Hybridoma cell strain generating anti-human NGAL specific monoclonal antibody, monoclonal antibody generated by same and application

A hybridoma cell line, monoclonal antibody technology, applied in the direction of anti-animal/human immunoglobulin, microorganism-based methods, microorganisms, etc., can solve the problems of difficult clinical application and high price

Inactive Publication Date: 2013-05-01
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 currently on the market for the detection of NGAL all use imported NGAL antib

Method used

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  • Hybridoma cell strain generating anti-human NGAL specific monoclonal antibody, monoclonal antibody generated by same and application
  • Hybridoma cell strain generating anti-human NGAL specific monoclonal antibody, monoclonal antibody generated by same and application
  • Hybridoma cell strain generating anti-human NGAL specific monoclonal antibody, monoclonal antibody generated by same and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of NGAL recombinant protein

[0039] 1. Entire gene acquisition of NGAL coding sequence

[0040] According to the NGAL gene sequence (NM_005564.3) provided by GeneBank, the pseudo-cloning sequence of the human neutrophil gelatinase-related lipocalin gene was determined, as shown in SEQ ID NO: 2:

[0041] Design NGAL full-length primers: shown in SEQ ID NOs: 3 and 4, respectively

[0042] SEQ ID NO: 3: NGAL-1: 5'-TAAGGTACCCATGCCCCTAGGTCTCCTG-3'

[0043] SEQ ID NO::4:NGAL-2:5'-GGTGGAATTTTAGCCGTCGATACACTG-3'

[0044] The primers respectively contain restriction sites: NGAL-1: Acc651; NGAL-2: EcoRI, synthesized by Invitrogen Company.

[0045] Total leukocyte RNA was extracted by Trizol Reagent method, leukocyte cDNA was obtained by reverse transcription, and human NGAL was obtained by PCR amplification. A PCR system was established, and PCR was performed at different annealing temperatures. Take 25 μL of PCR products for analysis by 1% TAE electr...

Embodiment 2

[0059] Example 2 Bioinformatics analysis and identification of immunogens

[0060] 1. Find the amino acid sequence of the NGAL protein from GENEBANK, which consists of 198 amino acid residues.

[0061] 2. Online bioinformatics glycosylation prediction analysis The 86th amino acid in NGAL protein may be glycosylated. In order to obtain the best specific monoclonal antibody that can recognize the natural protein, the full-length NGAL protein expressed in 293 cells was selected. As an immunogen, its amino acid sequence is the same as SEQ ID NO:1.

Embodiment 3

[0062] Example 3 Preparation of anti-NGAL monoclonal antibody

[0063] 1. Preparation of NGAL monoclonal antibody

[0064] The recombinant full-length NGAL protein was emulsified with an equal amount of Freund's complete adjuvant, and the antigen was emulsified by double-syringe mutual push method. After the emulsification was completed, 5 Balb / c mice of about 8 weeks of age were given basal immunization (100 μg / mouse) by subcutaneous multi-point injection and intraperitoneal injection. After 2 weeks, NGAL whole antigen was emulsified with the same amount of incomplete Freund's adjuvant, and 5 Balb / c mice were boosted by subcutaneous injection and intraperitoneal injection (100 μg / mouse); 2 Weeks later, the same method was used for an additional immunization, and 7 days later, the mice were sacrificed to remove the spleen, and the spleen cells were fused.

[0065] The fusion process is to mix spleen cells and myeloma cells (Sp2 / 0) at a ratio of 8:1, and use polyethylene gl...

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Abstract

The invention discloses a hybridoma cell strain generating an anti-human NGAL specific monoclonal antibody and a monoclonal antibody generated by the same. The hybridoma cell strain is generated in a way that after an amino acid sequence shown by SEQIDNO:1 immunizes a Balb/c rat, a spleen B lymph cell and a myeloma cell of the rat are fused for further cultivation. The antibody can be used for the immunity detection of the NGAL. Based on the invention, an immunity detection reagent for the NGAL can be developed and used for diagnosing and detecting acute kidney injury and other diseases.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to a hybridoma cell line and an anti-human neutrophil gelatinase-related lipocalin-specific monoclonal antibody produced therefrom. Background technique [0002] Kidney injury is one of the most common complications of various critical diseases in clinical practice. Although research on the pathophysiology and pathogenesis of kidney injury and clinical treatments have made great progress in recent decades, its morbidity and mortality remain high. It has been clear that early treatment is very important for the reversal of renal injury, and early diagnosis is the key to guiding clinical timely treatment. At present, the most commonly used clinical biochemical markers of renal injury are blood urea nitrogen, blood creatinine, and blood cystatin C. Urea is an important terminal small molecule metabolite of protein metabolism in the human body, and its concentration is ...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/18G01N33/577C12R1/91
Inventor 陈安胡川闽何莹易维京
Owner ARMY MEDICAL UNIV
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