CN102495207B - High-sensitivity enzyme-linked immunoassay method - Google Patents

Abstract

An enzyme-linked immunoassay method based on chemical oscillation kinetic detection. According to the specific binding characteristics of the analyte and antibody (antigen), under specific conditions, the corresponding enzyme-labeled antigen or antibody and the corresponding antibody or The antigen completes the process of adsorption and binding, which uses the catalytic speed of the labeled enzyme to the substrate to indirectly represent the concentration of the analyte. This method not only effectively overcomes the problem of insufficient sensitivity of conventional ELISA based on spectrophotometry, but also utilizes the principle of antibody-antigen specific binding to combine high-sensitivity chemical oscillation kinetic detection with specificity and The combination of enzyme-catalyzed kinetic analysis with sensitivity amplification overcomes the problems of low selectivity of chemical oscillation and incapability of qualitative determination, so that chemical oscillation combined with enzyme-linked immunoassay can qualitatively and quantitatively determine small molecules of medicine and pesticides, antibodies, antigens , specific proteins, nucleic acids, disease factors and other substances.

Description

A high-sensitivity enzyme-linked immunosorbent assay method

technical field

The invention relates to a high-sensitivity enzyme-linked immunosorbent assay method for detecting substrate changes based on chemical oscillation dynamics, belonging to the technical field of biochemical analysis methods.

Background technique

Since its founding by Engvall in 1971, enzyme-linked immunoassay (ELISA) has achieved rapid development due to its simplicity, rapidity, and non-radioactivity, and has been widely used in biomedical research and clinical disease diagnosis. Many important disease diagnostic markers use enzyme-linked immunoassays, such as anti-HAV, hepatitis B five items, anti-HCV, anti-HIV, syphilis antibodies, prenatal and postnatal TORCH series, antigens or antibodies of venereal pathogens, hormones, tumor markers, Autoantibodies, cytokines, HBV-DNA, HCV-RNA, genetic disease genes and tumor genes, etc. Enzyme-linked immunoassay provides life-critical experimental data for early detection of diseases, disease diagnosis, treatment plan formulation, curative effect evaluation, and blood testing. ELISA has also been widely used in pesticide residues, drug detection, and low-concentration chemical analysis.

The principle of the classic enzyme-linked immunosorbent assay method is that the analyte specifically adsorbs the active enzyme through the antigen-antibody system; the enzyme catalyzes the hydrolysis or redox reaction of the substrate and causes a change in the absorbance of the substrate; The change value of the absorbance of the substrate is detected at the wavelength. In an ideal double-sandwich method or indirect method quantitative detection, under specific reaction conditions, within a certain concentration range, the change value of absorbance is approximately proportional to the amount of the enzyme cross-linked on the antigen or antibody, that is, the is approximately proportional to the antigen or antibody concentration. For qualitative testing, the cut-off values for diagnostic results can be confirmed by statistical methods. Because the microplate reader is based on spectrophotometry, the minimum detection limit of the substrate is generally higher than 10 ^-5 mol/L by spectrophotometry. The detection limit is also much higher than that of the immunoradiological assay, that is, only high concentrations of antigens or antibodies can be detected by conventional enzyme-linked immunoassays. Due to the limitations of spectrophotometry on substrate detection methods, low-concentration diagnostic markers cannot be detected or accurately detected by classical enzyme-linked immunoassays, which also reduces the effectiveness of conventional enzyme-linked immunoassays in disease diagnosis to a certain extent. Sensitivity and scope of application.

Chemical oscillation refers to the regular periodic changes in the concentration of intermediates in time and space in an open chemical reaction system far from the equilibrium state. Chemical oscillations have attracted people to study this nonlinear chemical phenomenon from both theoretical and applied levels. In terms of theory, Belgian physical chemist Ilya Prigogine created the theory of dissipative structure, which explained the mechanism, conditions and laws of a system transforming from chaos to order. At the application level, a series of organic and inorganic substances have been successfully detected by using the chemical oscillation kinetic analysis method. In chemical oscillation, due to the complexity of chemical reaction and diffusion, certain physical quantities such as intermediate product concentration, electrode potential, etc. will change periodically with time, and the existence of specific trace additives will affect the oscillation reaction and generate related characteristic signals . In 1978, Tikhonova used a classical chemical oscillator in a closed system for Ru ²⁺ detection, and established a chemical oscillation kinetic detection method by analyzing the kinetic curve of the electrode potential changing with time during the oscillation process. Compared with the traditional method, this method has the advantages of simplicity, rapidity and high sensitivity. Thallium, mercury, silver ions, vitamin K ₃ , B ₂ , B ₆ have been measured, and there are also reports for the concentration of CO, Cl ₂ , ClO ₂ and other gases. Due to the instability of the classic chemical oscillation with metal ions as the catalyst in the closed system, the relative error of the measurement is relatively large. In 1995, Rafael proposed to change the closed system to a continuous flow stirred reactor, using analyte pulse perturbation Technology, inject different concentrations of analyte species into the oscillation system far away from the equilibrium state in a pulsed manner, observe the change of the characteristic signal of the oscillation reaction system and compare it with the standard characteristic signal, you can find the difference between the concentration of the analyte species and the characteristic signal relationship between variables. At present, this method has been used to determine gallic acid, caffeine, vitamin C in citrus juice, and cylanaldehyde in sugar. Using the analyte pulse perturbation technique, Liang Ju's detection limit for peroxynitrite anion was 2.25×10 ^-9 mol/L, and Guo's detection limit for furfural was 3×10 ^{− 9} mol L ^{− 11} . Although the sensitivity of chemical oscillation detection is very high, the chemical oscillation detection method is very sensitive to various admixtures and has poor selectivity, so the chemical oscillation detection method is mostly used for quantitative detection rather than qualitative detection.

Contents of the invention

The object of the present invention is, in order to overcome the insufficient sensitivity of conventional ELISA based on spectrophotometry, replace spectrophotometry with a new highly sensitive method for detecting substrate changes, which can greatly improve the analytical sensitivity; Due to the low selectivity of the simple chemical oscillation method, qualitative detection cannot be performed. If the high-sensitivity chemical oscillation kinetic detection and the enzyme-catalyzed kinetic analysis with specificity and sensitivity amplification are combined, the chemical oscillation selectivity will be overcome. Low, can not be qualitatively measured.

The technical solution of the present invention is that the present invention introduces high-frequency chemical oscillation kinetic detection into the ELISA detection system, skillfully combines high-sensitivity chemical oscillation detection and highly specific ELISA diagnostic technology, and uses chemical oscillation method Instead of spectrophotometry, detect changes in the concentration of substrates and their products in enzyme-linked immunoassays, thereby establishing an enzyme-linked immunological diagnostic technology based on chemical oscillation kinetic detection.

According to the characteristics of the specific binding between the test object and the antibody (antigen), the present invention uses the corresponding enzyme to label the antigen or antibody and the corresponding antibody or antigen to complete the adsorption and binding process at a certain temperature and time. The concentration of the analyte can be indirectly expressed by the catalytic speed of the substance, and the detection method of the concentration of the substrate or the enzymatic reaction product of the substrate is a chemical oscillation kinetic detection method. The qualitative determination principle of the enzyme-linked immunoassay technology based on chemical oscillation kinetic detection is that only when the analyte exists, the analyte will specifically bind the ELISA to the corresponding antibody (antigen), and the specific antibody or antigen will be adsorbed. The directly or indirectly linked enzyme will be adsorbed and catalyze the substrate reaction at a certain temperature and time. When the changed substrate is added to the chemical reaction oscillation system, the change of the substrate will cause the chemical oscillation characteristic value such as period , amplitude, intermediate product concentration, induction period and other changes. Compared with the blank, the change of the chemical oscillation characteristic value will reflect the presence or absence of the analyte. The principle of quantitative determination of enzyme-linked immunoassay technology based on chemical oscillation kinetic detection is that changes in the concentration of the analyte will cause a corresponding change in the binding amount of specific antibodies (antigens) that directly or indirectly bind to the analyte, which directly or indirectly The amount of the indirect link enzyme will change accordingly, and the catalytic reaction speed of the substrate will have a corresponding change. When the substrate is added to the chemical reaction oscillation system, the chemical oscillation characteristic values such as period, amplitude, intermediate product concentration, induction Changes in the period will reflect changes in substrate and product concentrations.

The present invention also utilizes the principle of antibody-antigen specific combination, and combines high-sensitivity chemical oscillation kinetic detection with enzyme-catalyzed kinetic analysis with specificity and sensitivity amplification, so that chemical oscillation detection can only detect the analyte , to avoid the interference of other impurities, to overcome the low selectivity of chemical oscillation, the problem of qualitative determination cannot be carried out, so that chemical oscillation can be used for qualitative and quantitative determination. Effective selection of ELISA enzymes, substrates, and chemical oscillation reaction systems can greatly improve the sensitivity of ELISA detection.

The ELISA method of the present invention may be an indirect method, a double-antibody sandwich method, a two-site one-step method, a competition method, a capture method, or a biotin-avidin method.

The enzyme detection system in the enzyme-linked immunosorbent adsorption method of the present invention can be: A, hydrogen peroxide/horseradish peroxidase system, the corresponding hydrogen donors are o-phenylenediamine, tetramethylbenzidine, aminosalicyl Acid, o-benzidine, 2,2-amino-2 (3-ethyl-thiazolinesulfonic acid-6) ammonium salt; B, alkaline phosphatase system, the substrate is phenol phosphate, Vitamin C phosphate, 4-nitrophenol phosphate (PNP) or naphthol-AS-Mx phosphate + diazonium salt; C, β-D-galactosidase system, the substrate is 4MuG or ONPG; D , Malate dehydrogenase system, the substrate is malic acid, E glucose oxidase system.

The enzyme detection system in the enzyme-linked immunosorbent adsorption method of the present invention can also be a multi-enzyme cascade amplification system instead of a single horseradish peroxidase or alkaline phosphatase system, so as to improve the catalysis of the enzyme to the final substrate. Response capabilities, such as biotin (structurally modified biotin) and avidin (structurally modified avidin) systems, AP, alcohol dehydrogenase, and diaphorase constitute an enzyme cascade substrate amplification system, and enzyme inhibition cascade amplification System, coagulation factor enzyme cascade amplification system.

The chemical oscillation involved in the present invention is B-Z oscillation reaction, B-L oscillation system, B-R oscillation reaction, copper catalyzed oscillation reaction, chlorine dioxide chemical oscillation reaction, peroxidase-oxidase biochemical oscillation system, permanganate oscillation reaction, liquid Membrane oscillation reaction system.

In the copper-catalyzed B-Z oscillation system, B-L oscillation system, and B-R oscillation reaction, the substrate can be a polyoxygen organic compound with an active methylene group, such as malonic acid, tartaric acid, and gallic acid, with a redox electrode potential below 1.0 V. , Pyrogallic acid, lactic acid, acetone, maleic acid, citric acid, malic acid, acetylacetone, ethyl acetoacetate, etc.

The catalyst in the chemical oscillation reaction of the present invention can be a transition metal ion or a transition metal ion complex with M ^(n+l)+ /M ^{n +} electrode potential between 1.00-1.51V, such as Ce ³⁺ /Ce ⁴⁺ (1.44V), Mn ²⁺ /Mn ³⁺ (1.44V), Ru(bpy) ₂ ²⁺ / Ru(bpy) ₃ ³⁺ , Fe(phen) ₂ ²⁺ /Fe(phen) ₃ ³⁺ and Cu ²⁺ , Ni ²⁺ and tetrazamacrocyclic complexes.

The measurement system of chemical oscillation can be a closed system; it can be an open system with continuous flow stirring, which uses the analyte pulse perturbation technology to inject different concentrations of analyte species into the oscillation system far away from the equilibrium state in a pulsed manner; it can also be Use the subcritical state system before the bifurcation for determination.

The measurement index of the chemical oscillation characteristic can be the change of the induction period, the change of the potential before and after the addition of the substrate, or the change of the oscillation period before and after the addition of the substrate.

Figure 1 is a schematic diagram of the principle of the enzyme-linked immunosorbent assay method based on chemical oscillation detection of substrates. It takes the most classic antibody-antigen-antibody sandwich method as an example to illustrate the principle of the detection method of the present invention. As shown in the figure, the specific antibody is immobilized on the stationary phase, which is the stationary phase antibody 1. After the completion, the excess antibody is washed away; the specimen to be tested is added to the specimen 2. If the specimen contains the antigen to be tested, it will be Combine specifically with the antibody on the stationary phase; wash away the non-binding part of the sample to be tested, add secondary antibody 3 labeled with enzyme 5, and bind to the antigen; wash away excess unbound secondary antibody, add For the catalyzed substrate 6, within a certain period of time, the enzyme 4 labeled on the secondary antibody will catalyze the substrate to form a catalyzed product 7. Inject the substrate catalyzed by the enzyme into a chemical oscillating reaction system, measure and compare the changes in the period, amplitude or induction period of the oscillating system, compare with the standard period, amplitude or induction period, calculate and verify the antigen concentration. At a certain temperature and time, the corresponding enzyme-labeled antigen or antibody is used to complete the adsorption and binding process with the corresponding antibody or antigen. It uses the catalytic speed of the labeled enzyme to the substrate to indirectly indicate the concentration of the diagnostic marker, and the substrate or The detection method of the concentration of the enzymatic reaction product is a chemical oscillation kinetic detection method.

The beneficial effect of the present invention compared with the prior art is that the present invention introduces high-frequency chemical oscillation dynamic detection into the enzyme-linked immunosorbent detection system, and skillfully combines high-sensitivity chemical oscillation detection and highly specific enzyme-linked immunological diagnostic technology , using the chemical oscillation method instead of the spectrophotometric method to detect the changes in the concentration of the substrate and its products in the enzyme-linked immunoassay, thereby establishing an enzyme-linked immunological diagnostic technology based on the detection of chemical oscillation kinetics. This method not only effectively overcomes the problem of insufficient sensitivity of the conventional enzyme-linked immunosorbent assay based on spectrophotometry, but also can greatly improve the analytical sensitivity by replacing the spectrophotometric method with a new highly sensitive method for detecting substrate changes; On the other hand, the present invention utilizes the principle of antibody-antigen specific binding, and combines high-sensitivity chemical oscillation kinetic detection with enzyme-catalyzed kinetic analysis with specificity and sensitivity amplification, so that chemical oscillation detection only Detecting the analyte avoids the interference of other impurities, overcomes the problem of low selectivity of chemical oscillation and cannot perform qualitative determination, and enables qualitative and quantitative determination of chemical oscillation. Effective selection of ELISA enzymes, substrates, and chemical oscillation reaction systems can greatly improve the sensitivity of ELISA detection.

The invention is suitable for qualitative and quantitative determination of small molecules of medicine and pesticides, antibodies, antigens, specific proteins, nucleic acids, disease factors and other substances.

Description of drawings

1 is a schematic diagram of the principle of an enzyme-linked immunosorbent assay method based on chemical oscillation detection of a substrate;

The numbers in the figure indicate: 1 is the stationary phase antibody; 2 is the analyte; 3 is the enzyme-linked antibody; 4 is the enzyme linked to the antibody; 5 is the enzyme; 6 is the substrate; 7 is the product catalyzed by the enzyme.

Detailed ways

Example 1

This embodiment uses an indirect method to measure hepatitis C virus (HCV) antibody: the antigen of the pathogen is coated to form a solid-phase antigen, and after treatment, clinical samples that may contain hepatitis C virus (HCV) antibody to be tested, such as serum samples, are added. , incubate, wash the plate, add horseradish peroxidase-labeled anti-human IgG antibody, incubate, wash the plate; add enzyme substrate hydrogen peroxide, incubate, use BZ (Belousov-Zhabotinsky) chemical oscillation kinetics to detect enzymatic activity The difference between the product and the original substrate. In the BZ system, the copper (Ⅱ) complex of fourteen-membered tetraazamacrocycle (Curtis ring) is used as the catalyst, and NaBrO ₄ -malic acid-[CuL](ClO ₄ ) ₂ -H ₂ SO ₄ is used as the chemical oscillation system , form a stable oscillation system at 22 degrees Celsius, add catalytic products to this system, and indirectly confirm the presence or absence of antibodies according to the periodic changes caused by chemical oscillations.

Example 2

In this example, the double-antibody sandwich method is used to determine hepatitis B virus e antigen (HBeAg): the HBeAg antibody is adsorbed on the solid phase surface; the antigen sample to be tested is added, incubated, and the plate is washed to form an antigen-antibody complex, and alkaline phosphoric acid is added Esterase ALP enzyme-labeled anti-human Ig antibody forms an antibody-antigen-antibody sandwich structure, adds vitamin C phosphate, a substrate that can be catalyzed by enzyme hydrolysis, incubates at 37 degrees for 30 minutes, and adds it to the flowing chemical oscillation system , the substrate vitamin C phosphate itself will not cause changes in the period, amplitude or induction period of the oscillation system, but the substrate vitamin C phosphate can cause periodic changes in the oscillation system to calculate the concentration of antigen or confirm the presence or absence of antigen.

Example 3

In this example, a competitive method is used to determine the small molecule hapten abamectin: the abamectin antibody is adsorbed on the surface of a solid phase carrier, and the enzyme-labeled antigen labeled with horseradish peroxidase and the abamectin sample to be tested are added , to compete for binding antibodies; for the control, only enzyme-labeled antigen was added, and substrate hydrogen peroxide was added. After incubating for 30 minutes, the product was added to the chemical shaking system [Ni(LA3)] ²⁺ , malonic acid, H ₃ P0 ₄ , and the amplitude changes of the two shaking systems were measured, and compared with the standard, the amplitude of adding only the antigen The change values were compared to calculate the concentration of the antigen abamectin. The minimum detection limit of this method can reach 2.1*10 ^-8 mol/L.

Example 4

In this example, streptavidin (streptavidin, SA) biotin method was used to measure human anti-tumor factor, the anti-tumor factor antibody was adsorbed on the surface of the solid phase carrier, the sample to be tested was added, incubated, washed, and labeled with biotin Anti-human Ig, incubated, washed, added streptavidin labeled with alkaline phosphatase (ALP), incubated, washed, added phosphate phenol ester as the substrate of the catalytic reaction, after catalytic hydrolysis, The _hydrolysis of _the substrate phenolic phosphate produces phenol _{that can} change the BZ chemical oscillation system _. In the Stirred Reactor (CSTR), using the analyte pulse perturbation technique (APP), the hydrolyzate of the enzyme-linked immunoreaction is injected into the oscillating system far away from the equilibrium state in a pulsed manner, and the periodic change of the chemical oscillation caused by the hydrolyzate can be used as The basis for the qualitative and quantitative analysis of human anti-tumor factors, the quantitative relationship between the change of cycle and the concentration of human anti-tumor factors can be described as delta T= A*log C +B, delta T is the change of chemical oscillation cycle, C is the human anti-tumor factor Concentration of immune factors.

Claims (5)

1. high-sensitivity enzyme-linked immunoassay method, it is characterized in that, described method combines chemical oscillation detection and enzyme-linked immunologic diagnosis technology, replace spectrophotometric method with the chemical oscillation method, the variation of substrate and production concentration thereof in the detection enzyme-linked immuno assay, thus foundation is based on the enzyme-linked immunologic diagnosis technology of chemical oscillation kinetic measurement; The characteristic that described method is combined with antibody or antigentic specificity according to measured object, in uniform temperature with in the time, finish the absorption cohesive process with corresponding enzyme-labelled antigen or antibody with corresponding antibody or antigen, the enzyme that it utilizes mark comes the concentration of secondary indication measured object to the catalysis speed of substrate, and the detection means of the enzymatic reaction product concentration of substrate or substrate is chemical oscillation kinetic measurement method;

Described Enzyme-linked Immunosorbent Assay determining adsorption analytical approach adopts indirect method, double antibody sandwich method, dibit point single stage method, competition law, prize law, avidin-biotin complex technology;

The detection system of enzyme is in the described Enzyme-linked Immunosorbent Assay absorption method: A, and hydrogen peroxide/horseradish peroxidase system, corresponding hydrogen donor are o-phenylenediamine, tetramethyl is for biphenylamine, aminosalicylic acid, adjacent biphenyl methylamine, 2,2-connects amido-2 (3-ethyl-and thiazoline sulfonic acid-6) ammonium salt; B, the alkaline phosphate ester enzyme system, substrate is phenol phosphate, vitamin C phosphoric ester, 4-nitrophenols phosphate (PNP) or naphthols-AS-Mx phosphate+diazo salt; C, the beta-D-galactosidase system, substrate is 4MuG or ONPG; D, the malate dehydrogenase enzyme system, substrate is malic acid; E, the glucose oxidase enzyme system;

The detection system of enzyme adopts multienzyme cascade amplification system to replace single horseradish peroxidase or alkaline phosphoric acid enzyme system in the described Enzyme-linked Immunosorbent Assay absorption method, to improve enzyme to the catalytic reaction ability of final substrate, described multienzyme cascade amplification system comprises biotin and Avidin system, AP and alcohol dehydrogenase, diaphorase constitutive enzyme cascade substrate amplification system, enzyme suppresses cascade amplification system, coagulation factors enzyme cascade amplification system;

The detection system of described chemical oscillation kinetic measurement is the B-Z oscillating reactions, B-L oscillation system, B-R oscillating reactions, copper catalysis oscillating reactions, chlorine dioxide chemical oscillating reaction, the biochemical oscillation system of Guo Yangization Mei – oxidase, the permanganate oscillating reactions, the liquid membrane oscillation reaction system;

2. a kind of high-sensitivity enzyme-linked immunoassay method according to claim 1, in the detection system of described chemical oscillation kinetic measurement, in the B-Z of copper catalysis oscillation system, B-L oscillation system, B-R oscillating reactions, substrate be the oxidation-reduction electrode electromotive force below 1.0 V, have the polyoxy organic compound of active methylene group: malonic acid, tartrate, gallic acid, pyrogallic acid, lactic acid, acetone, maleic acid, citric acid, malic acid, diacetone, ethyl acetoacetate.

3. a kind of high-sensitivity enzyme-linked immunoassay method according to claim 1, the catalyzer in the described chemical oscillating reaction is M ^(n+l)+/ M ^N+Transition metal ion or transition metal ion complex: the Ce of electrode potential between 1.00-1.51V ³⁺/ Ce ⁴⁺(1.44V), Mn ²⁺/ Mn ³⁺(1.44V), Ru (bpy) ₂ ²⁺/ Ru (bpy) ₃ ³⁺, Fe (phen) ₂ ²⁺/ Fe (phen) ₃ ³⁺And Cu ²⁺, Ni ²⁺With the macrocyclic tetraaza complex.

4. a kind of high-sensitivity enzyme-linked immunoassay method according to claim 1, the mensuration system of described chemical oscillation is closed system; Be the open system that continuous flow is stirred, it utilizes analyte pulsed perturbation technology, injects the analyzed species of variable concentrations in the oscillation system of far from equilibrium attitude in the mode of pulse; Residing subcritical state system is measured before utilizing fork.

5. a kind of high-sensitivity enzyme-linked immunoassay method according to claim 1, the change of the change that described chemical oscillation testing index is induction period, adding substrate front and back current potential or the change of the oscillation period of adding substrate front and back.

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