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Anti-human serum albumin monoclonal antibody

A technology of human serum albumin and monoclonal antibody, applied in the direction of anti-animal/human immunoglobulin, instruments, biochemical equipment and methods, etc., can solve the problem of high price, unknown binding site, and inability to provide mouse hybridoma cells Strain and other problems, to achieve high sensitivity and strong specificity

Active Publication Date: 2021-03-12
FORTUNEROCK (CHINA) CO LTD ALL RIGHTS RESERVED +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There are commercially available anti-human serum albumin monoclonal antibodies, but although these antibodies are specific, their binding sites on HSA are unknown, and they are expensive, and cannot be provided by mouse hybridoma cell lines.

Method used

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  • Anti-human serum albumin monoclonal antibody
  • Anti-human serum albumin monoclonal antibody
  • Anti-human serum albumin monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Computer software analysis confirms antigenic determinant and artificially synthesized antigenic determinant polypeptide

[0041] The amino acid sequences of human, bovine, monkey, mouse, sheep and rabbit serum albumin were analyzed for molecular structure, and the possible epitope amino acid polypeptide sequences were confirmed. These peptides (short peptides) are artificially selected and outsourced to synthesize the peptides.

[0042] The human serum albumin selected by artificial synthesis computer may be the amino acid sequence polypeptide-short peptide of the antigenic determinant, and after confirmation, the following amino acid sequence polypeptide is selected and synthesized to carry out the research on the preparation of anti-human serum albumin monoclonal antibody. The artificially synthesized polypeptide amino acid sequences are:

[0043] SEQ ID No. FR-1011: VRPEVDVMCTAFHDNEET (18aa, 140-157aa)

[0044] SEQ ID No. FR-1012: LLRLAKTYETTLEKCCAAAD (2...

Embodiment 2

[0052] Example 2 Immunization of mice and establishment of hybridoma cell lines

[0053] Mix and fully emulsify equal volumes of 200 μg of KLH-coupled short peptide + Freund’s complete adjuvant, and implement basic immunization on BalB / c mice by intraperitoneally injecting BalB / c mice with a volume of 0.2 ml per injection A short peptide was used to immunize five mice at the same time. For booster immunization, 100 μg of immune antigen coupled with KLH-coupled short peptide + incomplete Freund's adjuvant was mixed in equal volume and fully emulsified, and injected intraperitoneally once every 2 weeks, and the volume of each injection was 0.2 ml. Three days before cell fusion, 100 μg of KLH-coupled short peptide was mixed with 0.89% normal saline, and the mice were boosted by intraperitoneal injection. At the beginning of the second immunization, fundus blood of the mice was taken each time to measure the immune response to determine whether to continue the immunization. Gener...

Embodiment 3

[0054] Example 3 Preparation of mouse hybridoma cells

[0055] Preparation of NS-1 myeloma cells: select NS-1 cells in good growth state, wash the culture medium once, and gently blow down the NS-1 myeloma cells with 10ml culture medium.

[0056] Preparation of mouse splenocytes: Take the mice 3 days after the last booster immunization, remove the eyeballs and collect blood for the isolation of positive serum. The mice were killed by cervical dislocation, the abdominal cavity of the mice was opened aseptically, the spleen was taken out, washed with DMEM culture medium, and the connective tissue attached to the spleen was carefully removed. The spleen was then transferred to another plate containing DMEM medium. Squeeze with tweezers to fully release the mouse splenocytes to make a splenocyte suspension.

[0057] Cell fusion: Mix the NS-1 myeloma cells prepared above with splenocytes, centrifuge at 800×g for 5 minutes, and discard the supernatant. Gently shake the bottom of ...

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Abstract

According to the invention, a human serum albumin mature peptide amino acid sequence is analyzed by a computer, and is compared with mouse, cow, monkey, sheep and rabbit albumin mature peptide amino acid sequences to select a plurality of human serum albumin specific antigenic determinant amino acid sequence short peptides, wherein the amino acid lengths of the short peptides are respectively 15-35; an artificial synthetic oligopeptide is used for immunizing mice to obtain mouse hybridoma cell engineering strains capable of generating specific anti-human serum albumin monoclonal antibodies which do not have immune reaction with mice, cattle, monkeys, sheep and rabbits; and the antibody prepared from the cell strain has ultrahigh sensitivity and ultrahigh specificity to human serum albumin.

Description

technical field [0001] The invention belongs to the field of biotechnology. The present invention relates to analyzing the amino acid sequence of human serum albumin mature peptide by computer, comparing with the amino acid sequence of mouse, cow, monkey, sheep and rabbit dealbumin mature peptide, and selecting a plurality of human serum albumin specific epitope amino acid sequence short peptides , the amino acid length of the short peptide is 15-35 respectively. Artificially synthesized short peptides are used to immunize mice to obtain mouse hybridoma cell engineering strains that can produce specific anti-human serum albumin monoclonal antibodies that do not have immune reactions with mice, cows, monkeys, sheep, and rabbits. The antibody prepared by the cell line has super high sensitivity and super specificity to human serum albumin. Background technique [0002] Human serum albumin (HSA) is a soluble monomeric protein that constitutes half of the total protein in bloo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N5/20G01N33/68
CPCC07K16/18G01N33/68G01N2333/765
Inventor 于在林富岩杨小楠侯琼富俞淞陈颖
Owner FORTUNEROCK (CHINA) CO LTD ALL RIGHTS RESERVED
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