Anti-beta-interferon monoclonal antibody and its application

A monoclonal antibody, interferon technology, applied in the direction of anti-cytokine/lymphokine/interferon immunoglobulin, anti-animal/human immunoglobulin, biochemical equipment and methods, etc. Cloning antibodies, affecting drug efficacy, etc.

Inactive Publication Date: 2013-01-30
SHANGHAI INST OF IMMUNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In fact, the antibodies produced after the use of protein-type biopharmaceuticals are binding antibodies, which can affect the efficacy of biological drugs, or have no or little effect, and when the binding antibody has a functional part with biological drugs When binding efficacy, it is called neutralizing antibody. Once the neutralizing antibody is produced, it will greatly affect the efficacy of biological drugs. Therefore, a monoclonal antibody with neutralizing effect is prepared to form a competitive ELISA method for detecting neutralizing antibodies in patients. It has practical significance for judging the efficacy of protein gene drugs in vivo, but there is no such monoclonal antibody at present
[0006] At present, there are no ELISA detection kits targeting the IFN-β functional domain in the market, and the antibodies used in the few ELISA detection kits usually target the entire IFN-β molecule

Method used

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  • Anti-beta-interferon monoclonal antibody and its application
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  • Anti-beta-interferon monoclonal antibody and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0060] Example 1 Titer determination of anti-IFN-β monoclonal antibody

[0061] 1. Preparation and purification of ascites-type anti-IFN-β monoclonal antibody

[0062] 1. Preparation of ascites type monoclonal antibody

[0063] Take the bred female Babl / c mice and inject 0.5ml of medical liquid paraffin oil into the abdominal cavity. After 7-10 days, inject the four strains of hybridoma cells that have been expanded and cultured into the abdominal cavity of the Babl / c mice respectively. The cell injection concentration is 2×10 7 cell / mouse, each cell line injected 3 mice. Collect ascites after 10-14 days, the output of ascites is 4-10mL / monkey, centrifuge at 2000rpm for 5min, take the supernatant for purification and identification

[0064] 2. Purification of monoclonal antibodies

[0065] The ascites-type monoclonal antibody was roughly extracted by 50% ammonium sulfate salting-out method, and the ascites-type monoclonal antibody was refined by using a Protein A Sepharose...

Embodiment 2I

[0070] Identification of embodiment 2IFN monoclonal antibody

[0071]1. The results of monoclonal antibody screening and subclass identification and titer determination

[0072] Roche’s Ig Subclass Determination Kit was used to properly dilute the tested sample according to the requirements of the kit, and the monoclonal antibody Ig class contained in the culture supernatant of the above four highly positive hybridoma cells was detected, and the identification results were IgG 2 , IgG 2 , IgG 1 and IgG 3 , the light chains of which are all κ type; the ascites type monoclonal antibody titers prepared by 4 hybridomas were detected by indirect ELISA method, and the ELISA coating antigen was IFN-β (AVONEX TM ).

[0073] The steps for detecting the titer of ascitic fluid-type monoclonal antibody by indirect ELISA are as follows:

[0074] 1) Dilute the ascites-type monoclonal antibody sequentially with dilution buffer to a volume ratio of 1:100, 1:1000, 1:10000, 1:50000, 1:1000...

Embodiment 3I

[0093] The preliminary application of embodiment 3IFN monoclonal antibody

[0094] IFN Monoclonal Antibody Used to Purify IFN Biologics Expressed by Yeast Fermentation

[0095] The rhIFN-β expression vector 1pPICZaIFNS1-X33 constructed by the Shanghai Institute of Immunology was used to prepare recombinant human IFN-β through a 15L fermenter of B.BRAUN Company, and the fermented liquid containing rhIFN-β was pre-treated with 25% ammonium sulfate and 55% sulfuric acid After ammonium salting out, the rhIFN-β crude product was purified by Sepharose 4B activated by Ab-BI1 coupling, and compared with the purification method of blue spectrum chromatography (Blue S6FF).

[0096] (1) Immunoaffinity chromatography: Connect Ab-BI1 according to the instructions of activated Sepharose 4B, fill it into a chromatographic column with an inner diameter of 2.5 cm and a column length of 10 cm. After the column is equilibrated, take the crude rhIFN-β solution through ammonium sulfate Sample loa...

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Abstract

An anti-beta-interferon monoclonal antibody is generated by a hybridoma cell strain having a preservation number of CCTCC NO:C201121. A monoclonal antibody specifically combined with the beta-interferon having a sequence represented by SEQ ID No1 has a neutrality effect on the beta-interferon. The anti-beta-interferon monoclonal antibody can be used for the affinity chromatography purification and ELISA detection of the beta-interferon.

Description

technical field [0001] The present invention relates to a monoclonal antibody, in particular to an anti-beta-interferon monoclonal antibody, which can combine with the functional domain of beta-interferon and has a neutralizing effect on beta-interferon, and its purification and in vitro application in detection. Background technique [0002] β-interferon (interferon-β, IFN-β) is a type I membrane glycoprotein with a molecular weight of 20,500 Dalton, containing 166 amino acids. The isoelectric point of IFN-β prepared by genetic engineering recombination is pH8.9. IFN-β is sensitive to protease, and it can remain stable even after being treated at 56°C for 10 minutes at pH 2. IFN-β not only acts as a broad-spectrum antiviral and immunomodulator, but also has antitumor cell growth effects. Its broad-spectrum anti-virus is mainly reflected in: anti-DNA virus, pox virus, herpes virus, human papilloma virus, cytomegalovirus, adenovirus, RNA virus, orthomyxovirus and retroviru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/24C07K1/22C12N5/20G01N33/577G01N33/543C12R1/91
Inventor 余奇文张冬青张继英李宁丽马安伦席波聂红沈伯华沈天伟柏峻王利
Owner SHANGHAI INST OF IMMUNOLOGY
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