 345
nm eluted at approximately 1.4 mol/liter ammonium formate.
After the gradient, the column was washed with 600 ml of 1.8
mol/liter ammonium formate. Three 345 nm absorbing peaks
were eluted in this wash. Eluted unreacted sisomicin was
well separated from the last 345 nm absorbing peak.
The carbodiimide-activated reaction leads to the for-
mation of amide bonds between the carboxylic acid of 3-[7-
(3-carboxycoumarinoxy)]-galactoside and the primary amino
groups of sisomicin. The major peak of ~-galactosyl-umbell-
iferone-sisomicin (the last 345 nm absorbing peak) was used
in the present studies. Ammonium formate was removed by
lyophilization. Because the absorptivity of isolated label-
ed con~ugate
` 1
- 28 -
* Trade Mark
.
. -
11~8~380
`~ is currently unknown, the relative concentration is presented
A345 units One A345 unit is the quantity of
material contained in 1 ml of a solution that has an absorbance
of 1.0 at 345 nm when measured with a 1 cm light path.
B. Assay Procedure - Rate Assay
The principle of the assay is shown schematically in
Figure 1 of the drawings.
A reagent, prepared in 50 mmol/liter N,N-bis-(2-
hydroxyethyl~-glycine ~Bicine) buffer (pH 8.2, Nutritional
Biochemicals Corp., Cleveland, Ohio, U.S.A.), contained
~-galactosidase (25 ng protein/ml, Escheric~ia co~i - derived
enzyme, Grade IV, Sigma Chemical Co., St. Louis, r~issouri,
U.S.A.) and antiserum to gentamicin (prepared as described in
Nature Ne~ BioZ. 239: 214(1972) in an amount sufficient to
decrease the reaction rate in the final reagent to 20 to
30~ of the rate observed in the absence of antibody). One
unit (U) of the enzyme was defined as that amount which
hydrolyzed 1.0 ~mole of o-nitrophenyl-~-D-galactoside per
minute at pH 7.2 at 37C. The enzyme preparation used had a
specific activity of 745 U per milligram of protein.
To 2.0 ml aliquots of the reagent in a cuvette were added
1 ~1 aliquots of serum standards or unknown. After mixing,
5 ~1 of an aqueous solution of the labeled conjugate prepared
in part A (0.125 A345 units per ml) was added to each cuvette
and the rate of increase in fluorescence was monitored in
each for 2 to 3 minutes. All solutions were kept at 25C,
except the labeled conjugate which was kept in an ice bath.
- 29 -
,
.
~1~8~80
C. nesults - Rate Assay
The absorbance spectrum of the labeled conjugate,
~-galactosyl-umbelliferone-sisomicin, showed an abscrbance
maximum at 345 nm. When the conjugate was hydrolyzed with
bacterial ~-galactosidase to remove the galactose moiety, the
absorbance at 345 nm decreased and a new maximum appeared at
402 nm. The absorbance of the enzyme-treated conjugate was
1.46 times that of the untreated conjugate.
Analysis of the fluorescence spectrum of the conjugate
revealed a similar shift in the maximum wavelength. Before
enzyme treatment, the conjugate exhibited excitation and
emission maxima at 350 and 394 nm, respectively. After
hydrolysis with ~-galactosidase, a 15-fold increase in fluores-
cense was observed, with new excitation and emission maxima
of 409 and 445 nm, respectively. Hence, under the conditions
of the fluorescent assay (excitation and emission wavelengths
of 400 and 453) the unreacted conjugate contributed negligible
fluorescence. For all of the aminoglycoside antibiotic assays
reported herein, the excitation and emission wavelengths used
in the fluorometric measurements were approximately 400 and
450 nm, respectively.
The effect of antiserum to gentamicin on the ability
of the labeled conjugate to function as a substrate for
~-galactosidase was examined. Various amounts of antiserum
were added to 2.0 ml of buffered ~-galactosidase. The labeled
conjugate was added and the reaction rate determined using an
Aminco-Bowman Spectrofluorometer connected to a strip-chart re-
corder. Reaction rates are expressed in terms of recorder
units/minute. As the amount of antiserum increased, the
- 30 -
1:148{~80
\
reaction rate decreased as shown in Figure 2 of the drawings.
Based upon this experiment, an amount of antis~rum sufficient
to inhibit the reaction rate by 70 to 80~ was chosen for the
competitive binding reactions. The reaction between the anti-
body and the conjugate appeared to be complete in the time
required for mixing the reagents, because incubation of the
conjugate with the antibody before adding enzyme did not alter
the results.
For the standard curve, gentamicin standards were prepared
from 0 to 14 ~g/ml (mg/liter) in normal human serum and assayed
as described in part B above. Figure 3 of the drawings shows
the standard curve of the reaction rate related to gentamicin
concentration in serum standards. Reaction rate was calculated
for each standard as the percentage of the maximum reaction
rate in the absence of an-tiserum, after substraction of
fluorescence in the absence of drug in the standard. No
difference was observed for standards prepared in buffer com-
pared to standards prepared in serum. Varying the time of
incubation of the standards with the antibody/enzyme reagent
from 0.25 to 60 minutes before adding the labeled conjugate did
not alter the standard curve. Hence, the assay can be per-
formed as rapidly as the reagents can be mixed.
~, .
D. Assay Procedure - Fixed-Time Assay
A reagent was prepared~ by adding 140 ~1 of antiserum
to gentamicin (prepared as ln part B above - to inhibit *he
maximum reaction rate in the final reagent by 75~) to 40
milliliter (ml) of 0.05M Bicine buffer, pH 8.2. To 2.0 ~l-
aliquots of this reagent in a cuvette were added 7.5 ~1
aliquots of serum standards, After mixing, 40 ~1 of an
aqueous solution of the labeled conjugate prepared in part A
(0.013 A345 units per ml) were added to each cuvette. After
further mixing, 30 ~1 of ~-galactosidase solution (21 U/~l)
were added to each cuvette and the solutions again mixed.
After 20 minutes at room temperature, the resulting fluores-
cence for each cuvette was measured in the fluorometer and
expressed in terms of the instrument reading.
E, Results - Fixed-Time Assay
A standard curve generated by testing various standard
samples containing known concentrations of gentamicin
according to the preceding method is depicted in Figure 4
of the drawings.
EXAMPLE 2
S~somio?,n Assay
A. Preparation of glycone-dye-drug conjugate
The labeled conjugate used in this Example was that
prepared according to part A of Example 1.
`~ 1148~80
B. Assay Procedure
A reagent was prepared by adding 170 ~1 of antiserum to
gentamicin (prepared as in part B of Example 1 - to inhibit
the maximum reaction rate in the final reagent by 90~) and
150 ~1 of 0.1 mg/ml ~-galactosidase (6U) to 200 ml of 50 mM
Bicine buffer. To 2.0 ml aliquots of this reagent in a
cuvette were added 20 ~1 aliquots of aqueous standard siso-
micin solutions. After mixing, 20 ~1 of an aqueous solution
of the labeled conjugate (part A above - 0.032 A345 units per
ml) were added to each cuvette. Fluorescence was measured in
an Aminco-Bowman Spectrofluorometer and reaction rates calcu-
lated for each standard as in part C of Example 1.
C. Results
.
A standard curve generated by testing various standard
samples of sisomicin according to the preceding metho-d is
tepicted in Figure 5 of the drawings.
.
EXAMPLE 3
NetiZmicin Assay
A. Preparation of glycone-dye-drug conjugate
The labeled conjugate used in this Example was that
prepared according to part A of Example 1.
- 33 -
1148080
B. Assay Procedure
The procedure was ~he same as that described in part B
of Example 2 using aqueous netilmicin standards.
C. Results
A standard curve generated for the assay of`netilmicin
according to the above procedure is depicted in Figure 6
of the drawings.
EXAMPLE 4
Tobramycin Ass~y
A. Preparation of glycone-dye-drug conjugate
The reaction sequence and methodology for the preparation
of the labeled tobramycin conjugate were basically those of
Table 3 and part A of Example 1, respectively.
With 55 mg (135 ~mol) of the potassium salt of
~-[7-t3-carboxycoumarinoxy)]-D-galactoside was mixed 150 mg
~220 ~mol) of tobramycin (Eli Lilly ~ Co., Indianapolis,
Indiana U.S.A.) in l.5 ml of distilled water. The pH was
adjusted to 3.65 by the dropwise addition of lN hydrochloric
acid and the resulting solution cooled in an ice bath. To
initiate the coupling reaction, 30 mg ~160 ~mol) of
- 34 -
,
8~ 8~
l-ethyl-3(3-dime~hylaminopropyl) carbodiimide hydrochloride
were added. After overnight incubation of 4C, two drops of
lN sodium hydroxide were added to-give a pH of 6.1.
The product was purified by chromatography on carboxy-
methyl Sephadex gel ~Pharmacia Laboratories, Inc.) with
ammonium formate as eluant. After an initial wash with
O.O5M ammonium formate to remove unreacted galactoside,
1.5M ammonium formate was used to elute conjugated products.
Five peaks of material absorbing at 345 nm were eluted, with
the third peak being selected for use in this s.tudy.
B. Assay Procedure
A reagent was prepared by adding 150 ~1 of antiserum to
tobramycin (prepared as described in part B of Example 1 using
tobramycin in place of gentamicin in synt.hesis of the
immunogen - to inhibit the maximum Teaction rate in the
final reagent by 80%) and 250 ~1 of an aqueous solution of
~-galactosidase (4U/ml) to 100 ml of 0.05-M Bicine buffer,
pH 8.2. To 2.0 ml aliquots of this reagent in a cuvette
were added 10 ~1 aliquots of aqueous standard tobramycin
solutions followed by 20 ~1 of an aqueous solution of the
labeled conjugate prepared as in part A t0..03 A345 units per
ml). After measuring the rate of resulting fluorescence,
reaction rates were calculated for each standard as in part
C of Example 1.
- 35.- .
* Trade Mark
1!~,
~ .
~1~8~80
C. Results
A standard curve generated by testing various tobramycin
standard samples according to the preceding method is
depicted in Figure 7 of the drawings.
EXAMPLE 5
Kanamycin Assay
A. Preparation of glycone-dye-drug conjugate
The labeled conjugate used in this Example was that
prepared according to part A of Example 4.
B. Assay Procedure
The procedure was the same as that described in part B
- of Example 4 using aqueous kanamycin standards.
C. Results
A standard curve generated for the assay of kanamycin
according to the above procedure is depicted in Figure 8 of
the drawings.
- 36 -
~148~80
EXAMPLF 6
Ami kacin h B say
A. Preparation of glycone-dye-drug conjugate
The reaction sequence and methodology for the preparation
of the labeled amikacin conjugate were basically those of
Table 3 and part A of Example 1, respectively.
290 mg ~540 ~mol) of amikacin (Bristol Laboratories,
Syracuse, N.Y. U.S.A.) were mixed 110 mg ~270 ~mol) of the
potassium salt of ~-[7-(3-carboxycoumarinoxy)]-D^galactoside
in 3 ml of distilled water. The pH was adjusted to 4.1 by
addition of lN hydrochloric acid. After the solution had
been cooled in an ice bath, 55 mg (292 ~mol) of
l-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride
were added to initiate the reaction. After overnight incuba-
tion at 4C, the reaction mixture was chromatographed on
carboxymethyl Sephadex gel. After washing the column with
0.05M ammonium formate to remove unreacted galactoside, l.5M
ammonium formate was used to qlute the desired con~ugate.
Three peaks of material absorbing at 345 nm were obtained,
with the last peak being used for this study.
B. Assay Procedure
A reagent was prepared by adding 80 ~1 of antiserum to
amikacin ~prepared as described in part B of Example 1 using
amikacin in place of gentamicin in synthesis of the immunogen -
to inhibit the maximum reaction rate in the final reagent by
- 37 -
~L148~80
70%) and 60 ~1 of an aqueous solution of ~-galactosidase
(4U/ml) to 60 ml of 0.05M Bicine buffer, pH 8.2. To 2.0 ml
aliquots of this reagent in a cuvette were added 10 ~1 aliquots
of aqueous amikacin standard solutions followed by 20 ~1 of
an aqueous solution of the labeled conjugate prepared as in
part A (0.03 A345 units per ml). After measuring the rate of
resulting fluorescence, reaction rates were calculated for
each standard as in part C of Example 1.
C. Results
A standard curve generated by testing various amikacin
standard samples according to the preceding method is
depicted in Figur'e 9 of the drawings.
EXAMPL~ 7
DiphenyZhydant*on Assay
A. Preparation of glycone-dye-drug,conjugate
In a liter, 3-neck round bottom flask was placed 8.64 g
of a 50% suspension of sodium hydride (NaH) in mineral oil
~0.18 mol). The NaH was washed free of mineral oil with
hexane under an argon atmosphere. It was then suspended in
350 ml of dry dimethylformamide (DMF~ and stirr'ed whil'e a
solution of 34.4 g (0.173 mol) of N-(4-bromobutyl)phthalimide
in lS0 ml of dry DMF was added over a 20 minute period.
After stirring at room temperature for 18 hours, the reaction
- 3B -
1~8~)80
was diluted with 200 ml of H20 and the precipitate collected
and dried to yield 49 g of 2-[(4-N-phthalimido)butoxy]benzo-
phenone, mp 119-121C. A 1 g sample was recrystallized from
ethanol to give 740 mg of white needles, mp 121-122C.
A mixture of 22.4 g ~0.056 mol) of 2-[(4-N-phthalimido)
hutoxy]benzophenone, 4.15 g ~0.064 mol) of potassium
cyanide, 17.3 g (0.18 mol) of ammonium carbonate, 24 ml o
water, and 200 ml of DMF was placed in a steel autoclave
and heated at 110C for 4 days. The contents were cooled
and adsorbed onto 100 g of silica gel 60 and placed atop a
700 g column of silica gel made up in 9:1 (v:v) carbontetra-
chloride:acetone. Elution was with the same solvent and
fractions of approximately 20 ml volumes were collected.
Fractions 276-803 were combined and evaporated to give 4.65 g
of solid. Recrystallization from ethanol gave 2.65 of
5-[2-(4-N-formylamino)butoxyphenyl]-5-phenylhydantoin as a
white solid, mp 201-203C.
A solution of 3.5 g (9.4 mmol) of 5-[2-(4-N-formylamino)
butoxyphenyl]-S-phenylhydantoin in 100 ml of 1 N sodium
hydroxide was heated on the steam bath for 24 hours. The
solution was cooled and neutralized with carbondioxide until
precipitation ceased. The precipitate was filtered and
recrystallized twice, first from pyridine-2-propanol, then
from methanol to give 1.5 g of 5-[2-(4-aminobutoxy)phenyl]
-4-phenylhydantoin as fine white crystals, mp 235C (decomp).
A mixture of 808 mg (2 mmol) of the potassium salt of
7-~-galactosylcoumarin-3-carboxylic acid [Burd et aZj CZin.
Chem. 23:1402(1977)] and 20 ml of dry DMF was made and
.
- 39 -
1~48~80
cooled to 0C. To this mixture was added 216 mg (2 mmol)
of ethylchloroformate. After stirring for one hour at this
temperature, 638 mg (2 mmol~ of 5-~2-(4-aminobutoxy)phenyl]
-5-phenylhydantoin, 244 mg of 4-dimethylaminopyridine, and
5 ml of dry pyridine were added. After stirring for 5 hours,
the reaction was stored overnight at 0C, then adsorbed onto
7 g of silica gel 60. The impregnated silica gel was placed
atop a column of 200 g of silica gel 60 and the column eluted
with a gradient of 2 liters of ethyl acetate to 2 liters of
l:l (v:v) ethylacetate:ethanol. Ten ml fractions were
collected, Fractions 143-160 were combined to give approxi-
mately 200 mg of the labeled conjugate N-{4-[2-(5-phenylhydan-
toinyl-5)phenoxy]butyl}-7-~-galactosylcoumarin-3-carboxamide
as a glossy solid.
The solid was taken up in methanol and chromatographed
on Sephadex LH-20 (45 cm by 3.2 cm), eluting with methanol.
Seven ml fractions were collected. Fractions 30 to 40 were
combined and evaporated to give 100 mg of the desired labeled
conjugate as a pale, glossy solid.
Analysis: Calculated for C35H35N3O12 H2O: C,
H, 5.24; N, 4.95.
Found: C, 59.51; H, 5.04; N, 6.14.
[a]D = ~39-04 (c 1.0, methanol).
B. Assay Procedure
A reagent was prepared containing ~-galactosidase
(0.018 U/ml) and antiserum to diphenylhydantoin (raised
against o-caproyldiphenylhydantoin - in an amount sufficient
in the final reagent to decrease fluorescence to 10~ of that
- 40 -
1148~80
observed in the absence of antibody) in 50 mmolar Bicine buffer,
pH 8.2. To 3~0 ml aliquots of this reagent in a cuvette were
added 100 ~1 aliquots of diphenylhydantoin serum standards
followed by 100 ~1 aliquots of the labeled conjugate (part A
s above). After mixing, the reaction mixtures were incubated
20 minutes at room temperature and fluorescence measured in
each cuvette (excitation and emission wavelengths were 400
and 450 nm, respectively).
C. Results
A standard curve generated for the assay of diphenyl-
hydantoin according to the above procedure is depicted in
Figure 10 of the drawings.
The examples demonstrate that the use of the novel
labels of the present invention are more advantageous than
the prior labels used in speci~ic binding assays employing an
enzyme-cleavable substrate as label. With the present labels,
endogeneous enzyme activity of a serum sample and antibody
-induced hydrolysis of the cleavable linkage were found not
to be a source of potential error. Further, no background
hydrolysis of the labeled conjugate was observed.
- 41 -
