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High-sensitivity enzyme-linked immunoassay method

An enzyme-linked immunosorbent adsorption and high-sensitivity technology, which is applied in the direction of analyzing materials, using catalysis for chemical analysis, measuring devices, etc., to overcome the lack of sensitivity, improve the sensitivity of the analysis, and improve the sensitivity

Inactive Publication Date: 2012-06-13
EAST CHINA JIAOTONG UNIVERSITY
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0005] The object of the present invention is, in order to overcome the insufficient sensitivity problem of conventional ELISA based on spectrophotometry, use a kind of new highly sensitive detection substrate The analytical sensitivity can be greatly improved by changing the method instead of the spectrophotometric method; due to the low selectivity of the simple chemical oscillation method, qualitative detection cannot be carried out. The combination of enzyme-catalyzed kinetic analysis will be able to overcome the problems of low chemical oscillation selectivity and incapable of qualitative determination

Method used

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Examples

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Embodiment 1

[0027] 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, and use B-Z (Belousov-Zhabotinsky) chemical oscillation kinetics to detect enzymatic activity The difference between the product and the original substrate. In the B-Z system, the copper (II) complex of the fourteen-membered tetraazamacrocycle (Curtis ring) was used as the catalyst, and NaBrO 4 -Malic acid-[CuL](ClO 4 ) 2 -H 2 SO 4 It is a chemical oscillation system, forming a stable oscillation system at 22 degrees Celsius, adding catalytic products to the system, and indirectly confirming the presence or absence of an...

Embodiment 2

[0029] 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.

[0030]

[0031]

Embodiment 3

[0033] 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, add the product to the chemical shaking system [Ni(LA3)] 2+ , malonic acid, H 3 P0 4 In the method, the amplitude change of the two oscillation systems is measured, and compared with the standard amplitude change value of only adding antigen, the concentration of antigen abamectin is calculated, and the minimum detection limit of this method can reach 2.1*10 -8 mol / L.

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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 [0001] 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 [0002] 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 diagnos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558G01N31/10
Inventor 胡林徐文媛胡刚刘海燕卢明英吴海燕胡米微
Owner EAST CHINA JIAOTONG UNIVERSITY
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