Reagent kit

A kit and plate-type technology, applied in the field of enzyme-labeled immunoassay technology, can solve the problems of expensive, difficult to be widely used in clinical practice, and the sensitivity of general enzyme immunoassay methods cannot meet the requirements, and achieve low cost and low negative test results. bottom effect

Inactive Publication Date: 2003-03-12
SHANGHAI SECOND MEDICAL UNIV
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Problems solved by technology

Corresponding imported kits are used in China, which are expensive and difficult to be widely used in clinical practice
[0007] Under normal circumstances, the content of cardiac troponin I in the blood is very low (<0.1 μg / L), and the sensitivity of general enzyme immunoassay methods cannot meet this requirement

Method used

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Embodiment 1

[0019] Example 1 An antibody, antigen or immune complex was indirectly immobilized on a solid phase by a streptavidin-biotin capture system, and a plate-type streptavidin two-site sandwich one-step method was established. Among them, the plate-type streptavidin solid-phase two-site sandwich one-step method is to pre-coat the enzyme label plate with streptavidin, biotin-labeled anti-cTnI monoclonal antibody, and horseradish peroxidase to label another anti-cTnI antibody. cTnI monoclonal antibody.

[0020] Preparation of streptavidin solid phase: take 50μl streptavidin (1mg / ml), add 200μl distilled water to dilute, add 50μl 3mol / l acetic acid, leave at room temperature for 5min, adjust to pH 6.0 with 2mol / l Tris, and then use Coating buffer (0.05mol / l, pH 9.5 carbonate buffer) was diluted to 2μg / ml, added to polystyrene ELISA microplate, 120μl / well at 4°C overnight, washed 3 times with washing solution, added blocking solution (0.5 %BSA, 0.02mol / l, pH 7.2 PBS) at 37°C for 1 hou...

Embodiment 2

[0024] Two-site sandwich one-step ELISA method for quantitative detection of serum cTnI

[0025]Figure 1 is the operation flow chart of the plate-type streptavidin solid-phase two-site sandwich one-step ELISA for quantitative detection of serum cTnI created by the present invention. Or hold the test serum sample, then add 100 μl of biotinylated anti-TnI monoclonal antibody (about 2 μg / ml) and a mixture of horseradish peroxidase-labeled anti-TnI monoclonal antibody (pH 7.2, 0.02mol / l phosphate buffer). solution), react at 37°C for 90 minutes, and wash. Add o-phenylenediamine (OPD) substrate solution at 37° C. for 15 minutes, and add 2 mol / l sulfuric acid to stop the reaction. The absorbance value (A) at 490nm was measured on a microplate reader, with A490nm as the ordinate and each TnI concentration as the abscissa, and a standard curve was drawn.

[0026] Methodological examination:

[0027] Standard curve: use purified bovine cardiac muscle TnI antigen, after quantificatio...

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Abstract

A reagent kit for enzyme-linked immunodetection is characterized by that on the basis of ELISA method, the chain avidin-biotin capture system is used to indirectly fix the antibody, antigen, or immune complex to solid phase and the plate-type chain avidin dual-site sandwich one-step ELISA for quantitatively detecting the serum cTnI. Its advantages are simple method, high speed, and low-negative background.

Description

technical field [0001] The kit of the invention relates to biological technology, in particular to an enzyme-labeled immunoassay technology. The streptavidin and biotin capture technology is applied to the enzyme-linked immunology technology, and a plate-type streptavidin double-site sandwich one-step quantitative enzyme method is established. Streptavidin-ELISA was used to detect serum cTnI levels in patients. Background technique [0002] In my country, with the development of the national economy and the improvement of people's living standards, the morbidity and mortality of cardiovascular diseases are increasing year by year, so it is necessary to strengthen the diagnosis of early AMI. The diagnosis of ischemic heart disease is divided into three aspects: biochemical examination, ECG changes and persistent chest pain. In biochemical tests, the indicators considered to be effective in diagnosing myocardial ischemic injury are: troponin T (TnT) and I (TnI). TnT, TnI and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02G01N33/53G01N33/535G01N33/543G01N33/577
Inventor 郑佐娅王鸿利
Owner SHANGHAI SECOND MEDICAL UNIV
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