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Humanized anti-mycobacterium tuberculosis complex LAM monoclonal antibody and preparation and application thereof

A monoclonal antibody and antibody technology, applied in the direction of analytical materials, chemical instruments and methods, instruments, etc., can solve the problems of poor sensitivity and achieve high sensitivity and specificity

Active Publication Date: 2021-10-08
迪比康上海生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Alere LAM is a LAM detection kit based on colloidal gold. Due to the use of polyclonal antibodies for detection, the sensitivity is not good. Currently it is only recommended for tuberculosis screening in AIDS patients[6]

Method used

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  • Humanized anti-mycobacterium tuberculosis complex LAM monoclonal antibody and preparation and application thereof
  • Humanized anti-mycobacterium tuberculosis complex LAM monoclonal antibody and preparation and application thereof
  • Humanized anti-mycobacterium tuberculosis complex LAM monoclonal antibody and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Construction and screening of anti-LAM scFv phage antibody library

[0062] Peripheral blood was collected from 50 healthy people, mononuclear cells (PBMC) were separated, and each group of 5 people was mixed into a group, and cDNA was obtained by RNA extraction and reverse transcription. The variable regions (VH and VL) of the light and heavy chains of the antibody were amplified by PCR, respectively, and the VH and VL were spliced ​​into fused scFv fragments using the SOE-PCR method. Connect the scFv fragment to the expression vector, and transform it into E. coli by electric shock to obtain the antibody library. The library capacity of each group is not less than 10 9 . Add helper phage to the antibody library for infection, recover and obtain the phage display antibody library, the titer of each group is not less than 10 13 Pfu / mL. According to the phage titer, the phage display antibody libraries of each group were mixed to obtain 50 anti-LAM scFv phag...

Embodiment 2

[0064] Example 2 Amino acid sequence analysis of anti-LAM monoclonal antibody

[0065] Two anti-LAM monoclonal antibodies named A1 and A12 were obtained through screening and confirmation of the scFv phage antibody library, and the sequences of the light chain and heavy chain variable regions were obtained by sequencing.

[0066] For the A1 antibody, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.7, and its coding gene sequence is shown in SEQ ID NO.9;

[0067] The heavy chain variable region of the A1 antibody contains three complementary determining regions, namely CDR-H1, CDR-H2, and CDR-H3; the amino acid sequence of CDR-H1 is shown in SEQ ID NO.1, and the amino acid sequence of CDR-H2 is shown in SEQ ID As shown in NO.2, the amino acid sequence of CDR-H3 is shown in SEQ ID NO.3;

[0068] The amino acid sequence of the light chain variable region of the A1 antibody is shown in SEQ ID NO.8, and its coding gene sequence is shown in SEQ ID N...

Embodiment 3

[0079] Example 3 Recombinant Expression and Purification of Anti-LAM Monoclonal Antibody

[0080] The nucleotide sequence fragments containing antibody sequences A1 and A12 were amplified by PCR or artificially synthesized, and inserted into expression plasmid Vector-hIgG1 to construct IgG antibody heavy chain and light chain expression plasmids, respectively.

[0081] Use the Qiagen plasmid extraction kit to extract the antibody heavy chain and light chain expression plasmids, and jointly transiently transfect Expi293F cells or prepare their stable transfer cells. After 5-7 days of cell culture, the supernatant is purified by Protein G, and the antibody is harvested by dialysis Purify, concentrate by ultrafiltration, and measure antibody concentration by ultraviolet absorption. The expressed and purified complete LAM antibody was identified by SDS-PAGE, and both A1 and A12 were under reducing conditions ( figure 1 There are about two bands under the 1 swimming lane of ), und...

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Abstract

The invention discloses a nucleic acid sequence of a pair of humanized monoclonal antibodies specifically binding with mycobacterium tuberculosis complex (MTBC) lipoarabinomannan (LAM), and a preparation method and application of a recombinant antibody of the human monoclonal antibodies. The monoclonal antibody disclosed by the invention is obtained by screening and identifying a healthy human scFv phage antibody library inoculated with bacillus calmette guerin vaccine, an antibody sequence of the monoclonal antibody is obtained by sequencing, the antibody sequence is further cloned to an expression vector, then 293 cells are transfected, and then purification is carried out to obtain the monoclonal antibody. The recombinant antibody prepared by the invention can bind with MTBC LAM at high sensitivity, an immunochromatography reagent card for rapidly detecting LAM antigens is further developed based on monoclonal antibody pairing, MTBC and part of slowly growing mycobacteria split products can be specifically detected, and do not have cross reaction with rapidly growing mycobacteria. The immunochromatography reagent card developed by the invention can be directly used for qualitative detection (taking a colloidal gold reagent card as an example) or quantitative detection (taking a quantum dot fluorescent microsphere test paper card as an example) of a urine sample of a tuberculosis patient, and has relatively high detection sensitivity and extremely high specificity, is simple and convenient to operate, is short in time consumption, and has a wide clinical application prospect in the aspect of rapid diagnosis of tuberculosis.

Description

technical field [0001] The invention relates to the technical field of antibodies, in particular to a monoclonal antibody against mycobacterium tuberculosis complex LAM and its preparation and application. Background technique [0002] Tuberculosis is a major respiratory infectious disease caused by Mycobacterium tuberculosis (Mtb) infection, and it is one of the top ten causes of death in the world and the leading cause of death from a single infectious disease [1]. Rapid and accurate laboratory diagnosis of tuberculosis is the key to effective treatment and improved prognosis, and is the first step in ending tuberculosis. There are many detection methods for active tuberculosis. The classic smear microscopy method is simple and fast but has a high rate of missed detection. The detection cycle of Mtb culture method is too long and the sensitivity is not ideal. Geneexpert Mtb / RIF improves the detection sensitivity by specifically amplifying the Mtb gene, and shortens the de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44G01N33/53G01N33/577
CPCC07K16/44G01N33/5308G01N33/577C07K2317/21C07K2317/56C07K2317/565C07K2317/52
Inventor 贾晓龙黄欢胡静
Owner 迪比康上海生物科技有限公司
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