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ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting human anti-rabies virus antibody

A technology of rabies virus and kit, which is applied in the field of detection of antibodies in human blood samples, which can solve the problems of serious non-specific binding in ELISA detection, increased background of negative detection, low accuracy and sensitivity, and achieve small errors , reduce non-specific binding and improve accuracy

Inactive Publication Date: 2014-10-01
NCPC NEW DRUG RES & DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In daily clinical serum (serum) samples, a considerable proportion contains the above-mentioned various interfering substances to varying degrees, which leads to serious non-specific binding in ELISA detection, increased negative background of detection, and low accuracy and sensitivity.

Method used

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  • ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting human anti-rabies virus antibody
  • ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting human anti-rabies virus antibody
  • ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting human anti-rabies virus antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1, the preparation of the ELISA kit that detects human anti-rabies virus antibody

[0021] (1) Preparation of reagents

[0022] Coating buffer: 0.1M carbonate buffer, pH9.6, weighed Na 2 CO 3 3.18g, NaHCO 3 5.88g, dilute to 1000ml with ultrapure water.

[0023] PBS buffer (pH7.4): 0.02M PBS buffer, pH7.4, weighed Na 2 HPO 4 12H 2 O 5.16g, NaH 2 PO 4 2H 2 O 0.87g, NaCl 7.6g, dilute to 1000ml with ultrapure water.

[0024] Plate washing solution (PBST): PBS buffer (pH7.4) containing 0.05% Tween-20.

[0025] Blocking solution: PBS buffer (pH7.4) containing 1% BSA.

[0026] Eluent: 300 mmol / L acetic acid solution.

[0027] Neutralizing solution: 1mol / L Tris-HCl buffer (pH8.0) containing 2% BSA.

[0028] Stop solution: 2 mol / L H 2 SO 4 solution.

[0029] Substrate: TMB A, B chromogenic solution.

[0030] (2) Preparation method of NM57 mouse monoclonal antibody S-230 and S-271

[0031] The amino acid sequence of the S-230 light chain variable ...

Embodiment 2

[0038] Embodiment 2, use kit to detect the plasma concentration of human anti-rabies virus NM57 monoclonal antibody

[0039] (1) Capture-elute NM57 monoclonal antibody in serum (plasma) specimen

[0040] ① Capture target antibody: Add 40 μl of blocking solution to each well of the antibody capture plate, then add reference standards S1-S6, high, medium and low concentration quality controls, and serum samples to be tested, and incubate at 37°C for 2 hours with shaking. The preparation method of the reference standard S1~S6 is to dilute NM57 to 300 ng / ml with normal human serum to obtain S1, then double-dilution with normal human serum to obtain S2 (150 ng / ml), and sequentially double-dilute to S3 (75ng / ml). ml), S4 (37.5ng / ml), S5 (18.8ng / ml), S6 (9.38ng / ml); the preparation method of high, medium and low concentration quality control products is to dilute NM57 to 225 ng / ml with normal human serum Concentration quality control product X1, then diluted 4 times with normal huma...

Embodiment 3

[0050] Embodiment 3, test kit detection methodology verification result

[0051] (1) Accuracy:

[0052] Six batches of high, medium and low samples (X1, X2, X3) were tested, and the recovery rate was calculated.

[0053]

[0054] The test results showed that the recoveries of high, medium and low concentration standards were 90%-110%.

[0055] (2) Accuracy (repeatability)

[0056] In one batch of determination, carry out the determination of 6 repeated samples for the high, medium and low samples (X1, X2, X3), and calculate the CV% value.

[0057]

[0058] The test results showed that the intra-plate repeatability (intra-assay precision) of high, medium and low concentration standards was 5.2%-6.4%.

[0059] (3) Accuracy (intermediate precision)

[0060] Three batches of high, medium and low samples (X1, X2, X3) were measured, and the CV% value was calculated.

[0061]

[0062] The test results showed that the inter-plate repeatability (inter-assay precision) of ...

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Abstract

The invention relates to an ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting human anti-rabies virus antibody and a detecting method thereof. The kit comprises a capture antibody plate and a detection antibody plate. The method adopts the steps of capturing and eluting target antibody to remove the interference of intrinsic protein in a detection sample so as to reduce the probability of nonspecific binding during ELISA detection. The kit can be used for qualitatively or quantitatively detecting the human anti-rabies virus antibody in a human blood specimen, the detecting method is high in recovery rate, precision and linearity when the concentration of a sample is 12.7ng / ml -225ng / ml, and the kit can be used for accurately detecting samples with high specificity and high sensitivity.

Description

technical field [0001] The invention relates to the technical field of biological detection, and in particular to the detection of antibodies in samples containing human blood. Background technique [0002] In 1971, Engvall et al. in Sweden used cellulose and polypropylene test tubes as solid-phase carriers to adsorb antigens / antibodies respectively, and established Enzyme-linked Immunosorbent Assay (ELISA). In 1974, Voller et al. switched to polystyrene micro-reaction plates as solid-phase immunosorbent carriers, which enabled the popularization and application of the ELISA method, and developed the enzyme-labeled antibody technology for antigen localization into a method for the determination of trace substances in liquid specimens, and gradually It has become the most commonly used method in antigen antibody detection. It combines the immune reaction of the enzyme marker with the antigen-antibody complex and the catalytic amplification of the enzyme, which not only maint...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531
CPCG01N33/54393G01N33/56983G01N2333/145
Inventor 刘艳玲魏敬双王惠欣王辉李艳霞李丽敏段迎霞刘祥义刘晓志高健段宝玲
Owner NCPC NEW DRUG RES & DEV
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