CA1094477A - Assays utilizing localized electromagnetic radiation sources - Google Patents
Assays utilizing localized electromagnetic radiation sourcesInfo
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- CA1094477A CA1094477A CA277,702A CA277702A CA1094477A CA 1094477 A CA1094477 A CA 1094477A CA 277702 A CA277702 A CA 277702A CA 1094477 A CA1094477 A CA 1094477A
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- enzyme
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
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- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
ASSAYS UTILIZING LOCALIZED
ELECTROMAGNETIC RADIATION SOURCES
Abstract of the Disclosure Chemical species including biological materials such as enzymes, substrates, antibodies, antigens and the like, are determined in very small quantities (down to picomole ranges) through the use of electromagnetic radiation generating reactions such as those catalyzed by light emitting enzyme systems of the bacterial luciferase and fire-fly luciferase types. The sensitivity of the detection systems is increased by concentrating and localizing at least one component of the electromagnetic radiation generating reaction onto a solid support material. The component may thus be immobilized and solubilized. The localized, immobilized component is generally contacted with suitable reactants in a liquid environment in order to produce the electromagnetic signals in a localized area of the reaction environment, i.e., on the support material. The signals are thereby intensified and detected on suitable instrumentation. The electromagnetic radiation generating systems may also be coupled with other reactions that yield a necessary component of said systems, whereby chemical species associated with the coupled reactions may also be determined.
ELECTROMAGNETIC RADIATION SOURCES
Abstract of the Disclosure Chemical species including biological materials such as enzymes, substrates, antibodies, antigens and the like, are determined in very small quantities (down to picomole ranges) through the use of electromagnetic radiation generating reactions such as those catalyzed by light emitting enzyme systems of the bacterial luciferase and fire-fly luciferase types. The sensitivity of the detection systems is increased by concentrating and localizing at least one component of the electromagnetic radiation generating reaction onto a solid support material. The component may thus be immobilized and solubilized. The localized, immobilized component is generally contacted with suitable reactants in a liquid environment in order to produce the electromagnetic signals in a localized area of the reaction environment, i.e., on the support material. The signals are thereby intensified and detected on suitable instrumentation. The electromagnetic radiation generating systems may also be coupled with other reactions that yield a necessary component of said systems, whereby chemical species associated with the coupled reactions may also be determined.
Description
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
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 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_ ~`
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, 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 `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 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
- 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.
~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 .
.
. : ., 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.
-./ - . ., ......................... ~
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 10~4~!l77 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 ` ` `; ~ 10~77 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 ~0~4477 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.
' "
,.
~ , .
.~ . ' .
:, ~
. .
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 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_ ~`
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, 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 `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 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
- 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.
~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 .
.
. : ., 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.
-./ - . ., ......................... ~
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 10~4~!l77 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 ` ` `; ~ 10~77 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 ~0~4477 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.
' "
,.
~ , .
.~ . ' .
:, ~
. .
Claims (20)
1. 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 and a light generating 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 light generating enzyme and detecting light generated by said light emitting enzyme.
2. The assay method of claim 1 wherein said oxidoreduc-tase and said light generating enzymes are insolubilized by covalent bonding to the solid support.
3. The assay method of claim 1 wherein said oxidoreduc-tase and said light generating enzyme are localized on said support in intimate proximity to one another.
4. The method of claim 1 wherein the light is detected by a photometer.
5. The method of claim 2 wherein the solid support is aryl-amine glass.
6. The method of claim 1 wherein said first product is reduced nicotinamide adenine dinucleotide.
7. The method of claim 1 wherein said second enzyme is FMN oxidoreductase.
8. The method of claim 1 wherein said light generating enzyme is luciferase.
9. The method of claim 1 wherein said first product is reduced nicotinamide adenine dinucleotide, said light generating enzyme is flavin mononucleotide oxidoreductase, and said first product is reacted with flavin mononucleo-tide in the presence of the oxidoreductase to reduce the flavin mononucleotide, the light generating enzyme is luciferase, and the reduced flavin mononucleotide is reacted with a suitable substrate in the presence of the luciferase to oxidize the flavin mononucleotide and produce light in proportion to the quantity of reduced flavin mononucleotide.
10. A product useful for assaying biochemical species and chemical species associated with bioreactions comprising an insoluble support member, an enzyme 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.
11. The product of claim 10, wherein the insoluble support member and the enzyme retaining material are glass.
12. A product useful for coupling bio-reactions and assaying bio-materials, comprising a non-reactive elongated rod having an insoluble enzyme retaining material attached thereto, an oxidoreductase enzyme immobilized on said enzyme retaining material; and luciferase enzyme also immobilized on said enzyme retaining material.
13. The product of claim 12 wherein said insoluble enzyme retaining material is porous glass beads.
14. The product of claim 12, wherein said oxidoreductase is FMN oxidoreductase, and both said enzyme retaining material and said elongated rod are substantially transparent to light detectable by a photometer.
15. An assay method for chemical species that enter into enzymatic reaction with NAD or NADP as a co-factor to produce NADH or NADPH and one product thereof, comprising providing an elongate support member having porous glass beads affixed thereto, immobilizing an FMN oxidoreductase on said glass beads, also immobilizing bacterial lucifer-ase on said glass beads, contacting said elongate support member and the immobilized FMN oxidoreductase and bacterial luciferase with an aqueous solution including the NADH or NADPH, FMN, a long chain aldehyde, and oxygen, to thereby effect a reduction of FMN to FMNH2 and the oxidation of NADH or NADPH to NAD or NADP by the FMN oxidoreductase, and the subsequent reoxidation of the FMNH2 to FMN by the bacterial luciferase to generate light proportional to the amount of FMNH2 reoxidized, detecting and quantifying the generated light to measure the amount of FMNH2 produced by the oxidation of the NADH or NADPH, and calculating therefrom the amount of chemical species necessary to produce the initial NADH or NADPH.
16. The product of claim 10 wherein the enzyme retaining material is an agarose.
17. The product of claim 12, wherein the enzyme retaining material is an agarose.
18. The product of claim 14 wherein said enzyme retaining material has a high surface concentration of binding sites at which said enzymes are immobilized, and said immo-bilized enzymes are in intimate proximity to one another on said enzyme retaining material.
19. An assay method for chemical species that enter into enzymatic reaction with NAD or NADP as a co-factor to produce NADH or NADPH and one product thereof comprising:
providing a non-reactive elongated rod having an enzyme retaining material attached thereto, said rod and enzyme retaining material both being substantially trans-parent to light detectable by a photometer, said enzyme retaining material having immobilized thereon an FMN
oxidoreductase enzyme and a luciferase enzyme;
immersing said elongated rod in an aqueous solution including said NAD or NADP, to thereby contact said immobilized enzymes with said solution;
enclosing said aqueous solution and said immersed rod within a photometric sample chamber; and detecting a generated light flash by a photometer, said light flash generated by adding said chemical species to said aqueous solution either during said immersing step or during said enclosing step.
providing a non-reactive elongated rod having an enzyme retaining material attached thereto, said rod and enzyme retaining material both being substantially trans-parent to light detectable by a photometer, said enzyme retaining material having immobilized thereon an FMN
oxidoreductase enzyme and a luciferase enzyme;
immersing said elongated rod in an aqueous solution including said NAD or NADP, to thereby contact said immobilized enzymes with said solution;
enclosing said aqueous solution and said immersed rod within a photometric sample chamber; and detecting a generated light flash by a photometer, said light flash generated by adding said chemical species to said aqueous solution either during said immersing step or during said enclosing step.
20. The assay method as in claim 19 further comprising:
removing said elongated rod from immersion in said aqueous solution; and, rinsing said elongated rod with a solution sufficient to prepare said elongated rod for reuse, and simultaneously retaining said immobilized enzymes on said enzyme retaining material of said elongated rod.
removing said elongated rod from immersion in said aqueous solution; and, rinsing said elongated rod with a solution sufficient to prepare said elongated rod for reuse, and simultaneously retaining said immobilized enzymes on said enzyme retaining material of said elongated rod.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US75043676A | 1976-12-14 | 1976-12-14 | |
| US750,436 | 1976-12-14 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1094477A true CA1094477A (en) | 1981-01-27 |
Family
ID=25017870
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA277,702A Expired CA1094477A (en) | 1976-12-14 | 1977-05-04 | Assays utilizing localized electromagnetic radiation sources |
Country Status (5)
| Country | Link |
|---|---|
| JP (1) | JPS5374492A (en) |
| CA (1) | CA1094477A (en) |
| DE (1) | DE2722391A1 (en) |
| FR (1) | FR2374643A1 (en) |
| GB (1) | GB1571466A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150218613A1 (en) * | 2013-10-29 | 2015-08-06 | GeneWeave Biosciences, Inc. | Reagent cartridge and methods for detection of cells |
| US9546391B2 (en) | 2013-03-13 | 2017-01-17 | GeneWeave Biosciences, Inc. | Systems and methods for detection of cells using engineered transduction particles |
| US10351893B2 (en) | 2015-10-05 | 2019-07-16 | GeneWeave Biosciences, Inc. | Reagent cartridge for detection of cells |
| US11077444B2 (en) | 2017-05-23 | 2021-08-03 | Roche Molecular Systems, Inc. | Packaging for a molecular diagnostic cartridge |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1103050A (en) * | 1977-09-28 | 1981-06-16 | Anthony A. Bulich | Method for detecting toxic substances in liquid |
| DE2833723A1 (en) * | 1978-08-01 | 1980-02-21 | Boehringer Mannheim Gmbh | METHOD AND REAGENT FOR DETERMINING AN OXIDIZED PYRIDINE COENZYME |
| US4231754A (en) * | 1979-05-23 | 1980-11-04 | Miles Laboratories, Inc. | Chemiluminescent analytical device |
| CS238261B1 (en) * | 1983-07-28 | 1985-11-13 | Jiri Cerkasov | Enzymatic reactions execution and tracing method and device for application of this method |
| FR2562252B1 (en) * | 1984-03-27 | 1988-01-22 | Inst Nat Sante Rech Med | IMMUNOENZYMATIC ASSAY PROCESS WITHOUT SEPARATION STEP AND REAGENTS AND NECESSARY FOR ITS IMPLEMENTATION |
| DE3737649A1 (en) * | 1987-11-06 | 1989-05-24 | Inst Zellforschung Und Biolumi | Method for determining the luminescence of cell cultures, and device for carrying out the method |
| WO1995023967A1 (en) * | 1994-03-02 | 1995-09-08 | Biolytik Gesellschaft Für Bio-Sensitive Analytik Mbh | Method of rapidly detecting herbicidal active substances in water |
-
1977
- 1977-04-25 GB GB1701277A patent/GB1571466A/en not_active Expired
- 1977-05-04 CA CA277,702A patent/CA1094477A/en not_active Expired
- 1977-05-13 FR FR7714832A patent/FR2374643A1/en active Granted
- 1977-05-14 DE DE19772722391 patent/DE2722391A1/en not_active Ceased
- 1977-05-14 JP JP5487577A patent/JPS5374492A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9546391B2 (en) | 2013-03-13 | 2017-01-17 | GeneWeave Biosciences, Inc. | Systems and methods for detection of cells using engineered transduction particles |
| US10240212B2 (en) | 2013-03-13 | 2019-03-26 | GeneWeave Biosciences, Inc. | Systems and methods for detection of cells using engineered transduction particles |
| US20150218613A1 (en) * | 2013-10-29 | 2015-08-06 | GeneWeave Biosciences, Inc. | Reagent cartridge and methods for detection of cells |
| US9540675B2 (en) * | 2013-10-29 | 2017-01-10 | GeneWeave Biosciences, Inc. | Reagent cartridge and methods for detection of cells |
| US10125386B2 (en) | 2013-10-29 | 2018-11-13 | GeneWeave Biosciences, Inc. | Reagent cartridge and methods for detection of cells |
| WO2015164746A1 (en) * | 2014-04-24 | 2015-10-29 | GeneWeave Biosciences, Inc. | Reagent cartridge and methods for detection of cells |
| US10351893B2 (en) | 2015-10-05 | 2019-07-16 | GeneWeave Biosciences, Inc. | Reagent cartridge for detection of cells |
| US11077444B2 (en) | 2017-05-23 | 2021-08-03 | Roche Molecular Systems, Inc. | Packaging for a molecular diagnostic cartridge |
Also Published As
| Publication number | Publication date |
|---|---|
| FR2374643B1 (en) | 1984-07-06 |
| FR2374643A1 (en) | 1978-07-13 |
| GB1571466A (en) | 1980-07-16 |
| DE2722391A1 (en) | 1978-06-15 |
| JPS5374492A (en) | 1978-07-01 |
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