CN102495207A - High-sensitivity enzyme-linked immunoassay method - Google Patents
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- CN102495207A CN102495207A CN2011104390794A CN201110439079A CN102495207A CN 102495207 A CN102495207 A CN 102495207A CN 2011104390794 A CN2011104390794 A CN 2011104390794A CN 201110439079 A CN201110439079 A CN 201110439079A CN 102495207 A CN102495207 A CN 102495207A
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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 ELISA adsorption analysis 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, applies to fields such as biomedical research and clinical disease diagnosis widely at present.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 has analytically also obtained widespread use at residues of pesticides, drug test, low concentration chemistry article.
Classical enzyme linked immunosorbent assay analysis method principle is that measured object is through 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 ELIASA under specific wavelength, to detect the changing value of substrate absorbance.In desirable two sandwich methods or indirect method detection by quantitative; 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, promptly be approximated to direct ratio with antigen or AC to be detected.For qualitative detection, can confirm the cutoff value of diagnostic result through statistical method.Because of ELIASA is to detect the basis with the AAS, AAS generally is higher than 10 to the LDL 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: has only the antigen of high concentration or antibody to be detected by the enzymoimmunoassay of routine.Because of the limitation of AAS to the substrate detection method, the diagnosis marker of low concentration has been surveyed not or indeterminacy with classical enzymoimmunoassay, and this has also reduced susceptibility and the scope of application of conventional ELISA in medical diagnosis on disease to a certain extent.
Chemical oscillation is meant that in the open chemical reaction system away from equilibrium state, intermediate concentration presents cyclical variation clocklike on time and space.Chemical oscillation has attracted people from theoretical and two these nonlinear chemical phenomenons of aspect research 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 rule.At application, utilization chemical oscillation kinetic analysis has successfully carried 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 influence oscillating reactions and produce relevant characteristic signal.Tikhonova was used for Ru with the classical chemical oscillator in closed system in 1978
2+Detect,, set up chemical oscillation kinetic measurement method through to the time dependent kinetic curve analysis of electrode potential in the oscillatory process.With tradition relatively, this method have easy fast, advantage such as sensitivity height.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.Because of the classics in closed system is the chemical oscillation instability of catalyzer with the metallic ion; It is bigger to cause measuring relative error; Nineteen ninety-five Rafael proposes; Closed system is changed in the continuous flow stirred reactor, utilize analyte pulsed perturbation technology, in away from the oscillation system of equilibrium state, inject the analyte kind of variable concentrations with 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 analyte kind 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, the chemical oscillation detection method is very responsive to the multiple thing that adds, and poor selectivity is so the chemical oscillation detection method is used for detection by quantitative but not qualitative detection more.
Summary of the invention
The objective of the invention is, for overcoming based on the not enough problem of the conventional ELISA susceptibility of AAS, the method that changes with a kind of new high-sensitive detection substrate replaces AAS 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 AAS with the chemical oscillation method, the variation of substrate and production concentration thereof in the detection ELISA, thus set up enzyme-linked immunologic diagnosis technology based on the chemical oscillation kinetic measurement.
The characteristic that the present invention combines with antibody (antigen) specificity according to measured object; In uniform temperature with in the time; Accomplish the absorption cohesive process with corresponding enzyme-labelled antigen or antibody with corresponding antibody or antigen; The concentration that it utilizes the enzyme of mark that the catalysis speed of substrate is come the secondary indication measured object, and the detection means of the enzymatic reaction product concentration of substrate or substrate is a chemical oscillation kinetic measurement method.The qualitative determination principle technological based on the ELISA of chemical oscillation kinetic measurement does; When having only the measured object of existence; Determinand combines the enzyme linked immunological specificity with corresponding antibody (antigen); Antibodies specific or antigen will be adsorbed, and 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 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.The quantitative measurement principle technological based on the ELISA of chemical oscillation kinetic measurement does; The variation of measured object concentration will cause the corresponding change of specific antibody (antigen) binding capacity that directly or indirectly combines with measured object; Its amount that directly or indirectly connects enzyme will change thereupon; Catalytic reaction speed to substrate will produce change; 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 principle that the present invention has also utilized antibody-antigentic specificity to combine; Highly sensitive chemical oscillation kinetic measurement and the Dynamics of Enzyme Catalysis analysis with selectivity and sensitivity amplification are combined; Let 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 immunological absorption 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 architecture of enzyme can be in the enzyme linked immunological absorption absorption method of the present invention: A, and oxydol/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 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 a malic acid, E glucose oxidase enzyme system.
The detection architecture of enzyme also can be to adopt multienzyme cascade amplification system to replace single HRPO or alkaline phosphoric acid enzyme system in the enzyme linked immunological absorption absorption method of the present invention; To improve the catalytic reaction ability of enzyme to final substrate; Like 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, liquid film oscillating reactions 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 like 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 like 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 a closed system; Can be the open system that continuous flow is stirred, it utilizes analyte pulsed perturbation technology, in away from the oscillation system of equilibrium state, injects the analyte kind of variable concentrations with 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 that detects the enzyme linked immunosorbent assay analysis method of substrate based on chemical oscillation, and it is an example with the most classical antibody-Ag-Ab Sanming City smelting sandwich method, and detection side's ratio juris of the present invention is described.As shown in the figure, the antibody that will have selectivity is bonded on the stationary phase, is stationary phase antibody 1, accomplishes many Yus of Hou flush away antibody; Add determinand sample 2, if contain antigen to be measured, then can carry out selectivity with the antibody on the stationary phase and combine in the sample; The non-binding part of flush away sample to be measured adds the secondary antibodies 3 that is marked with enzyme 5, combines with antigen; The unnecessary unconjugated secondary antibodies of flush away, adding can be by enzymatic substrate 6, and in 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; Accomplish the absorption cohesive process with corresponding enzyme-labelled antigen or antibody with corresponding antibody or antigen; The concentration that it utilizes the enzyme of mark that the catalysis speed of substrate is come the secondary indication diagnosis marker, and the detection means of substrate or enzymatic reaction product concentration is a chemical oscillation kinetic measurement method.
The beneficial effect of the present invention and prior art comparison 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 AAS with the chemical oscillation method, the variation of substrate and production concentration thereof in the detection ELISA, thus set up enzyme-linked immunologic diagnosis technology based on the chemical oscillation kinetic measurement.This method had both effectively overcome based on the not enough problem of the conventional ELISA susceptibility of AAS, and the method that changes with a kind of new high-sensitive detection substrate replaces AAS can significantly improve sensitivity for analysis; On the other hand; The principle that the present invention has utilized antibody-antigentic specificity to combine again; Highly sensitive chemical oscillation kinetic measurement and the Dynamics of Enzyme Catalysis analysis with selectivity and sensitivity amplification are combined, let 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 that qualitative, quantitative measures materials such as medicine and agricultural chemicals micromolecule, antibody, antigen, specified protein, nucleic acid, disease factor.
Description of drawings
Fig. 1 is the principle schematic that detects the enzyme linked immunosorbent assay analysis method of substrate based on chemical oscillation;
Picture in picture number expression: the 1st, stationary phase antibody; The 2nd, determinand; The 3rd, the enzyme len antibody; The 4th, join the enzyme on antibody; The 5th, enzyme; The 6th, substrate; The 7th, the enzymatic product.
Embodiment
Embodiment 1
Present embodiment adopts indirect method to measure HCV (HCV) antibody: antigen coated with pathogen; Form solid phase antigen, handle clinical sample such as blood serum sample etc. that the back adding possibly contain HCV (HCV) antibody to be measured, incubation; Wash plate; Add the anti-human IgG antibody of horseradish peroxidase-labeled, incubation is washed plate; Add the zymolyte oxydol, incubation is with difference between B-Z (Belousov-Zhabotinsky) chemical oscillation kinetic measurement enzymatic prepn and the former substrate.Copper (II) complex with 14 Yuans four nitrogen heterocyclic rings (Curtis ring) in the B-Z system is a 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 hepatitis B e antigen (HBeAg): HBeAg antibody is adsorbed in solid phase surface; Add the determined antigen sample, incubation is washed plate; Form antigen-antibody complex, add antibody, form antibody-Ag-Ab Sanming City smelting sandwich structure with the anti-people Ig of alkaline phosphatase ALP enzyme target; Adding can be by the substrate phosphoric acid vitamin C of enzyme hydrolysis catalysis; Incubation is 30 minutes when 37 spend, and is added to mobile chemical oscillation system, and 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 micromolecule haptens AVM: AVM antibody is adsorbed on surface of solid phase carriers, adds the enzyme-labelled antigen and the AVM sample to be measured of the mark of horseradish peroxidase, the competition binding antibody; Contrast only adds enzyme-labelled antigen, adds the substrate oxydol.Incubation joined chemical oscillation system [Ni (LA3)] with product after 30 minutes
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 AVM concentration, and this method LDL can reach 2.1*10
-8Mol/L.
Embodiment 4
Present embodiment employing Streptavidin (streptavidin, SA) the biotin method is measured people's anti-tumor factor, and anti-tumor factor antibody is adsorbed on surface of solid phase carriers, adds testing sample; Incubation, washing, the anti-people Ig of biotin that added mark; Incubation, washing adds 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 continuous flow stirred reactor (CSTR); Utilize analyte pulsed perturbation technology (APP); In away from the oscillation system of equilibrium state, inject the hydrolysate of enzyme linked immunoassay with 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, and the 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 (6)
1. high sensitivity ELISA adsorption analysis method; It is characterized in that; Said method combines the enzyme-linked immunologic diagnosis technology of highly sensitive chemical oscillation detection and high degree of specificity dexterously; Replace AAS with the chemical oscillation method, the variation of substrate and production concentration thereof in the detection ELISA, thus set up enzyme-linked immunologic diagnosis technology based on the chemical oscillation kinetic measurement; The characteristic that said method combines with antibody or antigentic specificity according to measured object; In uniform temperature with in the time; Accomplish the absorption cohesive process with corresponding enzyme-labelled antigen or antibody with corresponding antibody or antigen; The concentration that it utilizes the enzyme of mark that the catalysis speed of substrate is come the secondary indication measured object, and the detection means of the enzymatic reaction product concentration of substrate or substrate is a chemical oscillation kinetic measurement method;
Said enzyme linked immunological absorption determining adsorption analytical approach can be indirect method, double antibody sandwich method, dibit point single stage method, competition law, prize law, avidin-biotin complex technology;
The detection architecture of enzyme can be in the said enzyme linked immunological absorption absorption method: A, and oxydol/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 a malic acid; E, the glucose oxidase enzyme system;
The detection architecture of enzyme also can adopt multienzyme cascade amplification system to replace single HRPO or alkaline phosphoric acid enzyme system in the said enzyme linked immunological absorption absorption method; To improve the catalytic reaction ability of enzyme to final substrate; Like 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 architecture of said 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, liquid film oscillating reactions system.
2. a kind of high sensitivity ELISA adsorption analysis method according to claim 1; Said method combines highly sensitive chemical oscillation kinetic measurement and the Dynamics of Enzyme Catalysis analysis with selectivity and sensitivity amplification; Let 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.
3. a kind of high sensitivity ELISA adsorption analysis method according to claim 1; In the detection architecture of said chemical oscillation kinetic measurement; 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 like malonic acid, tartrate, gallic acid, pyrogallic acid, lactic acid, acetone, maleic acid, citric acid, malic acid, diacetone, ethyl acetoacetate.
4. a kind of high sensitivity ELISA adsorption analysis method according to claim 1, the catalyzer in the said chemical oscillating reaction 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 like 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.
5. a kind of high sensitivity ELISA adsorption analysis method according to claim 1, the mensuration system of said chemical oscillation can be a closed system; Can be the open system that continuous flow is stirred, it utilizes analyte pulsed perturbation technology, in away from the oscillation system of equilibrium state, injects the analyte kind of variable concentrations with the mode of pulse; Residing subcritical state system is measured before also can utilizing fork.
6. a kind of high sensitivity ELISA adsorption analysis method according to claim 1, the testing index of said 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.
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| CN114994194A (en) * | 2022-05-09 | 2022-09-02 | 广东安纳检测技术有限公司 | Method and device for measuring malonic acid in water |
| CN114994194B (en) * | 2022-05-09 | 2024-04-09 | 广东安纳检测技术有限公司 | Method and device for measuring malonic acid in water |
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