Human beta2-microglobulin quantitative test card and clinical applications

A technology for quantitative detection of microglobulin, which is applied in anti-animal/human immunoglobulin, biological testing, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc. Belt or rear belt and other problems, to achieve the effect of convenient mass production and easy operation

Active Publication Date: 2019-05-07
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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  • Human beta2-microglobulin quantitative test card and clinical applications
  • Human beta2-microglobulin quantitative test card and clinical applications
  • Human beta2-microglobulin quantitative test card and clinical applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Anti-human β 2 -Preparation of Microglobulin Hybridoma Cell Line

[0041] 1. Animal immunity

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

[0043] 2. Cell Fusion

[0044] (1). Preparation of spleen cells

[0045] Immunize the mice, remove the eyeballs and take blood, place them in 75% (v / v) alcohol for 10 minutes after being killed by severed cervical vertebrae, take out their spleens in a sterile operating table, place them in a cell screen, and gri...

Embodiment 2

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

[0056] The sequence of the antibody variable region of the hybridoma cell lines B12 and B17 was determined.

[0057] a. RNA extraction: refer to the instructions of the cell total RNA extraction kit (purchased from Roche) to extract total RNA from the hybridoma cell lines B12 and B17 and immediately perform reverse transcription;

[0058] b. RNA reverse transcription into DNA: refer to Thermo Scientific Reverted First strand cDNA Synthesis Kit (purchased from Thermo Company) to reverse transcription of the total RNA extracted in the previous step to prepare cDNA, which is stored frozen at -20°C for use;

[0059] c. PCR amplification and recovery of variable region sequences: the cDNA obtained in the previous step is used as a template, and the murine IgG subtype monoclonal antibody variable region sequence universal primers are used as primers for the heavy and light chain variable region sequ...

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 , C-terminal introduces six histidines, completes gene synthesis, and uses Pichia pastoris expression system for recombinant expression of single-chain antibody. The expressed antibodies were named antibody BM12 and BM17, respectively. The recombinant expression of the aforementioned single-chain antibody is specifically as follows:

[0065] 1. Construction of expression plasmid of single chain antibody gene

[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 sequence is shown in SEQ ID NO: 18 shown. The single-...

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Abstract

The present invention relates to a human beta2-microglobulin quantitative test card. The invention prepares a plurality of antibodies and performs paired screening to obtain a group of antibody combinations (BM12 and BM17) having sensitivity and specificity which can meet requirements of the quantitative test card. At the same time, the test card is convenient for mass production, and can meet theneeds of large-scale clinical applications in the future. The antibody combination is debugged and optimized in the detection system to obtain a colloidal gold immunochromatographic quantitative testcard for human beta2-microglobulin which is easy to operate, sensitive, specific and has related detection performance meeting the detection of human blood or urine samples.

Description

Technical field [0001] The present invention belongs to the field of biotechnology, and specifically relates to two kinds of anti-human β 2 -Microglobulin antibody and its preparation method and the above-mentioned antibody in human β 2 -Application in quantitative detection of microglobulin. Background technique [0002] β 2 -Microglobulin (β 2 -microglobulin, β 2 -MG) is an endogenous small molecular 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 immune response ; It is the β chain part of the human leukocyte antigen on the cell surface. The molecule contains disulfide bonds and does not contain sugar. It is similar in structure to immunoglobulin. [0003] β 2 -MG is widely present in plasma, urine, cerebrospinal fluid, saliva and colostrum. Serum beta 2 -MG synthesis speed and the amount of cell membrane release are very con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C12N15/81C07K16/28
Inventor 马永丁娜
Owner ZONHON BIOPHARMA INST
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