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Chimeric antibody of anti-cardiolipin/beta2 glycoprotein I complex

A chimeric antibody and anticardiolipin technology, applied in the biological field, can solve the problems of kit detection data fluctuation, shortage of serum raw material supply, inconsistent result interpretation, etc.

Inactive Publication Date: 2016-07-27
SHANGHAI KEXIN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Considering biosafety issues, major hospitals implement strict management on serum samples, resulting in a shortage of serum raw materials in the production process of ELISA kits
On the other hand, the clinically collected serum raw materials have serious individual differences, which often lead to fluctuations in the detection data of the kit and inconsistencies in the interpretation of results during use
This is an unavoidable problem in the application of ELISA kits produced by clinical serum

Method used

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  • Chimeric antibody of anti-cardiolipin/beta2 glycoprotein I complex
  • Chimeric antibody of anti-cardiolipin/beta2 glycoprotein I complex
  • Chimeric antibody of anti-cardiolipin/beta2 glycoprotein I complex

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Construction and expression of anti-cardiolipin / β2 glycoprotein I complex chimeric antibody IgG type eukaryotic expression vector (both heavy chain and light chain genes were synthesized by Shanghai Hanyu Biotechnology Co., Ltd.):

[0037] The amino acid sequence of the antibody heavy chain variable region is SEQ ID NO: 1, the coding DNA sequence used in this embodiment is SEQ ID NO: 2; the amino acid sequence of the antibody IgG heavy chain is SEQ ID NO: 3, the coding DNA sequence used in this embodiment The DNA sequence is SEQ ID NO:4.

[0038] SEQ ID NO: 1

[0039] VQLQESGAELVKPGASVKLSCKASGYTFTSHWMHWVKLRPGQGFEWIGENNPRNGDTNFNEKFKRKATLTVEKSSNTAYMQLNSLTSEDSAVYYCTIWSFYYGFDYWGQGTSLTVSSAKTT

[0040] SEQ ID NO: 2

[0041] GTGCAGCTGCAGGAGTCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCCTCTGGCTACACCTTCACCAGCCACTGGATGCACTGGGTGAAGCTGAGGCCTGGACAAGGCTTTGAGTGGATTGGAGAGAATAATCCTCGCAATGGTGATACTAACTTCAATGAGAAGTTCAAGAGAAAGGCCACACTGACTGTAGAGAAATCCTCCAACACAGCCTACATGCAGCTCAACAGCCT...

Embodiment 2

[0059] Functional identification of chimeric antibody against cardiolipin / β2GP1 complex

[0060] Antibody affinity identification:

[0061] The cardiolipin / β2GP1 complex (mixing cardiolipin and β2 glycoprotein I at an equimolar ratio of 1:2) was coated on an ELISA plate at a concentration of 2 μg / ml cardiolipin (working volume 30ul), and statically placed at 4°C. Leave overnight. Wash 3 times with PBS (PBST) containing 0.05% Tween20. Block with 0.1% BSA for 1 hour at room temperature. Wash 3 times with PBST, prepare the antibody prepared in Example 1 to a concentration of 4 μg / ml, and perform 3-fold serial dilution (diluent: PBST+1%BSA+0.05%ProClin), a total of 8 gradients, each well Add 30 μl and let stand at room temperature for 1 hour. Wash 3 times with PBST, add 30 μl 1:4000 diluted horseradish peroxidase-labeled goat anti-human IgG Fc, and let stand at room temperature for 1 hour. Wash 4 times with PBST, add TMB for color development, and use 2M H 2 SO 4 Stop the r...

Embodiment 3

[0063] Application of anti-cardiolipin / β2GP1 complex chimeric antibody in ELISA detection kit

[0064] Material preparation for ELISA applications:

[0065] The antibody prepared in Example 1 was diluted with antibody dilution buffer (PBST+1%BSA+0.05%ProClin) and calibrated with a commercial ELISA kit to obtain the relative RU value of the cardiolipin antibody relative to human serum.

[0066] The quality control serum in ELISA application is prepared from clinically positive serum of cardiolipin antibody IgG, 100RU / ml.

[0067] The precision serum in ELISA application is the cardiolipin antibody IgG clinical serum sample (60RU / ml and 150RU / ml).

[0068] The clinical serum samples (20 cases positive and 20 cases negative) used in the evaluation of ELISA application were collected from clinical serum samples.

[0069] The above quality control serum, precision serum, and clinical serum samples all conform to the biosafety testing regulations, and the IgG value of cardiolipin ...

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Abstract

The invention relates to a biological technique, and in particular to a chimeric antibody of an anti-cardiolipin / beta2 glycoprotein I complex as well as a preparation method and application thereof. The amino acid sequence of an antibody light chain variable region of the chimeric antibody of the anti-cardiolipin / beta2 glycoprotein I complex is as shown in SEQ ID NO:5 or a conservative variation sequence thereof, and the amino acid sequence of an antibody heavy chain variable region of the chimeric antibody is as shown in SEQ ID NO:1 or a conservative variation sequence thereof. By using a hybridoma technique, the inventor of the invention obtains a hybridoma cell strain of a mouse anti-cardiolipin / beta2 glycoprotein I complex antibody, a variable region sequence of the antibody is cloned, furthermore the chimeric antibody of the anti-cardiolipin / beta2 glycoprotein I complex, which is prepared by using a gene engineering technique, has good affinity with the anti-cardiolipin / beta2 glycoprotein I complex, and the chimeric antibody can be used in an ELISA detection kit.

Description

technical field [0001] The invention relates to biotechnology, in particular to a chimeric antibody against cardiolipin / β2 glycoprotein I complex and its preparation method and application. Background technique [0002] The clinical signs of antiphospholipid syndrome (APS) are not fully clear, the main clinical manifestations are: thrombosis, habitual abortion, thrombocytopenia and neuropsychiatric symptoms. According to current guidelines, lupus anticoagulant, anticardiolipin antibodies, and β2-glycoprotein I antibodies are the main serological clinical tests for antiphospholipid antibodies. If all three items are positive, it indicates that the patient or healthy person is at high risk of antiphospholipid antibody syndrome, and if only one item is positive, the risk is low. [0003] Studies have shown that these three inspection items are very sensitive to external factors, and it is difficult to standardize the detection methods. Lupus anticoagulant is most associated w...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13G01N33/68G01N33/543
CPCC07K16/18C07K2317/24C07K2317/32C07K2317/56
Inventor 王红勋郭延荣范磊孙祥明包骏
Owner SHANGHAI KEXIN BIOTECH
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