Anti-D-dimer monoclonal antibody and application thereof

A monoclonal antibody, dimer technology, applied in the direction of anti-coagulation factor immunoglobulin, anti-animal/human immunoglobulin, biochemical equipment and methods, etc. Qualitative and other problems, to achieve the effect of good stability, high sensitivity and high diagnostic consistency

Active Publication Date: 2011-04-13
上海贝西生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has low accuracy and can only be used qualitatively, so it is suitable for emergency
3) Chromogenic substrat...

Method used

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  • Anti-D-dimer monoclonal antibody and application thereof
  • Anti-D-dimer monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: Preparation of anti-D-dimer monoclonal antibody

[0017] 1. Animal immunity

[0018] Take female BALB / c mice aged 6-8 weeks, and inject pure D-dimer with a concentration of 5mg / ml for the first immunization, and inject 1ml of the total amount through the abdominal cavity and under the armpit of the limbs; Methods Booster immunization once, a total of 3 immunizations, on the 4th day after the last immunization, blood was collected from the eyeballs of mice after three immunizations, the serum was separated by centrifugation, and mice with high titer were selected by ELISA method for fusion.

[0019] 2. Preparation of hybridoma cell lines

[0020] The splenocytes from the mice that had completed the immunization process were prepared to be fused with mouse myeloma cell SP2 / 0, and the feeder cells were prepared the day before the fusion; the mouse splenocytes were aseptically taken during fusion, and the splenocytes were mixed with SP2 / 0 cells at a ratio of 10:...

Embodiment 2

[0026] Example 2: Specific identification of monoclonal antibodies

[0027] Materials: Fibrinogen and its fragments X, Y, D, E and plasma.

[0028] Methods: ELISA method was used for detection, and D-dimer was used as positive control.

[0029] Results: Fibrinogen and its fragments X, Y, D, E and plasma were all negative.

[0030] Explanation: The anti-D-dimer monoclonal antibody of the present invention is highly specific to D-dimer.

Embodiment 3

[0031] Example 3: Cloning and sequencing of variable region sequences of monoclonal antibodies

[0032] Using 5'RACE (Rapid Amplification of cDNA Ends, rapid amplification of cDNA ends) technology, the variable region sequence of the functional antibody was cloned from the hybridoma cell line 6B8D11H12 secreting anti-D-dimer monoclonal antibody. The steps can be briefly described as follows: an antisense gene-specific primer (GSP1) is used to synthesize the first strand of cDNA, after the first strand of cDNA is purified, terminal deoxynucleotidy transferase (Terminal deoxynucleotidy transferase, TdT) is used to generate A synthetic homopolynucleotide anchor sequence is added to the 3' end of the The cDNA is amplified using a second nested gene-specific primer (GSP2) and an anchor primer that anneals to the homopolynucleotide tail. The specific experimental process is as follows:

[0033] Material:

[0034] -Anti-D-dimer monoclonal antibody secreted by hybridoma cell line 6...

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PUM

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Abstract

The invention discloses an anti-D-dimer monoclonal antibody. The amino acid residue sequence of the light chain of the antibody is shown in SEQ ID No.1, and the amino acid residue sequence of the heavy chain of the antibody is shown in SEQ ID No.2; and the antibody is secreted by a hybridoma cell strain 6B8D11H12 the preservation number of which is CCTCC NO.C201060. The anti-D-dimer monoclonal antibody can be used in preparing various D-dimer aspartame assay kits. By utilizing the anti-D-dimer monoclonal antibody disclosed by the invention and a D-dimer aspartame assay kit prepared by gold immunochromatographic assay(GICA), the detection sensitivity is up to 96.08%, the detection specificity is up to 94.79%, the detection accuracy is up to 95.67%, the Kappa detection value is up to 0.901 which is greater than 0.75, and the diagnosis is more consistent with clinical diagnosis, thus the anti-D-dimer monoclonal antibody can be used for aided diagnosis on thrombotic diseases in clinical applications.

Description

technical field [0001] The invention relates to an anti-D-dimer monoclonal antibody and its application, belonging to the field of biotechnology. Background technique [0002] D-dimer is the end product of cross-linked fibrin after the action of plasmin. In the coagulation process, after thrombin hydrolyzes fibrinogen, it releases fibrin peptide A (FPA) and peptide B (FPB) successively, and the rest is soluble fibrin monomer (SFM). , SFM transforms into fibrin, and then blood coagulates. The process is completed after a series of cross-linking. After that, the fibrin formed is stable and generally insoluble, but can be degraded by plasmin. Cross-linked fibrin is in fibrinolysis During the enzymatic degradation process, several polymers are gradually formed, among which D-dimer is one of its specific products, and its molecular weight is 184,000-202,000. In pathological conditions, the dynamic balance of coagulation and fibrinolysis is disrupted, and the tendency of coagula...

Claims

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Application Information

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IPC IPC(8): C07K16/36C12N5/20G01N33/577C12R1/91
Inventor 黄波周季余黄桂民
Owner 上海贝西生物科技有限公司
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