Note: Descriptions are shown in the official language in which they were submitted.
Background o~ the Invention
The present invention relates to biochemical
diagno~tic and assay methods and more specifically to the
de~l~rm~af~O~
B ~kYr}4~LM} o~ very small quantities o~ chemical species
involved in life processes, e.g., enzymeq and enzyme
substrates, antigenq and antibodies, etc. This invention
specifically relates to those analytical methods in which a
. chemical species to be determined, generally a bio-material,
109~4'77
is coupled through intermediate reactions or reacts directly
in an electromagnetic signal-generating system in which ~he
species, or its progeny in the case of intermediate reactions,
is converted into an end product ~ith the concomitant release
of electromagnetic radiation.
Life processes involve a staggering variety of bio-
chemical reactions, all interrelated,- and occurring either
simultaneously, or in carefully regulated sequences. Many
processes that may involve relatively massive amounts of
materials, e.g. metabolic processes, may, in turn, be
regulated by minute amQunts of bio-materials, e.g. enzymes or
hormones. In other instances, malfunctions and/or diseases of
the organism may release extremely small amounts of bio-
materials from their normal environments into other systems of
the organismO The detection and quantification of these bio-
materials, both in their normal environment and in abnormal
environments can yield a great amount of information
concerning the functioning of both major and minor systems in
the organism, and can indicata system malfunction and/or
disease, as well as invasion by foreign bodies such as
bacteria or viruses. Such a bio-material is thus generally
defined as any chemical compound found in living organisms.
In recent decades, various techniques have been
developed for determining very small quantities of bio-
materials. These techniques may utilize, for instance,radioactive tracer techniques, fluorometric techniques,
colored dye development, bioluminescence, chemiluminescence,
etc. Such techniques depend on inherent characteristics of
the materials of interest that give rise to signals that can
be detected on suitable instrumentation; or by combining or
10~l~4~7
associating ma~erials that generate, or can be induced to
generate such signals, with the molecular species of interest.
The particular analytical technique to which this
invention specifically relates involves electro~agnetic
radiation generating reactions, more particuarly those
electromagnetic radiation generating sys~ems in which light is
produced either by the reaction of a bio-material T,7ith a
protein or by the enzyme-potenti~ted reaction of the material
with a second chemical species. Such systems derived from
living systems and which invol~e proteins, including enzymes,
are defined herein as bioluminescent reactions. They haYe
been extensively discussed in the literature. For exa~ple,
see Johnson et al in Photophysiology, Vol. 7, pages 275-334
(1972). The sources of reagents for sucn reactions as well as
purification techniques for the reagents are well known. By
"electromagnet1c radiation generating" applieant means
chemical systems which emit electromagnetic radiation upon the
reaction of the system components with one another, whether or
not the reaction requires a catalyst, whereby at least one
product is yielded which was not a component of the unreacted
system.
The electromagnetic radiation produced by
bioluminescent reaction systems is, directly proportional to
the amount of the reaction limiting component available for
entering into the reaction. By way of illustration, light is
B produced when the enzyme 9 luciferas~9 ~cts to oxidize the
substrate, fire-fly luciferin, in the presence of co-factor,
adenosine triphosphate (ATP) and oxygen. The reaction may be
summarized:
Luciferin + ATP ~ 2 luciferase, oxidized Luciferin
AMP ~ H20 + P P
LIGHT ~ C02
1~944'77
Where AMP is adenosine monophosphate, and P-P is diphosphate.
The oxi~ation of each luciferin molecule yields a specific
quantity of light, with the total light yield being directly
proportional to the number of molecules oxidized. ThusJ a
measurement of the light yield indicates the number ~f
luciferin, ATP, or oxygen molecules entering into the
reaction, depending upon which of the three components is in
molar excess, or the activity of the catalyst, luciferase.
If ATP is the least abundant species, then the
reaction will cease when all the ATP is used up; or, if oxygen
is the least abundant, then when all the oxygen is used up.
By the same token, the activity of the luciferase can be
ascertained, if its activity is the limiting factor in the
reaction process. The most accurate and complete results are
obtained by ensuring a molar excess of all the components of
the bioluminescent system other than the one of unknown
concentration or activity to which the assay is directed.
Similar considerations apply in the use of the
bacterial luciferase system for analysis of chemical species,
e.g. bio-materials. Here, bacterial luciferase catalyzes the
oxidation of reduced flavin mononucleotide with oxygen in the
presence of a long carbon chain aldehyde to yield light, among
other products. This system is typically employed to
determine reduced flavin mononucleotide~ The flavin
mononucleotide may be the product of a reaction or series of
reactions in which the flavin mononucleotide is eventually
produced in a quantity that is directly proportional to a
chemical species reacted at the first of the series. For
example, dehydrogenases such as flavin mononucleotide
oxidoreductase will oxidize the reduced form of nicotine
adenine dinucleotide, which in turn may be produced by other
109~477
dehydrogenases, to reduced flavin mononucleotide. The product
flavin mononucleotide is then employed as the limiting
component in the bacterial luciferase system. The light so
generated is a measure of the original reduced nicotine
adenine dinucleotide. A multiplicity of reactions of this
nature may be coupled together to yield a product which is
determinable by a bioluminescent reaction. As a consequence,
any chemical species which can be reacted to eventually yield
a stoichiometrically equivalent quantity of ATP or reduced
flavin mononucleotide may be assayed, respectively, by the
fire-fly and bacterial luciferase systems. The prior art has
employed the foregoing bioluminescent reactions in qualitative
- or quantitative coupled and direct assays. For example, see
Hammerstedt, "Analytical Biochemistry" 52 :449-455 (1973);
Brolin et al., "Analytical Bioche~istry" 39 :441-453 (1971);
and Mansberg, U.S. Patent No. 3,679,312. In those cases where
these assays have heretofore been conducted in a liquid
environment all of the reagents were in solution and thus
distributed homogeneously throughout.
When assaying for very low quantities of chemical
speciss, or very low activities of enzymes~ the quantity of
electromagnetic radiation generated by processes such as noted
above, is correspondingly small. In addition, since the
reactions have heretofore been carried out in solution, the
2~ radiation emitting components are dilute and the radiation is
emitted throughout a volume whereby the radiation intensity is
lower than if high concentrations of reagents could be
loyed. This adversely affects the assay sensitivity. In
addition, the larger and more opaque the volume of liquids,
3~ the greater is the possibility of self-absorption of the
emitted radiation before it can leave the solution and be
detected by suitable instrumentation. Finally, prior
~09~77
techniques measure the radiation as light emitted from
a transparent container. However, irregularities in the
container wall will scatter the light unpredictably, thus
introducing variation into the assay.
Another detriment of conducting electromagnetic
radiation assays in solution is the loss of costly
reactants such as, for instance, enzymes and co-enzymes.
Generally, there is no simple means of recovering such
materials from solution, and they must, therefore,
be discarded and replaced by new reactants for each
successive assay.
In order to conserve costly enzyme materials and
recover them for subsequent use, it has become well known
in the art to immobilize various enzymes to insoluble
support members or to one another so that the material
is not lost or leached into solution during the reaction
processes. See, for instance, U.S. Patent Nos. 3,925,157
to Hamsher; 3,930,950 to Royer; 3,959,079 to Mareschi et
al; 3,542,662 to Hicks et al, all of which describe various
means and materials for attaching enzymes to support
materials. H. H. Weetall has reviewed the chemistry of
enzyme immobilization in "Analytical Chemistry," Volume
46, pages 602A et. se~., (1974) and the applications of
immobilized enzymes has been discussed in "Analytical
Chemistry," Volume 48, pages 544 A et. seq. (1976). The
prior art has, however, not disclosed immobilizing bio-
luminescent proteins such as the luciferases so that they
can be recovered from the test solution and used over.
Similarly, it is heretofore unknown to immobilize flavin
mononucleotide oxidoreductase.
Summary of the Invention
In the prior art analytical processes a chemical
.......
.
` 109 ~4~7
species is assayed by reacting said species with an
electromagnetic radiation generating system distributed
substantially homogeneously throughout a reaction
environment, followed by detection of the generated
radiation as a measure of said species. In the present
invention, the prior art analytical processes are improved
by immobilizing at least one component of the electro-
magnetic radiation generating system within a localized
region of the reaction environment. Hence, the generated
radiation is concentrated at a single point or region of
emission rather than throughout the entire environment in
which the reaction takes place.
An important object of the invention is to provide an
improved assay method for enzymes or enzyme substrates.
According to one aspect of the invention there is
provided an assay method for enzymes or enzyme substrates
which comprises (1) providing at least one first enzyme
(2) reacting said first enzyme with the substrate whereby
a first product is formed in proportion to the first
enzyme or said substrate, (3) providing an oxidoreductase
qen~ n~
and a light ~ enzyme, both the oxidoreductase
and the light generating enzyme being insolubilized upon
an inert solid support, (4) reacting said first product
with such oxidoreductase to produce a second product,
said second product being a component which affects the
emission of light by said Iight generating enzyme and
detecting light generated by said light emitting enzyme.
According to another aspect of the invention there
is provided a product useful for assaying biochemical
species and chemical species associated with bioreac-
tions comprising an insoluble support member, an enzyme
-- 7 --
B
`
.
- 109~77
retaining material integral with said support member,
luciferase enzyme covalently bound to said enzyme
retaining material, and FMN oxidoreductase also covalently
bound to said enzyme retaining material in admixture with
said luciferase.
It is an advantage of the invention, at least in its
preferred forms, that it can immobilize onto a solid
support at least one component of an electromagnetic
radiation generating bioluminescent system.
It is anot~er advantage of the invention, at least
in its preferred forms, that it can provide a method
for concentrating and intensifying the electromagnetic
radiation emitted during the course of chemiluminescence.
It is still another advantage of the invention, at
least in its preferred forms, that it can immobilize and
concentrate bioluminescent systems that emit visible light
during reaction with suitable substrates in a liquid
environment.
It is yet another advantage of the invention, at
least in its preferred forms, that it can provide methods
for assaying very small quantities of chemical species
by coupling said species, or reaction products from said
species, with bioluminescent reactions, wherein at least
one bioluminescence generating component is concentrated
and immobilized on a solid support.
It is still another advantage of the invention, at
least in its preferred forms, that it can covalently
bond bacterial luciferase and flavin mononucleotide
oxidoreductase enzymes while retaining sufficient
enzyme activity to employ the enzymes in assays.
It is an additional advantage of the invention, at
.~
'10~477
least in its preferred forms, that it can provide an
article for introducing at least one component of a
chemiluminescent system into a liquid environment
without loss of said component into the environment.
It is a further advantage of the invention, at
least in its preferred forms, that it can eliminate the
variation in che~iluminescent and bioluminescent assays
performed in a liquid environment which is opaque or
variably opaque to the electromagnetic radiation generated
by said chemiluminescent and bioluminescent systems or
wherein the walls of the liquid container are irregular.
Detailed Description of the Invention
The electromagnetic radiation generating component is
generally immobilized on a support which is insol~ble in
the reaction environment, ordinarily liquid solutions, and
particularly aqueous solutions. However, the component
may be treated to render it insoluble without the use of a
support, for example, by polymerizing the component. It
is preferred to bond the component to the support in such
a fashion that the component will only leach into solution
in insignificant quantity. This is highly important in
ensuring the reliability of the assays when using an
immobilized component over a multiplicity of tests since
otherwise the net activity of the component in the test
will decrease steadily over use, and the results so
obtained will change unless standards are prepared with
impractical frequency. Of course, this loss in activity
would also lower the sensitivity of the assay. Hence,
it is preferred to covalently bond the component to the
support.
- 8a -
, . ,
109~
In the case of the bacterial luciferase syste~, for
example, FMN can be insolubilized upon a support according to
the method of Waters et al; ~Biochem. Biophys. Research Comm.'
57 (4)~ 2-1158 (1974). I have found that such insoluble
FMN can be reduced by FMN oxidoreductase acting upon reduced
pyridine nucleotide. The insolubilized, reduced FMN will in
turn participate in the ordinary soluble bacterial luciferase
syste~. However, just as in the case of insolubilized
luciferase, light is released only at the site of the
insolubilized reduced-FMN. In sum, the localized,
concentrated release of light which forms the basis of this
invention is best obtained by covalently bonding one or more
of the electromagnetic radiation generating system components
to an insoluble support.
The means of attachment to the support may be any
one of a number of known methods that have been used to
îmmobilize enzymes and similar bio-materials. It is only
necessary that the attachment procedure does not impair the
functionality of the immobilized component. It is also
advantageous to have as much as possible of the component
concentrated on the surface of the support material so that,
(1) it will be readily accessible to the other reaction
components, and t2) the emitted radiation will not be masked
or absorbed by the support material. Radiation transparent
support materials, such as glass, are particularly suitable
- and are preferred for use in the invention procedures.
The support material is most conveniently in the
form of rods, strips or similar shapes that may be immersed
into reaction solutions, and easily handled, cleaned, and
stored for subsequent use and re-use.
109~477
It is most usual in the case of biolum-nescen~
systems to im~obilize enzymes, since they are suscep'ible 'o
multiple re~se and are, most gener2lly, the cos~lies'
component of the bioluminescen~ syst~ms. Enzymes may bs
immobilized and insolubilized by suitable well-kno-m
techniques. An extensive review ol such techniques as well as
suppor' materials is set forth in ".Iethods in Enzymolo~y",
Academic Press, 1974.
The support may be selec~ed ~rom a large number of
materials. The basic properties of the support are, ~1) an
ability to immobilize or "fix" a component of the
electromagnetic radiation generating sys~em by either physical
or chemical bonding means without (2) interfering with the
activity Or the "fixed" component. The support should also
(3) be capable of immobilizing or concentrating a relatively
large amount of the component over as limited a surface or
volume aæ possible. Thus, it shoul~ have a high surface
concentration of blnding sitesc Also, i~ is desirable to use
porous or convoluted surfaces.
A great number of materials are suitable, among
which are synthetic organic polymers æuch as acrylics,
polyacrylamides, polyacrylic acids~ methacrylates, styrenes,
B nylons, etc.; carbohydrate polymers such as Sep~adex~
~Tr~.Je ~Y1a~3
Sepharos~, Agarose and derivatives; all types of cellulosics,
including cellulose products and the~r derivativ2s; and
miscellaneous materials such as silicas, insoluble prot~ins,
clays, resins, starches and the likec However, the prererred
materials are those materials that are op~ically transparent
and interfere to a minimum extent with the transmission of the
3o electromagnsfvic radiation generated ~rom the immobilized
components "f~xed" upon their surface. Porous glasses,
especially those of the arylamin~ or alkylamine typ~s
--10_
~`
109
av~ilable from Corning Glass, Biological Products Div., are
highly suitable for use as support material. Such porous
glasses react with the enzymes that comprise bioluminesc~nt
systems to provide strong, non-leaching covalent bonds; they
are inert and stable over extended periods of use; and they
are transparent to the emitted radiation.
The porous glasses are available in the form of fine
loose beads. For the purposes of the invention, it is
desirable to immobilize the component onto rods or sticks in
order to concentrate and localize the emitted radiation to the
greatest extent possible. The immobilization of the component
in rod form also facilitates insertion of the immobilized
component into standard cuvets.
In an embodiment of the invention, an optical fiber
15 bundle i~ used as the support. Such fibers, ~cnown also as
~'light pipes~', are readily available commercially. The
electromagnetic radiation generating component is immobilized
onto a light receiving and conducting sur~ace o~ the fiber or
fiber bundle. This permits a simple immersion of the fiber
surface into the test sample and detection of generated
radiation by conducting the radiation throu~h the fiber to a
radiation detecting element at same location distant from the
test sample. Thus, the detection of radiation is not af~ected
by sample opacity or irregularities in the sample container.
Chemical species not directly involved in the
radiation producing reaction may also be assayed through
reaction coupling techniques as described above wherein the
product of one reaction is utilized as a reactant in a
~ubsequent reaction. A final reaction product is utilized an
an essential and limiting reactant in the radiation producing
reaction to determine and control the amount and intensit~ of
radiation emitted from the final reaction. The concentration
~c 109~q7
or activity of the original species can then be calculated
from the radiation emitted in the final reaction.
If the immobilized radiation generating co-,nponent is
subject to chemical reversibility to its form prior to
radiation generation or if it undergoes no net change in
structure during the radiation emitting reaction as is,
respectively, the usual case with coenzymes and enzymes,
repeated use is possible. Thus, the costly component is
conserved and repetitive assays expedited.
The concentration and localization techniques can be
applied to various types of electromagnetic radiation
generating systems. Bioluminescent syst~ms such as the fire-
fly luci~erin-luciferase-ATP reaction or the bacterial
luciferase reaction involving flavin coenzymes are
particularly adaptable to the present techniques. The
bacterial luciferase system is additionally valuable in that
they are readily coupled to oxidoreductase reactions,
especially those utilizing nicotinamide adenine dinucleotide
(NAD+), and/or nicotinamide adenine dinucleotide pho~phate
(NADP+), which are involved in a great number o~ bio-systems.
The fire-fly luciferin-luciferase reaction is also especially
valuable since it is readily coupled to the ATP coenzyme
producing systems that are broadly involved in bio-energy
transfer systems.
Similarly, chemiluminescent systems may be readily
employed in the method of this invention. For example,
luminol can be entrapped within or covalently bound to an
insoluble matrix and then used in a conventional assay for
- oxidizing agents such as hydrogen peroxide. Again, the
emitted light is generated in the same reaction that is used
to detect and indicate the chemical species being tested for,
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109~77
and the light is generated in a highly concentrated form at a
localized point with the re~ction environ~nt.
The basic principles of the invention May be better
understood by considering the following specific detection
system:
A number of bacterial species, e.g., Photobacterium
fischeri, Photobacterium phosphoreum, and Beneckea harveyi, are
kno~rn to generate visible light. It has been determined that
this light generation involves the specific reaction of the
bacterial enzyme, luciferase, with a co-factor, flavin
mononucleo~ide (commonly abbreviated, FMN) to produce light.
More specifically5 the light producing reaction
occurs when luciferase catalyses the oxidation of the reduced
form of co-factor, flavin mononucleotide, FMNH2, to the
oxidized form, FMN, in the presence of a lon~ chain aldehyde
substrate and oxygen. The reaction may be written:
bacterial
RCH0 +2FMNH2 ~ 22 luciferase ~ 2 FMN ~ RCOOH ~ Hz
H20 + LIGHT
Where RCH0 may be any long chain aldehyde having from about 8
14 carbon atoms. Decanal, tridecanal, dodecanal, undecanal~
ekc. are suitable aldehydes for the ~ubstrate.
The light emitted in the reaction is directly
proportional to the number of molecules undergoin~ reaction.
Measurement of the emitted light, therefore, indicates the
least abundant molecular species present as the substrate or co
~actor; or should the luciferase be the reaction limiting
factor, then the light emitted is an indication of the enzyme's
activity.
3~ The bacterial luciferase is immobilized on arylamine
porous glass beads. The beads have been previously glued on a
thin glass rod. Any suitable glue material is used to tightly
-13-
`i ~ 10~77
adhere the beads to the rod. A standard epoxy glue is useful
for thi~s purpose. The luciferase is coupled to the porous
glass beads utilizing a diazoti~ation procedure like that
disclosed in the publication "Methods in Enzymology", the
Academic Press, Ne~ York, pages 59-72. Briefly, the high
silica porous glasses contain nitro-aryl groups ~ormed by the
amide coupling of nitrobenzoyl chloride thereto~ Thè nitro-
aryl are then reduced to amino-aryl groups by either sodium
dithionite or LiAlH2. The amino-aryl group are activate~ by
diazotization to provide coupling sites for the luciferase.
The luciferase in a buffered aqueous solution (pH7) is then
placed into contac~ with the beaded rods for 16 hours to effect
coupling of the enzyme to the porous glass. The excess,
uncoupled enzyme is then washed from the rods and the rods are
stored in buffer solution at reduced temperature (~ C) for
subsequent use. If carefully handled, and thoroughly rinsed
after each use, the rods with the immobilized luciferase may be
reused an indefinite number of times *ithout significantly
affecting the enzyme activity.
The same immobilization technique may be employe~ for
other bioluminescent enzymes, and proteins.
In order to conduct an assay, the rod with
immobilized luciferase is dipped into a solution containing all
the other components or reactants necessary to produce the
radiation generating reaction except for the species being
assayed. The species is provided, if at allg by the test
sample. Since at least one of the essential components is
immobilized on the support, the radiation generating reaction
takes place directly on the support surface. It is, therefore,
only necessary to enclose the reaction mixture and immersed rod
within the confines of a photometer sample chamber while the
radiation generating reaction takes place. All of the soluble
~' 'It'
10~4~77
components can be combined, the sample chamber closed and the
rod immersed, whereupon a flash occurs. Alternatively, it is
preferred to immerse the rod in a solution which is complete
but for one or more reagents, or sample, followed by closing
the sample chamber. Addition of the missing reagent or the
sample will then produce a flash. Suitable electronic
circuitry may then be utilized to measure the peak or total
radiation emitted from the reaction. The radiation intensity
or total radiation emitted measures the quantity of the least
abundant molecular species necessary for the radiation
emitting reaction; or alternately, the activity in the case of
enzymes or other catalytic materials.
The radiation which is emitted by the test system of
this invention may be determined by the Aminco Chem-Glo~
Photometer. This highly accurate and sensitive instru~ent is
conveniently employed with the method and article of this
invention. The instrument is equipped with a reaction chamber
that holds cuvets for the reaction, as well as ports for the
injection of various components while the sample is contained
in the instrument.
Suitable apparatus is also commercially available
for recording the radiation output detected by the photometer.
Turning to the fire-fly luciferase reaction
discussed above, fire-fly luciferase requires ATP for the
light emitting reaction. Fire-fly luciferase requires ATP for
the light emitting reaction~ ATP, in turn~ is a universal
; energy source in a vast number of bio reactions, and its
presence, or absence, in such systems is a unique measure of
many bio-reaction reactants and products.
Typical ATP producing systems are, by way of
illustration; sugar synthesis systems wherein phosphoenol
-15-
10~ ~477
pyruvate in the pre~ence of co-factor adenosine diphosphate
and the enzyme pyruvate kinase yields pyruvate and adenosine
trip~osphate (ATP). Other systems are muscle contraction
systems, wherein creatine phosphate is converted into creatine
while its co-factor adenosine diphosphate converts to ATP in
the presence of the enzyme, creatine phospho-kinase. ATP
assays can also, for instance, be use~ul in determining
bacterial content in urine, waste products, wine, beer, milk,
and, in general, bioma~s measurements. Hence, the measurement
of ATP in any bio-system can be utilized as a measure of ATP
co-factors, substrates9 and related enzymes.
It has been noted before that ATP is a co-enzyme in
the light producing luciferin-luciferase reaction. As a
consequence, the light generated from a luciferin~luciferase
reaCtion will assay ATP quantitatively wherein the ATP is the
limiting component in the reaction and qualitatively,
otherwise. An assay of ATP, in turn may be used to calculate
the abundance of chemical species which yield or metabolize
ATP.
In a similar manner, the bacterial luciferase
reaction may be coupled back to a vast number of bio-
reactions. Consider the following coupled reactions~
(1) Bio-material to be assayèd ~ NAD (or NADP~
Enzyme
NADH (or NADPH) + Product
~
t2) NADH (or NADPH) + FMN
NAD: FMN OXIDOREDUCTASE
FMNH2 + NAD (or NADP)
(3) RCHO ~ FMNH2 + 2
Immobilized
Bacterial Luciferase
FMN + RCOOH ~ H2O + H22 + LIGHT
-16-
- 10~4477
~here NAD refers to nicotinamide ad~nine dinucleotide, NADP
refers to nicotinamide adenine dinucleotide phosphate, and
NADH and NADPH are the reduced form3, respectively. FMN,
FMNH2, RCHO, and RCOOH have been defined hereinbefore.
Reaction (3) has been set forth before and defines
the bacterial luciferase light producing reaction that is
measured according to the principal method of the invention.
Reaction (2) is an oxidation-reduction reaction which is
catalyzed by the NAD:FMN oxidoreductase that is obtained by
kno~ln methods from bioluminescent b~cteria such as Beneckea
harveyi. For example, the oxidoreductase is separated from
the bacterial luciferase during the purification thereof by
well-known chromatographic techniques. Thus, when luciferase
is purified by chromotography on DEAE-Sephadex, the reductase
elutes before the luciferase and may be collected as a
separate fraction. Reaction (1) is any of a large number of
bio-reactions in which NAD (or NADP) are necessary co-factors.
A few examples of such NAD or NADP requiring reactions are.
Alcohol + NAD (or NADP) alcohol dehydrogenase
adehydes + NADH ~ or NADPH)
2~3-Butanediol + NAD butanediol dehydrogenase
acetoin + NADH
glycerol ~ NAD glycerol dehydrogenase
dihydroxyacetone ~ NADH
xylitol ~ NAD (or NADP) D-Xylulo9e reductase
(L-xylulose reductase)
D-xylulose (L-xylulose) + NADH
galactitol + NAD galactitol dehydrogenase
D-tagatose ~ NADH
~ glucuronate dehydrogenase
L-gulonate ~ NADP ~
D-glucuronate ~ NADPH
-17~
1094~77
alditol ~ NADp aldose reductase ald
glycollate + NAD glYOxylate reductase
glyoxylate + NADH
L-lactate ~ N~D lactate dehydrogenase
_,~
pyruvate + NADH
L malate + NAD malate dehydrogenase oxal~acet te NADH
-
~-O-glucose ~ NAD (or NADP) glucose de`nydrogenase
D-glucone- ~ -lactone + NADH (or NADPH)
andros~erone + NAD (or NADP) 3~ ~ -hydroxy steroid
~ dehydrogenase
androstane -3, 17-dione f NADH (or NADPH)
2o-dihydrocortisone ~ NAD cOrtisone reductase
cortisone + NADH
- pyridoxin + NADP pyridoxin dehydrogenase
pyridoxal ~ NADPH
mannitOl ~ NAD mannitl dehydrogenase
~ .
fructose + NADH
aldehyde ~ NAD ~ H20 adehyde dehydrogenase
acid + NADH -
Many other similar NAD or NADP co-factor reactions are
known and the above are merely illustrative.
In any event, it is clear that a graat number of bio-
reactions produce NAD or NADP in the reduced state. If such
reactions (1) are coupled into the NAD:FMN oxidoreductase or
NADP:FMN oxidoreductase reaction (2)l it is apparent the ~MNH2
will be produced in accordance with the quantity of NADH (or
NADPH) available from reaction (1). If the FM~H2 produced by
reaction (2) is thereupon introduced into reaction (3), the
bacterial luciferase reaction~ the light produced thereby will
be proportional to the original quantity of pyridine
nucleotide; and h.ence, to the dehydrogenase enzyme or its
3 substrate which is to be determined.
-18-
~0~ 77
Coupling the radlation producing reaction into
precursor reactions as noted above leads to a variation of the
immobilization procedures of the invention. Specifically, it
is often advantageous to concentrate and immobilize two or
more essential components for a series of reactions on a
single support member. Such technique permits the direct
coupling of reactions of the types (2) and ~3) noted above.
In such a technique, the desired ~MN oxidoreductase
is immobili~ed on the same support as the luciferase. This
yields the additional advantage of this invention that the
highly oxidation labile FMNH2 yielded by the NADH-FMN reaction
is produced in extremely close proximity to the luciferase and
thus, it is directly and immediately available to enter into
the luciferase reaction. Manipulative steps are thereby
reduced and losses or spurious re-oxidation of the FMNH2 by
the sample components or contaminants are avoided. In such
specialized uses, dehydrogenases, for example, can also be
bound to the support.
The following example will illustrate a double
immobilization of two enzymes on a single support.
Example:
10-15 mgs. fine beads of activated arylamine glass
were glued to 1.7 mm. diameter glass rods 4 cm. long. The
glass rods were first dipped into Duro E~Pox.E 5 glue and then
rolled into the porous glass beads. The rods and adherent
beads were allowed to dry overnightO The luciferase and
reductase enzymes (isolated frome Beneckea harveyi) were mixed
in the ratio of 1 mg. luciferase t-o 1.5 mgs. reductase of
which 0.5 ml. aqueous solution was contacted with the rods and
activated beads for 16 hours. The solution was buffered at pH
7.0 with O.lM phosphate. The rods were then washed with 25
.
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.
. : .,
109~77
mls. cold lM sodium chloride followed by lO0 mls. cold
distilled water to remove any unbound enzymes. The rods were
then incubated overnight in l~ bovine serum albumin (BSA) in
the phosphate buffer containing 5 x lO 4.~1 dithiothreitol
(DTT). The rods were then stored in p'nosp'nate buffer
containing the same amount of DTT at 4 C.
The bound enzymes were assayed, and TABLE I below
give typical results for the binding of the enzymes to the
porous glass beads and their apparent activities.
-
./ - . ., ......................... ~
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TABL.E I: BINDING OF LUCIFERASE A~D F~lN:R~DUCTASE TO GLASS RODS
FMN
Luciferase Reduction Coupled mgs. Protein
Relati~e Light umoles assay ml
Units/~l NADH Oxid. Relative
per ml per min Light units/ml
(~ Original 6 - ~ _
Mixture 7~0 x 10 .293 4.2 x 105 2.56
(B) Supernatant 2 x 106 .100 2 x 104 1.25
(C) Rods 2.5 x 103 .020 1.2 x 104 1.31
~ of Rods
Apparent
Activity 0.05~ 10.3 3.0 51
.
(A) Enzymatic activities of a mixture of soluble luci~erzse-reductase
prior to coupling to the beads, original mixture. (B~ After the
coupling procedure the mixture was again assayed, supernatant. (C)
The amount of activity associated with the rods was also assayed. The
percent of activity as assayed on the rods was based on the initial
total activity in the original mixture. Luciferase was assayed by
injection of FMNH2o FMN:Reductase was assayed by disappearance of
absorbance at 340 nm and the coupled assay is the light obtained upon
injection of NADH~
The enzymes, both those in solution and those immobilized on
the porous glass were assayed as follows~ All soluble enzyme assays
were performed at 23 C. Luci~erase was assayed b~ injection O.lcc
FMNH2, catalytically reduced with H2 over platinized asbestos, into a
solution containing luciferase, decanal and 0u1% BSA in O.lM phosphate
buffer pH 7Ø Final concentration of the reactants were: 2.3 x 10 ~-
M FMNH2 and 0.0005% decanal and 0.08 ug luciferase per ml. Light
intensity was measured in an Aminco Chem-Glo ~3 Photometer and recorded
on an Aminco Recorder. The peak intensity was linear with respect to
added luciferase in the ran~e of .08 ug to 8 ug per ml ~sing this
instrument. Immobili~ed luciferase was assayed using the same
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concentrations of substrates. The rod containing the glass
beads was placed in a test tube in the photometer and FMNH2
was injected.
Soluble FMN:reductase was assayed by measuring the
rate of disappearance of absorption at 340 nm in a Cary Model
14 recordin~ spectrophotometer. The reaction was initi~ted by
adding N~DH to 1 ~1 of 0.015 M phosphate buffer pH 7.0 -
containing 7 x 10-5M ethylenediamine tetraacetic acid, 0.4 mgs
reductase and FMN. Final concentrations were 2 x 10-4M NADH,
1.3 x 10 4M FMN. When the immobilized enzyme was assayed the
rod containing the enzyme was dipped in~o the cuve~ which was
mixed for 1 minute intervals, then removed and the 0~ 340
measured. This assay was linear for at least 3 minutes.
The coupled assay was measured by peak light
intensity obtained following injection of NAD(P)H into Q.5 ml
of O.lM phosphate buffer pH 7.0 containing 7.5 ug reductase, 5
ug luciferase, and 2.3 x 10 6M FMN and 0.0005~ decanal. When
the immobilized enzyme was being assayed, the rod was immersed
in the solution containing FMN and aldehyde. NAD(P)H was
injected into the solution~
The immobilized enzymes exhibited linearity in peak
light intensity as a function of either NADH or NADPH
concentration. Linearity with NADH was obtained in the range
of 1 x 10 12 moles to 5 x 10 8 moles9 and ~or NADPH in the
range o~ 1 x 10 11 moles to 2 x 10 7 moles. The bound enzymes
were stable and reusable.
The methods and techniques o~ the invention may be
applied to assaying ligand-receptor-interactions, in
particular, antigen-antibody binding~
3 More specifically, U.S. Patent NoO 3,817,837 to-
Rubenstein et al, issued June 18, 1974, describes a means for
assaying ligands wherein enzymes are bound to the ligand to
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provide an "enzyme-bound-ligandll. Enzym~tic activity of the
bound enzyme ma~ be inhibited when the '~enzyme-bound-ligands"
are contacted with receptor molecules. Binding of the ligand
by the receptor inhibits the activity of the enzyme bound to
the ligand in inverse proportion to the amount of native
ligand that is provided by a test sample. A determination of
the enzyme activity is thus a measure of the sample ligand.
It will be apparent that the binding enzyme may be selected
from those groups of enzymes that require NAD or NADP or ATP
as co-factors. In such event, the ligand bound enzyme is
reacted with a suitable substrate and co-factor to produce
NADH, NADPH, or ATP. The N~DH, NADPH or ATP ~hus produced may
then he coupled into the light producing luciferase reactions
in the identical manner as noted above to provide an assay
means for the enzyme~bound-ligand.
In a similar vein, immuno-assay procedures that rely
upon enzyme determinations may be coupled into the immobilized
light-producing reaction of the invention~ For instance, U.S.
Patent No. 3,791,932 to Schuurs et al, issued February 12
1974 discloses a procedure for determining ligands or
receptor~ which comprises reacting the component to be assayed
w~th its binding partner in an insolubilized form, thereafter
separating the solid phase from the liquid phase, and then
reacting the solid phase with a determined amount of a
coupling product of the substance to be determined with an
enzyme. The activity of the enzyme distributed between the
insolubilized and supernatant material is then determined as a
measure of the antigen or antibody in the test sample. As in
the case of the Rubenstein et al procedure, it is obvious that
3 a properly selected enzyme can be reacted with a substrate
which will yield a product determinable by the present
invention method. The enzyme reactiQn products, can be then
10~4q7
coupled into the immobilized radiation producing enzyme
reactions of the present invention to assay the product.
As an additional embodiment of the invention, it is
kno~n to detect bacteria in fluid samples through the reaction
of iron porphyrins, such as~ peroxidase, cytochrome, catalase
contained in microbial cells, with luminol (5-amino-2, 3-
dihydro-l, 4-phthalazine-dione) to produce visible light.
See, for instance, Picciolo, et al, Goddard Space Flig`nt
Center publication X-726-76-212, dated September 1976,
entitled ~Applications of Luminescent Systems To Infectious
Disease Methodology", pages 69 et. seq.
In such systems chemiluminescence is produced by the
reaction of luminol with hydrogen peroxide in aqueous alkaline
solution in the presence of an oxidizing activating agent such
as ferricyanide, hypochlorite, or a chelated transition metal
such as iron or copper. In the bacterial detection system,
the iron porphyrins are considered as activators for luminol
chemiluminescence.
Such a chemiluminescent system is adaptable to the
methods of the invertion by concentrating~ localizing and
immobilizing the luminol on suitable support materials. The
luminol may be absorbed on a support material such as those
previously referred to herein.
The localized immobilized luminol, ~ill generate a
concentrated light emission upon the activator-catalyzed
reaction with hydrogen peroxide. This emission may be
conventionally detected using the aforementioned photometer as
a measure of activator, hence bacterial, presenceO
Although the description, supra9 discloses and
describes a number of specific examples of the methods and
techniques of the present invention, it will be understood
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that the invention is not to be limited thereby. All
extensions or variations of the invention as will be apparent
to those skilled in the art are considered to be encompassed
by the invention disclosed herein and in accordance with the
claims appended hereto.
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