CN102495207B - High-sensitivity enzyme-linked immunoassay method - Google Patents
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- CN102495207B CN102495207B CN 201110439079 CN201110439079A CN102495207B CN 102495207 B CN102495207 B CN 102495207B CN 201110439079 CN201110439079 CN 201110439079 CN 201110439079 A CN201110439079 A CN 201110439079A CN 102495207 B CN102495207 B CN 102495207B
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Abstract
The invention discloses a high-sensitivity enzyme-linked immunoassay method. The method comprises the following steps of: according to a characteristic of specific binding of a measured object and an antibody (antigen), marking the antigen or the antibody and the corresponding antibody or antigen by a corresponding enzyme under a specific condition so as to achieve an absorption combination process, and indirectly representing the concentration of the measured object by the catalytic speed of the marked enzyme about a substrate. According to the high-sensitivity enzyme-linked immunoassay method provided by the invention, a problem of insufficient sensitivity of a regular enzyme-linked immunoassay based on a spectrophotometry is effectively overcome, and the high-sensitivity chemical oscillation dynamics detection and enzymatic dynamics analysis with specificity and flexibility amplification action are combined by utilizing an antibody-antigen specificity combination principle, so that the problems of low chemical oscillation selectivity and incapability of carrying out qualitative determination are overcome, thus the chemical oscillation in combination with the enzyme-linked immunoassay method can be used for qualitative and quantitative determination of substances such as medicine and pesticide micromolecules, antibody, antigen, specific protein, nucleic acid, disease factors and the like.
Description
Technical field
The present invention relates to a kind of high-sensitivity enzyme-linked immunoassay method that changes based on chemical oscillation kinetic measurement substrate, belong to the Methods Biochem Anal technical field.
Background technology
Enzymoimmunoassay since founding in 1971, has obtained hypergrowth with its characteristics such as easy, quick, "dead" from Engvall, now applies to widely the fields such as biomedical research and clinical disease diagnosis.A lot of important disease diagnosis marker have adopted enzyme-linked immunoassay, as survey anti-HAV, five indexes of hepatitis b, anti-HCV, anti-HIV, antigen or antibody, hormone, tumor markers, autoantibody, cell factor, H BV-DNA, HCV-RNA, hereditary disease gene and the oncogene etc. of syphilis antibody, prenatal and postnatal care TORCH series, venereal disease pathogen.Enzyme-linked immunoassay is disease early detection, medical diagnosis on disease, and therapeutic scheme formulation, therapeutic evaluation and blood testing etc. provide life-critical experimental data.Enzymoimmunoassay is analyzed and also is widely applied at residues of pesticides, drug test, low concentration chemistry product.
Classical enzyme linked immunosorbent assay analysis method principle is that measured object is by Ag-Ab systemic characteristic ground adsorption activity enzyme; Substrate for enzymatic activity is hydrolyzed or redox reaction and cause the variation of substrate absorbance; Utilize the changing value of microplate reader detection substrate absorbance under specific wavelength.In desirable two sandwich methods or the quantitative detection of indirect method, under special reaction condition, in the finite concentration scope, the changing value of absorbance with crosslinked on antigen or antibody the amount of substance of enzyme become to be similar to direct ratio, namely be approximated to direct ratio with detected antigen or antibody concentration.For qualitative detection, can confirm by statistical method the cutoff value of diagnostic result.Basic as detecting take spectrophotometric method because of microplate reader, spectrophotometric method generally is higher than 10 to the lowest detectable limit of substrate
-5Mol/L, even consider the enzymatic reaction number with thousand times amplification, its detectability also far above the detectability of immune radiating method, that is: only has the antigen of high concentration or antibody to be detected by the enzymoimmunoassay of routine.Because of the limitation of spectrophotometric method to the substrate detection method, the diagnosis marker of low concentration is not surveyed or indeterminacy with classical enzymoimmunoassay, and this has also reduced susceptibility and the scope of application of conventional euzymelinked immunosorbent assay (ELISA) in medical diagnosis on disease to a certain extent.
Chemical oscillation refers to that in the open chemical reaction system of far from equilibrium attitude, intermediate concentration presents regular cyclical variation in time and space.Chemical oscillation has attracted people from theoretical and two these nonlinear chemical phenomenons of layer viewpoint of application.Aspect theoretical, Belgian physical chemist Yi Liyapuligaojin has founded the theory of dissipative structures, has explained that a system is from the unordered mechanism to orderly conversion of chaos, condition and regularity.At application, use the chemical oscillation kinetic analysis successfully to carry out the detection of a series of organism and inorganics.In chemical oscillation, because of the complicacy of chemical reaction and diffusion, some physical quantity such as concentration of intermediate products, electrode potential etc. can change in time and periodically, and the existence that specific trace adds thing will affect oscillating reactions and produce relevant characteristic signal.Tikhonova was used for Ru with the classical chemical oscillator in closed system in 1978
2+Detect, by to the time dependent kinetic curve analysis of electrode potential in the oscillatory process, set up chemical oscillation kinetic measurement method.With tradition relatively, the method have easy fast, the advantage such as sensitivity is high.Thallium, mercury, silver ion have been measured, vitamin K
3, B
2, B
6, also be useful on CO, Cl
2, ClO
2Report etc. gas concentration.The chemical oscillation take metallic ion as catalyzer of the classics of cause in closed system is unstable, it is larger to cause measuring relative error, nineteen ninety-five Rafael proposes, closed system is changed in Batchwise reactor, utilize analyte pulsed perturbation technology, inject the analyzed species of variable concentrations in the oscillation system of far from equilibrium attitude in the mode of pulse, observe the oscillating reactions system characteristic signal variation and compare with the standard feature signal, just can find the relation between the variable quantity of analyzed species concentration and characteristic signal.Present this method has been measured the vitamin C in gallic acid, caffeine, the citrus juice, the fragrant blue aldehyde in the sugar etc.Operation analysis thing pulsed perturbation technology beam chrysanthemum reaches 2.25 * 10 to peroxynitrite negative ion detectability
-9Mol/L, Guo reaches 3 * 10 to the detectability of furfural
9Mol L
11Although the chemical oscillation detection sensitivity is very high, to add thing very responsive to multiple for the chemical oscillation detection method, and poor selectivity is so the chemical oscillation detection method is multiplex in quantitative detection but not qualitative detection.
Summary of the invention
The objective of the invention is, for overcoming the problem based on the conventional euzymelinked immunosorbent assay (ELISA) susceptibility deficiency of spectrophotometric method, the method that changes with a kind of new high-sensitive detection substrate replaces spectrophotometric method can significantly improve sensitivity for analysis; Because of simple chemical oscillation method selectivity low, can't carry out qualitative detection, if highly sensitive chemical oscillation kinetic measurement and the Dynamics of Enzyme Catalysis analysis with selectivity and sensitivity amplification are combined, can overcome the problem that the chemical oscillation selectivity is low, can not carry out qualitative determination.
Technical scheme of the present invention is, the present invention introduces the enzyme linked immunosorbent detection system with high frequency chemical oscillation kinetic measurement, the enzyme-linked immunologic diagnosis technology of highly sensitive chemical oscillation detection and high degree of specificity is combined dexterously, 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 present invention is according to the characteristic of measured object and antibody (antigen) specific binding, 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.Qualitative determination principle based on the enzyme-linked immuno assay technology of chemical oscillation kinetic measurement is, when only having the measured object of existence, determinand is with enzyme linked immunological specificity and corresponding antibody (antigen) combination, specific antibodies or antigen will be adsorbed, its enzyme that directly or indirectly connects will be adsorbed, catalytic substrate reaction in certain temperature and time, when the substrate that changes joins in the chemical reaction oscillation system, the variation of substrate will cause the variations such as chemical oscillation eigenwert such as cycle, amplitude, intermediate production concentration, induction period.With the blank contrast, the change of chemical oscillation eigenwert will reflect whether there is determinand.Quantitative measurement principle based on the enzyme-linked immuno assay technology of chemical oscillation kinetic measurement is, the variation of measured object concentration will cause the corresponding change of specific antibody (antigen) binding capacity of directly or indirectly being combined with measured object, its amount that directly or indirectly connects enzyme will change thereupon, catalytic to substrate will produce corresponding the variation, when substrate joins in the chemical reaction oscillation system, chemical oscillation eigenwert such as cycle, amplitude, intermediate production concentration, the change of induction period will reflect the variation of substrate and production concentration.
The present invention has also utilized the principle of antibody-antigentic specificity combination, highly sensitive chemical oscillation kinetic measurement and the Dynamics of Enzyme Catalysis analysis with selectivity and sensitivity amplification are combined, allow chemical oscillation detect and only detect determinand, avoided the interference of other impurity, overcome the problem that the chemical oscillation selectivity is low, can not carry out qualitative determination, made chemical oscillation can carry out qualitative, quantitative and measure.Enzyme, substrate and the chemical oscillating reaction system of the enzyme linked immunological of effectively selecting can increase substantially the sensitivity of enzyme linked immunosorbent detection.
Enzyme-linked Immunosorbent Assay absorption method of the present invention can be indirect method, double antibody sandwich method, dibit point single stage method, competition law, prize law, avidin-biotin complex technology.
The detection system of enzyme can be in the Enzyme-linked Immunosorbent Assay absorption method of the present invention: A, hydrogen peroxide/horseradish peroxidase system, corresponding hydrogen donor is o-phenylenediamine, tetramethyl is for biphenylamine, aminosalicylic acid, adjacent biphenyl methylamine, 2,2-connect 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 ester (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 glucose oxidase enzyme system.
The detection system of enzyme also can be to adopt multienzyme cascade amplification system to replace single horseradish peroxidase or alkaline phosphoric acid enzyme system in the Enzyme-linked Immunosorbent Assay absorption method of the present invention, to improve enzyme to the catalytic reaction ability of final substrate, such as biotin (biotin of structure of modification) and Avidin (Avidin of structure of modification) system, AP and alcohol dehydrogenase, diaphorase constitutive enzyme cascade substrate amplification system, enzyme suppresses cascade amplification system, coagulation factors enzyme cascade amplification system.
Chemical oscillation involved in the present invention 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.
In the B-Z of copper catalysis oscillation system, B-L oscillation system, B-R oscillating reactions, substrate can be that the oxidation-reduction electrode electromotive force is below 1.0 V, polyoxy organic compound with active methylene group is such as malonic acid, tartrate, gallic acid, pyrogallic acid, lactic acid, acetone, maleic acid, citric acid, malic acid, diacetone, ethyl acetoacetate etc.
Catalyzer in chemical oscillating reaction of the present invention can be M
(n+l)+/ M
N+Transition metal ion or the transition metal ion complex of electrode potential between 1.00-1.51V is 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+With the macrocyclic tetraaza complex.
The mensuration system of chemical oscillation can be closed system; Can 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 also can utilizing fork.
The testing index of chemical oscillation characteristics can be the change of induction period, the change that adds substrate front and back current potential or the change of the oscillation period before and after the adding substrate.
Fig. 1 is the principle schematic based on the enzyme linked immunosorbent assay analysis method of chemical oscillation detection substrate, and it illustrates detection side's ratio juris of the present invention take the most classical antibody-Ag-Ab Sanming City smelting sandwich method as example.As shown in the figure, the antibody that will have a selectivity is bonded to fixingly to be gone up mutually, is fixing phase antibody 1, finishes many Yus of Hou flush away antibody; Add determinand sample 2, if contain antigen to be measured in the sample, then can carry out selectivity with the fixing antibody of going up mutually and be combined; The non-binding part of flush away sample to be measured adds the secondary antibodies 3 that is marked with enzyme 5, is combined with antigen; The unnecessary unconjugated secondary antibodies of flush away, adding can be by enzymatic substrate 6, and within the regular hour, the enzyme 4 that is marked on the secondary antibodies will carry out catalysis to substrate, form catalysate 7.The substrate that enzymatic is produced is expelled in the chemical oscillating reaction system, measures the relatively variation of cycle, amplitude or the induction period of this oscillation system, compares with normal period, amplitude or induction period, calculates also calibrating antigen concentration.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 diagnosis marker to the catalysis speed of substrate, and the detection means of substrate or enzymatic reaction product concentration is chemical oscillation kinetic measurement method.
The present invention's beneficial effect compared with the prior art is, the present invention introduces the enzyme linked immunosorbent detection system with high frequency chemical oscillation kinetic measurement, the enzyme-linked immunologic diagnosis technology of highly sensitive chemical oscillation detection and high degree of specificity is combined dexterously, 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.This method had both effectively overcome the problem based on the conventional euzymelinked immunosorbent assay (ELISA) susceptibility deficiency of spectrophotometric method, and the method that changes with a kind of new high-sensitive detection substrate replaces spectrophotometric method can significantly improve sensitivity for analysis; On the other hand, the present invention has utilized again the principle of antibody-antigentic specificity combination, highly sensitive chemical oscillation kinetic measurement and the Dynamics of Enzyme Catalysis analysis with selectivity and sensitivity amplification are combined, allow chemical oscillation detect and only detect determinand, avoided the interference of other impurity, overcome the problem that the chemical oscillation selectivity is low, can not carry out qualitative determination, made chemical oscillation can carry out qualitative, quantitative and measure.Enzyme, substrate and the chemical oscillating reaction system of the enzyme linked immunological of effectively selecting can increase substantially the sensitivity of enzyme linked immunosorbent detection.
The present invention is applicable to qualitative, quantitative and measures the materials such as medicine and pesticide molecule, antibody, antigen, specified protein, nucleic acid, disease factor.
Description of drawings
Fig. 1 is the principle schematic based on the enzyme linked immunosorbent assay analysis method of chemical oscillation detection substrate;
Picture in picture number expression: the 1st, fixing phase antibody; The 2nd, determinand; The 3rd, the enzyme len antibody; The 4th, be associated in the enzyme on the antibody; The 5th, enzyme; The 6th, substrate; The 7th, the enzymatic product.
Embodiment
Embodiment 1
Present embodiment adopts indirect Determination hepatitis C virus (HCV) antibody: antigen coated with pathogen, form solid phase antigen, adding may contain the clinical sample of hepatitis C virus (HCV) antibody to be measured such as blood serum sample etc. after processing, incubation, wash plate, add the anti-human IgG antibody of horseradish peroxidase-labeled, incubation is washed plate; Add the zymolyte hydrogen peroxide, incubation is with B-Z (Belousov-Zhabotinsky) chemical oscillation kinetic measurement enzymatic preparation and former substrate difference.In the B-Z system take copper (II) complex of 14 Yuans four nitrogen heterocyclic rings (Curtis ring) as catalyzer, with NaBrO
4-malic acid-[CuL] (ClO
4)
2-H
2SO
4Be the chemical oscillation system, form the stable oscillation stationary vibration system at 22 degree Celsius, catalysate is added body series, according to the variation in caused cycle of chemical oscillation, whether indirect acknowledgment antibody exists.
Embodiment 2
Present embodiment adopts double antibody sandwich method to measure HBeAg (HBeAg): HBeAg antibody is adsorbed in solid phase surface; Add the determined antigen sample, incubation, wash plate, form antigen-antibody complex, add with the anti-human Ig antibody of alkaline phosphatase ALP enzyme target, form antibody-Ag-Ab Sanming City smelting sandwich structure, adding can be by the substrate phosphoric acid vitamin C of enzyme hydrolysis catalysis, incubation is 30 minutes when 37 spend, be added to mobile chemical oscillation system, substrate phosphoric acid vitamin C itself can not cause that cycle, amplitude or variation induction period of oscillation system change, but substrate phosphoric acid vitamin C can cause the cycle variation of oscillation system, calculates antigen concentration or confirms that whether antigen exists.
Embodiment 3
Present embodiment adopts competition law to measure little molecule haptens Avermectin: Avermectin antibody is adsorbed on surface of solid phase carriers, adds enzyme-labelled antigen and the Avermectin sample to be measured of the mark of horseradish peroxidase, the competition binding antibody; Contrast only adds enzyme-labelled antigen, adds the substrate hydrogen peroxide.Incubation after 30 minutes joins product chemical oscillation system [Ni (LA3)]
2+, malonic acid, H
3P0
4In, measure the amplitude variations of two oscillation systems, and with standard, the amplitude variations value that only adds antigen compares, and calculates antigen Avermectin concentration, and this method lowest detectable limit can reach 2.1*10
-8Mol/L.
Embodiment 4
Present embodiment adopts Streptavidin (streptavidin, SA) the biotin method is measured people's anti-tumor factor, anti-tumor factor antibody is adsorbed on surface of solid phase carriers, add testing sample, incubation, washing, the added mark anti-human Ig of biotin, incubation, washing, add with the Streptavidin behind alkaline phosphatase (ALP) mark incubation, washing, adding the phosphoric acid phenol ester is the substrate of catalytic reaction, behind catalyzing hydrolysis, the hydrolysis of substrate phosphoric acid phenol ester has generated the phenol that B-Z chemical oscillation system is changed, and reaction product is joined NaBrO
4-malic acid-[NiL] (ClO
4)
2-H
2SO
4Current system in, in Batchwise reactor (CSTR), utilize analyte pulsed perturbation technology (APP), inject the hydrolysate of enzyme linked immunoassay in the oscillation system of far from equilibrium attitude in the mode of pulse, hydrolysate causes that the cycle of chemical oscillation changes the foundation that can be used as people's anti-tumor factor qualitative and quantitative analysis, quantitative relationship between the variation in cycle and the people's anti-tumor factor concentration can be described as delta T=A*log C+B, delta T is the variation in chemical oscillation cycle, and C is the concentration of people's antineoplastic immune factor.
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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