Hybridoma cell strain and monoclonal antibody of anti-heart-type fatty acid-binding protein generated by same

A hybridoma cell line, monoclonal antibody technology, applied in the direction of anti-animal/human immunoglobulin, microorganisms, biological testing, etc., can solve the problems of high price and difficult experimentation

Inactive Publication Date: 2013-04-17
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The enzyme-linked immunosorbent immunoassay (ELISA) kits sold on the market for the detection of H-FABP mostly use H-FABP antibodies imported f

Method used

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  • Hybridoma cell strain and monoclonal antibody of anti-heart-type fatty acid-binding protein generated by same
  • Hybridoma cell strain and monoclonal antibody of anti-heart-type fatty acid-binding protein generated by same
  • Hybridoma cell strain and monoclonal antibody of anti-heart-type fatty acid-binding protein generated by same

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Experimental program
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Embodiment 1

[0040] The preparation of embodiment 1.H-FABP recombinant protein

[0041] 1) Obtain the whole gene of H-FABP coding sequence

[0042] According to the H-FABP gene sequence (NM_004102) provided by GeneBank, use the software to compare with the liver type, small intestine type, brain type, silver snow disease-related type and adipocyte type fatty acid binding protein gene sequence, observe the homology, and determine the heart type The pseudo-cloned sequence of the fatty acid binding protein gene (SEQ ID NO: 1):

[0043] ATGGTGGACG CTTTCCTGGG CACCTGGAAG CTAGTGGACA GCAAGAATTT CGATGACTAC 60

[0044] ATGAAGTCAC TCGGTGTGGG TTTTGCTACC AGGCAGGTGG CCAGCATGAC CAAGCCTACC 120

[0045] ACAATCATCG AAAAGAATGG GGACATTCTC ACCCTAAAAA CACACAGCAC CTTCAAGAAC 180

[0046] ACAGAGATCA GCTTTAAGTT GGGGGTGGAG TTCGATGAGA CAACAGCAGA TGACAGGAAG 240

[0047] GTCAAGTCCA TTGTGACACT GGATGGAGGG AAACTTGTTC ACCTGCAGAA ATGGGACGGG 300

[0048] CAAGAGACCA CACTTGTGCG GGAGCTAATT GATGGAAAAC TCATCCTGAC ACTCACCCAC ...

Embodiment 2

[0076] Example 2. Bioinformatics analysis and determination of detection targets

[0077] 1) Find the amino acid sequence of H-FABP protein from GENEBANK, which consists of 133 amino acid residues.

[0078] 2) Analyze the potential B cell epitope region in the H-FABP protein online, comprehensively consider the secondary structure, antigenicity, hydrophilicity, accessibility and flexibility of the protein, analyze and score each segment, and select the one with the highest score A region is used as a candidate B cell epitope region, which is 32aa-47aa (peptide 1, the amino acid sequence is represented by SEQ ID NO: 2: QVASMTKPTTIIEKNG)

Embodiment 3

[0079] Example 3. Preparation of anti-H-FABP monoclonal antibody

[0080] 1) Synthesis and cross-linking of epitope peptides

[0081] H-FABP B-cell epitope peptide 1 was chemically synthesized, purified and cross-linked with BSA to obtain H-FABP-peptide 1-BSA

[0082] 2) Preparation of H-FABP monoclonal antibody

[0083] The cross-linked product of the synthesized H-FABP epitope peptide and BSA and the recombinant protein H-FABP were emulsified with an equal amount of Freund's complete adjuvant, and the antigen emulsification was carried out by double syringes. After the emulsification was completed, 5 Balb / c mice aged about 8 weeks were given basic immunization (100 μg / ml) by multi-point subcutaneous injection in limbs and intraperitoneal injection. Two weeks later, emulsify the H-FABP whole antigen with the same amount of Freund's incomplete adjuvant, and immunize 5 Balb / c mice by multi-point subcutaneous injection in limbs and intraperitoneal injection (100 μg / ml); Two w...

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Abstract

The invention discloses hybridoma cell strains 1-F10 (preservation number: C20122139 and preservation unit: Wuhan University) and 3-H5 (preservation number: C20122140 and preservation unit: Wuhan University), and a monoclonal antibody of an anti-heart-type fatty acid-binding protein (H-FABP) generated by the same. The hybridoma cell strains 1-F10 and 3-H5 are prepared through predicating epitopes in the H-FABP and corresponding antibodies for detection can be secreted; and the subtypes of the monoclonal antibodies of the anti-H-FABP, which are secreted by the hybridoma cell strains 1-F10 and 3-H5, are IgG2a and IgG2b. The antibodies generated by the cell strains 3-H5 and 1-F10 are respectively used as a capturing antibody and a detection antibody, so as to successfully detect the difference of the H-FABP in blood serums of healthy people and myocardial infarction patients, and develop a diagnostic reagent which has good specificity and high accuracy, and can diagnose acute myocardial infarction in early stage.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to a hybridoma cell line and a monoclonal antibody against human heart-type fatty acid binding protein produced therefrom. Background technique [0002] Cardiovascular disease, especially coronary heart disease, seriously threatens human life and health. Acute myocardial infarction (AMI) is the main type of cardiovascular disease, with a sudden onset and a mortality rate of about 30% in the acute phase. AMI is a common acute and critical disease of coronary heart disease. Early diagnosis and reperfusion therapy are the key to reducing mortality and improving prognosis. The detection of dynamic changes in electrocardiogram and myocardial enzymes is an important auxiliary examination method in clinical practice. However, for AMI patients with non-specific changes in electrocardiogram, myocardial markers become an important condition for diagnosis. At present, the rese...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/18G01N33/68G01N33/577
Inventor 李淑慧胡川闽易维京韩起
Owner ARMY MEDICAL UNIV
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