WO2001025788A1 - Non-separation heterogenous assay for biological substances - Google Patents

Non-separation heterogenous assay for biological substances Download PDF

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Publication number
WO2001025788A1
WO2001025788A1 PCT/CA2000/001153 CA0001153W WO0125788A1 WO 2001025788 A1 WO2001025788 A1 WO 2001025788A1 CA 0001153 W CA0001153 W CA 0001153W WO 0125788 A1 WO0125788 A1 WO 0125788A1
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reactant
label
enzyme
substrate
vessel
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PCT/CA2000/001153
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French (fr)
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Zhibo Gan
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Zhibo Gan
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Priority to AU76363/00A priority Critical patent/AU7636300A/en
Publication of WO2001025788A1 publication Critical patent/WO2001025788A1/en

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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
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

  • the invention relates to the field of binding assays.
  • Binding assays comprise a variety of methods that utilize the specific reaction between a ligand and corresponding binding agent such as protein. Immunoassay, receptor binding assays and lectin binding assay are examples of the binding assays. Immunoassay is by far the most common form which has been developed into an extremely versatile analytic technique with a diverse range of assay protocols.
  • Both RIA and ELISA involve the coating and separation of labeled and unlabeled antigen or antibody after the binding. These procedures inevitably use heterogenous phase and require separation of antigen and/or antibody unbound to the surface, this type of immunoassay, therefore, is called “heterogenous” immunoassay.
  • Rubenstein et al (1972) developed a new immunoassay using an enzyme as a label in which the antigen-antibody reaction and its measurement are performed in solution without the need of prior separation of the free and antibody-bound components and without the use of solid phase. This type of separation- free immunoassay is called "homogenous" immunoassay.
  • Immunoassays have been widely used in human clinical tests and therapeutics, agriculture, food, veterinary and environmental diagnostics. In most cases, immunoassays are effective and valid (Cleaveland, J. S. et al, 1990), but in some cases they are not suitable, for example, in the determination of enzyme activity. This occurs because the binding assays for antibody and antigen (enzyme) can only be used to measure the concentration of an antigen (enzyme) but not its activity. It is important to know the catalytic activity of an enzyme and not just the amount of the enzyme as a given amount of the enzyme may have a widely varying activity depending on reaction conditions.
  • This present invention is for a method referred to as non-separation heterogenous assay that greatly simplifies the detection, identification, measurement of concentration and activity of biological substances. It is based on the change of the label signal due to the distribution of the label between a solid surface and liquid in a vessel after completion of the reaction among reactants.
  • the method involves the coating of a reactant (labeled or unlabeled) onto a surface, addition of a sample with or without a competitor labeled using a label tag or unlabeled. The change of the label signal can be directly measured.
  • the present invention relates to a new method for the detection, identification, measurement of concentration and activity of biological substances, which is based on the change of the label signal due to the uneven distribution of the label between surface and liquid in a vessel after completion of the reaction among reactants.
  • An assay in which the surface of a vessel is coated with reactant 1 which is a binding ligand for reactant 3 being the labeled form of reactant 2.
  • Competitive binding reactions between reactant 2 and reactant 3 for reactant 1 or between reactant 2 and reactant 1 for reactant 3 are initiated when an unknown amount of reactant 2 and a known amount of reactant 3 are added.
  • the change of the label signal of the reactant 3 in the reaction vessel can be directly measured without an additional step and is directly proportional to the amount of reactant 2.
  • reactant 4 which is an inhibitor of reactant 2.
  • the change of the label signal of the reactant 3 in the reaction vessel is directly proportional to the amount of reactant 4 (inhibitor). 2.
  • reactant 3 which is an inhibitor of reactant 2.
  • the change of the label signal is reciprocally proportional to the amount of reactant 3 (inhibitor).
  • Example 1 The detection of DNA hybridization Materials: Single stranded deoxyribonucleic acid (ssDNA), fluorescence labeled complementary ssDNA (fluo-cDNA), sample double stranded DNA (dsDNA) and a microplate.
  • ssDNA Single stranded deoxyribonucleic acid
  • fluo-cDNA fluorescence labeled complementary ssDNA
  • dsDNA sample double stranded DNA
  • ssDNA is added into the wells of a microplate containing spacer and incubated for the covalent end-linkage of DNA.
  • DNA assay (1). A series of concentrations of dsDNA in a buffer (50 ul/well) and sample are added to the wells of the microplate immobilized with ssDNA.
  • the change of the fluorescent intensity of the fluo-cDNA in the wells of the microplate is determined using a fluorometer and is directly proportional to the amount of dsDNA.
  • Example 2 Competitive assay for an antigen Materials: An antigen, fluorescence labeled antibody, sample containing the antigen and a microplate. Method:
  • the change of the fluorescent intensity of the fluo-antibody in the wells of the microplate is determined using a fluorometer and is directly proportional to the amount of the antigen in the sample.
  • protease assay A series of concentrations of a protease in a buffer (100 ul/well) and sample are added to the wells of the coated microplate and incubated at room temperature for 30 min. The change of the fluorescent intensity of the label in the wells of the microplate is measured with a fluorometer and is directly proportional to the activity of the protease. 3. Protease inhibitor assay:
  • a fixed concentration of the protease in the buffer (50 ul/well) is added to the wells containing inhibitor and the controls and incubated at room temperature for 1 h.
  • the change of the fluorescent intensity of the label in the wells of the microplate is measured with a fluorometer and is reciprocally proportional to the amount of the inhibitor.

Abstract

This present invention is for a method referrred to as non-separation heterogenous assay that greatly simplifies the detection, identification, measurement of concentration and activity of biological substances. It is based on the change of the label signal due to the distribution of the label between a solid surface and liquid in a vessel after completion of the reaction among reactants. The method involves the coating of a reactant (labeled or unlabeled) onto a surface, addition of a sample with or without a competitor labeled using a label tag or unlabeled. The change of the label signal can be directly measured.

Description

Non-separation heterogenous assay for biological substances
TECHNICAL FIELD
The invention relates to the field of binding assays.
BACKGROUND ART
Binding assays comprise a variety of methods that utilize the specific reaction between a ligand and corresponding binding agent such as protein. Immunoassay, receptor binding assays and lectin binding assay are examples of the binding assays. Immunoassay is by far the most common form which has been developed into an extremely versatile analytic technique with a diverse range of assay protocols.
The first quantitative precipitation test and purified antibody for the first time was developed by Heidelberger (1939). The practicability of this principle received wide attention and led to its exploitation in simple immunoassays that could measure single antigen systems. The diffusion of antibodies and antigen in agar gel (Oudin, 1946), immuno-electrophoresis (Grabar and Williams, 1953), antiglobulin test (Coombs, 1945), fluorescent labeled antibodies (Coons, 1941) etc has been used for the assay. A milestone in sensitive assays was radioisotopic labeling techniques for antibodies and antigens (Farr, 1958). The efforts formed the basis of the radioimmunoassay (RIA) which has been rapidly adapted by researchers and clinical laboratories. Nakane and Pierce (1966) demonstrated that enzyme could be coupled to antibody or antigen. The importance of this discovery is reflected in the now widespread application of chromogenic, fluorogenic, luminescent signals for the measurements with a similar sensitivity to that of RIA. These discoveries led to the development of enzyme linked immunosorbent assay (ELISA) by Engvall and Perlmann (1971) which is widely used for researches and clinical tests at present.
Both RIA and ELISA involve the coating and separation of labeled and unlabeled antigen or antibody after the binding. These procedures inevitably use heterogenous phase and require separation of antigen and/or antibody unbound to the surface, this type of immunoassay, therefore, is called "heterogenous" immunoassay. Rubenstein et al (1972) developed a new immunoassay using an enzyme as a label in which the antigen-antibody reaction and its measurement are performed in solution without the need of prior separation of the free and antibody-bound components and without the use of solid phase. This type of separation- free immunoassay is called "homogenous" immunoassay.
Most enzyme assays are carried out for the purpose of estimating the amount or activity of an enzyme present in a cell, tissue, other preparation, or as an essential part of an investigation involving the purification of an enzyme. The current assay methods have been developed based on the physical, chemical and immunological properties where they can be detected using photometric, radiometric, high performance liquid chromatographic, electrochemical assays, etc. (Eisenthal, R. and Danson, M. J., 1993). Although the methods basically fulfill the many essential requirements for routine analysis, there are, among those, the varying disadvantages of low sensitivity (Brenda Oppert et al, 1997), multiple steps (Twining, S. S., 1994) and steps that are tedious and time-consuming (Fields, R., 1976). Immunoassays have been widely used in human clinical tests and therapeutics, agriculture, food, veterinary and environmental diagnostics. In most cases, immunoassays are effective and valid (Cleaveland, J. S. et al, 1990), but in some cases they are not suitable, for example, in the determination of enzyme activity. This occurs because the binding assays for antibody and antigen (enzyme) can only be used to measure the concentration of an antigen (enzyme) but not its activity. It is important to know the catalytic activity of an enzyme and not just the amount of the enzyme as a given amount of the enzyme may have a widely varying activity depending on reaction conditions.
It is important for developing a method to reduce the step of the measurement procedure.
DISCLOSURE OF THE INVENTION
This present invention is for a method referred to as non-separation heterogenous assay that greatly simplifies the detection, identification, measurement of concentration and activity of biological substances. It is based on the change of the label signal due to the distribution of the label between a solid surface and liquid in a vessel after completion of the reaction among reactants. The method involves the coating of a reactant (labeled or unlabeled) onto a surface, addition of a sample with or without a competitor labeled using a label tag or unlabeled. The change of the label signal can be directly measured.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a new method for the detection, identification, measurement of concentration and activity of biological substances, which is based on the change of the label signal due to the uneven distribution of the label between surface and liquid in a vessel after completion of the reaction among reactants. 1. An assay in which the surface of a vessel is coated with reactant 1 which is a binding ligand for reactant 3 being the labeled form of reactant 2. Competitive binding reactions between reactant 2 and reactant 3 for reactant 1 or between reactant 2 and reactant 1 for reactant 3 are initiated when an unknown amount of reactant 2 and a known amount of reactant 3 are added. The change of the label signal of the reactant 3 in the reaction vessel can be directly measured without an additional step and is directly proportional to the amount of reactant 2. The reactions of the competitive binding between reactant 2 and reactant 3 for reactant 1 or between reactant 2 and reactant 1 for reactant 3 is interfered with by reactant 4 which is an inhibitor of reactant 2. The change of the label signal of the reactant 3 in the reaction vessel is directly proportional to the amount of reactant 4 (inhibitor). 2. An assay in which the surface of a vessel is coated with reactant 1 which is a labeled substrate for reactant 2 having a biological activity. The reaction is initiated when the reactant 2 is added. The change of the label signal in the reaction vessel can be directly measured and is proportional to the activity of reactant 2.
The reaction of hydrolyzation of reactant 1 coated on the surface of the vessel by reactant 2 is interfered with by reactant 3 which is an inhibitor of reactant 2. The change of the label signal is reciprocally proportional to the amount of reactant 3 (inhibitor).
BEST MODES FOR CARRYING OUT THE INVENTION
EXAMPLES
The following examples are for an illustrative purpose only, and not to limit the scope of the invention. Example 1 The detection of DNA hybridization Materials: Single stranded deoxyribonucleic acid (ssDNA), fluorescence labeled complementary ssDNA (fluo-cDNA), sample double stranded DNA (dsDNA) and a microplate. Method:
1. Immobilization of ssDNA: ssDNA is added into the wells of a microplate containing spacer and incubated for the covalent end-linkage of DNA.
2. DNA assay: (1). A series of concentrations of dsDNA in a buffer (50 ul/well) and sample are added to the wells of the microplate immobilized with ssDNA.
(2). A fixed amount of the fluo-cDNA (50 ul/well) is added to each well containing dsDNA and the control.
(3). Competitive hybridization between the fluo-cDNA and the dsDNA with the immobilized ssDNA are initiated by incubating at 85° C for a while and cool to room temperature for 1 h.
(4). The change of the fluorescent intensity of the fluo-cDNA in the wells of the microplate is determined using a fluorometer and is directly proportional to the amount of dsDNA.
Example 2 Competitive assay for an antigen Materials: An antigen, fluorescence labeled antibody, sample containing the antigen and a microplate. Method:
1. Coating of antigen: The antigen (100 ul/well) is added into the wells of a microplate and incubated at 37° C for 3 h. Then the microplate is washed 3 times using PBST. 2. Antigen assay:
(1). A series of concentrations of the antigen in a buffer (50 ul/well) are added to the wells of the coated microplate.
(2). A fixed amount of the fluo-antibody (50 ul/well) is added to each well containing the antigen and the control.
(3). Competitive binding reactions between the immobilized antigen and the free antigen (competitor) to the fluo-antibody are initiated by adding the fluo- antibody and incubated at 37° C for 2 h.
(4). The change of the fluorescent intensity of the fluo-antibody in the wells of the microplate is determined using a fluorometer and is directly proportional to the amount of the antigen in the sample.
Example 3 Fluorescent assay for protease and protease inhibitor Materials: Protease, protease inhibitor, fluorescence labeled casein and a microplate. Method:
1. Coating of a fluo-casein: fluo-casein (100 ul/well) in PBS (pH 7.2) is added into the wells of a microplate and incubated at 37° C for 3 h and then the microplate is washed 3 times using PBST. 2. Protease activity assay:
A series of concentrations of a protease in a buffer (100 ul/well) and sample are added to the wells of the coated microplate and incubated at room temperature for 30 min. The change of the fluorescent intensity of the label in the wells of the microplate is measured with a fluorometer and is directly proportional to the activity of the protease. 3. Protease inhibitor assay:
(1). Varying amounts of the protease inhibitor in the buffer (50 ul/well) and sample are added to the wells of the coated microplate. Negative and positive controls are included.
(2). A fixed concentration of the protease in the buffer (50 ul/well) is added to the wells containing inhibitor and the controls and incubated at room temperature for 1 h. The change of the fluorescent intensity of the label in the wells of the microplate is measured with a fluorometer and is reciprocally proportional to the amount of the inhibitor.
REFERENCES CITED
Brenda Oppert, Karl J. Kramer and William H. McGaughey 1997 Rapid microplate assay for substrate and inhibitor of protease mixtures. BioTechniques 23: 70-72.
Cleaveland, J.S. et al 1990 A microtiter-based assay for the detection of protein tyrosine kinase activity. Analytical Biochemistry 190: 249-253.
Coombs, R. R. A., Mourant, A. E. and Race, R. R. 1945 Certain properties of antiserums prepared against human serum and its various protein fractions. Their use in the detection of sensitization of huma res cells with incimolete Rh antibody and on the nature of this antibody. Br. J. Exp. Pathol. 26: 255-263.
Coons, A. H., Creech, H. J. and Jones, R. H. 1941 Immunological properties of an antibody containing a fluorescent group. Proc. Soc. Exp. Biol. Med. 47: 200-202.
Eisenthal, R. and Danson, M. J. 1993 Enzyme Assays: a practical approach. IRL Press at Oxford University, Oxford.
Engall, E. and Perlmann, P. 1971 Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8: 871-874.
Farr, R. S. 1958 A quantitative immunochemical measure of the primary interaction between I* BSA and antibody. J. Infect. Dis. 103: 239-262.
Fields, R. 1972 The rapid determination of amino groups with TNBS. Methods in Enzymology 25B: 464-468. Graber, P. and Williams, C. A. 1953 Method permitting the simultaneous study of electrophoretic and immunochemical properties of a mixture of proteins. Application to blood serum. Biochem. Biophys. Acta 10: 193-194.
Heidelberger, M. 1939 Chemical aspects of the precipitin and agglutinin reactions. Chem. Rev. 24: 323-343.
Nakane, P. K., and Pierce, G. B. 1966 Enzyme-labeled antibodies preparation and application for the localization of antigens. J. Histochem. Cytochem. 14: 929-931.
Oudin, J. 1946 Method of immunochemical analysis by specific precipitation in gelled medium. C. R. Acad. Sci. Paris 222: 1 15-1 16.
Rubenstein. K. E., Schneider, R. S. and Ullman, E. F. 1972 "Homogenous" enzyme immunoassay. A new immunochemical technique. Biochem. Biophys. Res. Commun. 47: 846-851.
Twining, S. S. 1994 Fluorescein isothrocyanate-labeled casein assay for proteolytic enzymes. Analytical Biochemistry 143: 30-34.

Claims

1. A competitive method for measuring the amount of a biological substance utilizing a competitive binding between the biological substances comprising: a. the surface of a vessel coated with reactant 1 b. a known amount of reactant 3 linked with a label and an unknown amount of reactant 2. The competitive reactions existing between reactant 2 and reactant 3 to bind to reactant 1 or between reactant 1 and reactant 2 to bind to reactant 3 c. determining the change of the label signal of reactant 3 in the reaction vessel wherein the intensity of the label signal in the vessel is directly proportional to the amount of reactant 2
2. The method of claim 1 wherein said reactant 1 is a receptor, said reactant 2 is a receptor binding ligand and said reactant 3 is the labeled form of reactant 2.
3. The method of claim 1 wherein said reactant 1 is a receptor binding ligand, reactant 2 is a receptor and said reactant 3 is the labeled form of reactant 2.
4. The method of claim 1 wherein said reactant 1 is a lectin, said reactant 2 is lectin binding ligand and said reactant 3 is the labeled form of reactant 2.
5. The method of claim 1 wherein said reactant 1 is a lectin binding ligand, said reactant 2 is a lectin and said reactant 3 is the labeled form of reactant 2. 6. The method of claim 1 wherein said reactant 1 is an enzyme, said reactant 2 is an inhibitor and said reactant 3 is the labeled form of reactant 2 7 The method of claim 1 wherein said reactant 1 is an inhibitor, said reactant 2 is an enzyme and said reactant 3 is the labeled form of reactant 2. 8. The method of claim 1 wherein said reactant 1 is an antigen, said reactant 2 is an antibody and said reactant 3 is the labeled form of reactant 2
9 The method of claim 1 wherein said reactant 1 is an antibody, reactant 2 is an antigen and said reactant 3 is the labeled form of reactant 2.
10. The method of claim 1 wherein said reactant 1 is a single stranded DNA (ssDNA), said reactant 2 is DNA containing complementary sequence of ssDNA and said reactant 3 is the labeled complementary ssDNA.
1 1. The method of claim 1 wherein said label is selected from the group of: fluorescent label, luminescent label, chromogenic label, and enzyme.
12. A method for detecting the biological activity of a biological substance utilizing the degradation of a substrate comprising: a. the surface of a vessel coated with reactant 1 linked with a label. b. addition of reactant 2 which has biological activity into the reaction vessel, said reactant 1 being hydrolyzed due to the activity of said reactant 2. c. measuring the intensity of the label signal in the reaction vessel, wherein the change of the label signal in the vessel is directly proportional to the biological activity of said reactant 2.
13. The method of claim 12 wherein said reactant 2 is an enzyme and said reactant 1 is a substrate for the enzyme.
14. The method of claim 13 wherein said substrate is a polymeric or an oligomeric substrate.
15. The method of claim 13 wherein said enzyme is an enzyme that is able to cleave the substrate. 16. The method of claim 14 wherein said polymeric substrate is selected from the group of: carbohydrate, DNA, RNA, protein, PEG, or polypeptide.
17. The method of claim 14 wherein said oligomeric substrate is selected from the group of: peptide, oligosaccharide, or oligonucleotide.
18. The method of claim 13 wherein said enzyme is a protease or proteinase and said substrate is a protein.
19. The method of claim 13 wherein said enzyme is a carbohydrate hydrolase and said substrate is a carbohydrate.
20. The method of claim 13 wherein said enzyme is a DNase and said substrate is a DNA.
21. The method of claim 13 wherein said enzyme is a RNase and said substrate is a RNA. 22. The method of claim 13 wherein said enzyme is a peptidase and said substrate is a peptide.
23. The method of claim 13 wherein said enzyme is an oligosaccharide hydrolase and said substrate is an oligosaccharide.
24. The method of claim 12 wherein said label is selected from the group of: fluorescent label, luminescent label, chromogenic label, and enzyme.
25. A method for detecting the amount of an inhibitor to a biological substance comprising: a. the surface of a vessel coated with reactant 1 linked with a label. b. addition of a known amount of reactant 2 which has biological activity, an unknown amount of reactant 3 being an inhibitor of reactant 2 into the reaction vessel. The cleavage of reactant 1 by reactant 2 being inhibited due to the activity of reactant 3. c. measuring the intensity of the label signal in the reaction vessel, wherein the change of the label signal in the vessel is reciprocally proportional to the amount of reactant 3.
PCT/CA2000/001153 1999-10-04 2000-10-03 Non-separation heterogenous assay for biological substances WO2001025788A1 (en)

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CA 2286414 CA2286414A1 (en) 1999-10-04 1999-10-04 Non-separation heterogenous assay for biological substance

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