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Anti-human beta2-MG antibody and application thereof

An antibody and sequence technology, which is applied in the application field of the above-mentioned antibody in the quantitative detection of human β2-microglobulin, can solve the problems of pre-band or post-band, the influence of measurement results, etc., and achieves the effect of simple operation and convenient mass production.

Active Publication Date: 2019-04-05
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the ratio of antigen and antibody is different, such as the phenomenon of anterior zone or posterior zone, it will have a greater impact on the measurement results.

Method used

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  • Anti-human beta2-MG antibody and application thereof
  • Anti-human beta2-MG antibody and application thereof
  • Anti-human beta2-MG antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Anti-human beta 2 - Preparation of microglobulin hybridoma cell lines

[0041] 1. Animal immunization

[0042] recombinant human beta 2 -MG (recombinantly expressed in Escherichia coli, produced by our company) was used to immunize BALB / c female mice (purchased from Changzhou Cavens Experimental Animal Co., Ltd.) according to the general immunization procedure. For specific immunization conditions, please refer to the "Experimental Guidelines for Antibody Preparation and Use". The serum titer of immunized mice was tracked by indirect ELISA method, and the immunized mouse with the highest serum titer was selected for fusion experiment of mouse splenocytes and mouse myeloma cells.

[0043] 2. Cell Fusion

[0044] (1). Preparation of spleen cells

[0045] Take the immunized mice, remove their eyeballs, take blood, put them to death by breaking the cervical spine, soak them in 75% (v / v) alcohol for 10 minutes, take out their spleens in a sterile operating ta...

Embodiment 2

[0055] Example 2. Determination of the variable region sequence of the hybridoma cell line antibody

[0056] The variable region sequences of the above-mentioned hybridoma cell lines B12 and B17 antibodies were determined.

[0057] a. Extraction of RNA: Extract the total RNA of the above-mentioned hybridoma cell lines B12 and B17 with reference to the instructions of the Total Cell RNA Extraction Kit (purchased from Roche Company) and perform reverse transcription immediately;

[0058] b. Reverse transcription of RNA into DNA: Refer to Thermo Scientific Reverted First strand cDNASynthesis Kit (purchased from Thermo Company) to reverse-transcribe the total RNA extracted in the previous step to obtain cDNA, and freeze it at -20°C for later use;

[0059] c. PCR amplification and recovery of the variable region sequence: the cDNA obtained in the above step is used as a template, and the variable region sequence of the heavy chain and light chain is sequenced with the general prime...

Embodiment 3

[0063] Example 3. Recombinant expression and purification of single-chain antibody

[0064] According to the sequencing results in Example 2, a connecting peptide (GGGGS) was added between the antibody heavy chain and light chain variable regions of the hybridoma cell lines B12 and B17 3 , six histidines were introduced into the C-terminus, the whole gene was synthesized, and the recombinant expression of the single-chain antibody was performed with the Pichia pastoris expression system. The expressed antibodies were named as antibodies BM12 and BM17, respectively. The recombinant expression of the above-mentioned single-chain antibody is specifically as follows:

[0065] 1. Construction of expression plasmids for single-chain antibody genes

[0066] The gene sequence of the single-chain antibody BM12 is shown in SEQ ID NO:19, and the amino acid sequence is shown in SEQ ID NO:17; the gene sequence of the single-chain antibody BM17 is shown in SEQ ID NO:20, and the amino acid...

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Abstract

The invention relates to a novel anti-human beta2-microglobulin antibody and application thereof, and belongs to the field of immunochemistry. Various antibodies are prepared by the invention, matching screening is performed, and an antibody combination (BM12 and BM17) with the sensitivity and the specificity meeting the requirements is obtained; meanwhile, the mass production is convenient; the requirements of large-scale clinic application in future can be met. The antibody combination is subjected to detection system debugging and optimization work to achieve the effect of simple and convenient operation. The industrialized application of colloidal gold rapid test paper card of human beta2-microglobulin with the sensitivity, specificity and relevant detection performance meeting the requirements of human-clinic sample detection can be realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to two anti-human beta 2 - Microglobulin antibody and its preparation method and above-mentioned antibody in human β 2 - Application in quantitative detection of microglobulin. Background technique [0002] beta 2 - Microglobulin (β 2 -microglobulin, beta 2 -MG) is an endogenous small molecule globulin produced by lymphocytes, platelets, and polymorphonuclear leukocytes, with a molecular weight of 11.8kDa; a single-chain polypeptide composed of 99 amino acids, which plays an important role in the immune response ; It is the β-chain part of human leukocyte antigen on the cell surface. It contains disulfide bonds in the molecule and does not contain sugar. It is similar in structure to immunoglobulin. [0003] beta 2 -MG widely exists in plasma, urine, cerebrospinal fluid, saliva and colostrum. Serum beta 2 -The rate of synthesis of MG and the amount released from the cell membran...

Claims

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Application Information

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IPC IPC(8): C07K16/18G01N33/68G01N33/558
CPCG01N33/558G01N33/68C07K16/18C07K2317/56G01N2333/4713
Inventor 马永赵利利王安良
Owner ZONHON BIOPHARMA INST
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