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Macaque IFN-gamma (interferon-gamma) monoclonal antibody hybridoma as well as preparation method and application thereof

A monoclonal antibody, hybridoma cell technology, applied in biochemical equipment and methods, material testing products, instruments, etc., can solve the problems of expensive and difficult to buy kits

Active Publication Date: 2015-04-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this kit is too expensive, and it costs about RMB 200 per sample to test, and it is difficult to buy

Method used

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  • Macaque IFN-gamma (interferon-gamma) monoclonal antibody hybridoma as well as preparation method and application thereof
  • Macaque IFN-gamma (interferon-gamma) monoclonal antibody hybridoma as well as preparation method and application thereof
  • Macaque IFN-gamma (interferon-gamma) monoclonal antibody hybridoma as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Establishment of cynomolgus IFN-γ monoclonal antibody.

[0047] 1. Construction of the expression plasmid pET-30a-MmIFN-γ of rhesus monkey IFN-γ:

[0048] 1. Acquisition and amplification of Rhesus monkey IFN-γcDNA:

[0049] Aseptically take the whole blood of local rhesus monkeys, anticoagulated with heparin, take 10mL whole blood, add Con A 60μg / mL at 37℃ and 5% CO 2 Cultivate 16h in the environment. Centrifuge at 3000g for 5min, discard the supernatant, add red blood cell lysate in a volume of 1:3, centrifuge at 5000g for 5min, repeat 2 to 3 times, and finally wash the white blood cells with PBS.

[0050] According to the method provided by RNA-SoIV Reagent, the total RNA of rhesus monkey peripheral blood leukocytes was extracted as a template for RT-PCR. According to the macaque IFN-γ (MmIFN-γ) mRNA sequence published on GenBank (GenBank accession No: AY376145.1), a pair of primers was designed for the sequence after the signal peptide was removed: upstream prim...

Embodiment 2

[0224] Example 2. Establishment of double antibody sandwich enzyme-linked immunosorbent assay (ELISA)

[0225] An application in the ELISA detection method of macaque IFN-γ, the steps are:

[0226] 1. Purification of MmIFN-γ polyclonal antibody:

[0227] Using the obtained purified MmIFN-γ antigen as an immunogen, 5 rabbits were immunized, and the rabbit serum with the highest titer was taken and purified by the saturated ammonium sulfate method.

[0228] 1) Collect blood from the heart, collect hyperimmune serum, and place it at 37°C for 1 hour to inactivate complement. Allow the serum to separate out naturally at 4°C overnight.

[0229] 2) Centrifuge the sample to be extracted at 13000r / min for 30min at 4°C or room temperature, and collect the supernatant.

[0230] 3) Take about 20 mL of the supernatant and mix it with an equal volume of normal saline, slowly add 40 mL of saturated ammonium sulfate dropwise with stirring, and react for 3 hours or overnight at 4°C to fully precipitate ...

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Abstract

The invention discloses a macaque IFN-gamma (interferon-gamma) monoclonal antibody hybridoma as well as a preparation method and application thereof. According to the invention, the preparation method comprises the following steps: performing RNA extract on ConA irritated macaque whole blood hemocyte, carrying out RT-PCR (reverse transcription-polymerase chain reaction), thus obtaining total cDNA, and amplifying total cDNA to obtain macaque IFN-gamma genes; obtaining IFN-gamma through vector in vitro expression and purification; after the purified macaque IFN-gamma is used for immunizing a mouse, separating the immunized mouse spleen B cell and using a hybridoma technique to obtain a hybridoma which effectively expresses IFN-gamma monoclonal antibody, MmIFN-gamma2F6 with CCTCC NO:C201235. The macaque IFN-gamma monoclonal antibody can be specifically bonded with the macaque IFN-gamma so as to be used for detecting macaque IFN-gamma in supernatant after macaque whole blood antigenic stimulation. The macaque IFN-gamma monoclonal antibody can be specifically bonded with the natural macaque IFN-gamma, the sensitiveness can be 125pg / ml when the macaque IFN-gamma monoclonal antibody is used for specific detection of macaque IFN-gamma sensitiveness, so that the sensitiveness required in the tuberculosis detection can be achieved.

Description

Technical field [0001] The present invention relates to the detection field of macaque IFN-γ, and more specifically to a hybridoma cell expressing a macaque IFN-γ monoclonal antibody (abbreviated as monoclonal antibody), and also to a macaque IFN-γ monoclonal antibody hybridoma cell The preparation method also relates to the application of a macaque IFN-γ monoclonal antibody cell in the detection of macaque tuberculosis. Background technique [0002] Tuberculosis is a chronic wasting infectious disease that is zoonotic. It is currently the infectious disease with the highest mortality rate, and it is also one of the major infectious diseases that are monitored worldwide. Tuberculosis is caused by the Mycobacterium tuberculosis complex, which is mainly transmitted through the respiratory system, but also through the digestive system. Mycobacterium tuberculosis and Mycobacterium bovis are the most important pathogenic bacteria. Among them, Mycobacterium bovis has a wide spectrum o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20G01N33/68G01N33/577
Inventor 郭爱珍姜勇陈颖钰张策郝丽影邹新峰陈焕春
Owner HUAZHONG AGRI UNIV