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Anti-human igm monoclonal antibody, its hybridoma cell line and application

A technology of hybridoma cell lines and monoclonal antibodies, which is applied in the fields of instruments, peptides, immunoglobulins, etc., can solve the problem of systematically evaluating cross-reactivity, stability, detection sensitivity and specificity, and whether antibodies are suitable for preparation Problems such as unclear in vitro diagnostic reagents and low specificity of polyclonal antibodies have been achieved to achieve excellent long-term and thermal stability, good immune effect, loose storage conditions and operating conditions

Active Publication Date: 2019-06-04
SICHUAN ANKERUI NEW MATERIAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In recent years, there have been some studies on anti-human IgM monoclonal antibodies. For example, a patent document WO 2010026758A1 discloses an anti-human IgM monoclonal antibody. This patent aims to solve the problem of low specificity of polyclonal antibodies, and focuses on the The nature of the monoclonal antibody-induced agglutination between human IgM and its effect of inhibiting non-specific reactions were verified, but no attention was paid to the systematic evaluation of the anti-human IgM monoclonal antibody when it was used as an in vitro diagnostic reagent (eg, antibody titer , cross-reactivity, stability, detection sensitivity and specificity, etc.), therefore, whether the antibody is suitable for the preparation of in vitro diagnostic reagents is not clear
[0005] Another example is the anti-human IgM monoclonal antibody of Xiamen Bosheng Biotechnology Co., Ltd., which is a common commercial anti-human IgM monoclonal antibody, but its stability is not good, so it has shortcomings when used in immunodiagnostic antibodies

Method used

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  • Anti-human igm monoclonal antibody, its hybridoma cell line and application
  • Anti-human igm monoclonal antibody, its hybridoma cell line and application
  • Anti-human igm monoclonal antibody, its hybridoma cell line and application

Examples

Experimental program
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Effect test

Embodiment 1

[0065] The immunization of embodiment 1 mice

[0066] Human IgM extracted from blood sources (Sichuan Mike Bio-New Material Technology Co., Ltd., batch number 150127) was diluted to 3.0 mg / ml with normal saline, mixed with an equal volume of Freund's complete adjuvant (Sigma Company, product number SLBF-9338V), and used The 1ml syringe is emulsified into an oily emulsion until the oily emulsion dripped into the water does not disperse and the emulsification can be stopped. The emulsion is subcutaneously administered to BALB / c mice in the armpits of limbs at a dose of 160 μl / only (Chengdu Dashuo Experimental Animal Center, 6 weeks) 21 days after the first immunization, human IgM was mixed with incomplete Freund's adjuvant (Sigma Company, product number SLBM9367V) in equal volumes and then emulsified. The immunization dose was 80 μl per mouse, and boosted every other week thereafter. After immunization once, the tail blood was collected before each immunization, the serum was se...

Embodiment 2

[0067] The preparation of embodiment 2 hybridoma cell lines

[0068] 2-1 Preparation of feeder cells

[0069] Peritoneal macrophages of normal 10-week-old BALB / c mice were used as feeder cells. One day before the fusion, BALB / c was sacrificed by pulling the blood from the eyes, soaking in 0.1% bromogeramine for 1 minute, then soaking in 75% alcohol for 1 minute, and cutting the abdominal skin with scissors under the aseptic operation in the ultra-clean bench to expose the peritoneum. Inject 6ml of RPMI1640 basic culture solution into the abdominal cavity with a syringe, then take it out with a dropper, then add 4ml of RPMI1640 basic culture solution to wash repeatedly, recover the washing solution, centrifuge at 1000rpm for 5 minutes to leave the precipitate, and use RPMI1640 culture solution with 20% newborn bovine serum Resuspend, adjust cell concentration to 3.2×10 5 cells / ml, added to 96-well plate, 100 μl / well, 37°C, 5% CO 2 nourish.

[0070] 2-2 Preparation of immune...

Embodiment 3

[0080] The preparation of embodiment 3 monoclonal antibody

[0081] Select 6-8 weeks healthy BALB / c mice, inject 0.5ml liquid paraffin (Tianjin Kemiou) into each mouse intraperitoneally, and inject 1.6×10 6 a hybridoma cell. Ascites can be produced 7 to 10 days after inoculation of the cells. Closely observe the health status and signs of ascites of the animals. When ascites is as large as possible, and before the mice are about to die, the mice are killed, and the ascites is sucked into the test tube with a dropper. One mouse 1 ~ 5ml ascites can be obtained. The collected ascites was centrifuged to obtain the supernatant, and a small sample was taken and stored in a -20°C refrigerator. The ascitic fluid was saturated and precipitated with ammonium sulfate, and then purified with a protein A affinity chromatography column. The purity of this antibody (referred to as IgM-Ab5) was detected by SDS-PAGE to be greater than 90%, and the electrophoresis results were as follows: fi...

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Abstract

The invention relates to a hybridoma cell strain and a secreted monoclonal antibody thereof; the antibody can be specifically combined with a human IgM, and can be used for in vitro detection of the human IgM, and particularly suitable for early diagnosis of rubella virus infection. The invention further relates to a kit comprising the hybridoma cell strain or the monoclonal antibody.

Description

technical field [0001] The present invention relates to a monoclonal antibody, in particular to an anti-human IgM monoclonal antibody. Background technique [0002] Immunodiagnostic reagents are one of the main types of in vitro diagnostic reagents. It utilizes the specific reaction of the combination of antigen and antibody to perform qualitative or quantitative diagnosis. Whether it is technology or market, this type of reagent is the fastest growing among all diagnostic reagent products at present. [0003] After pathogenic microorganisms infect the human body, IgM antibodies are first produced during the stimulation-induced humoral immune response. Early diagnosis of infectious diseases is the prerequisite for early and effective treatment, especially for infectious diseases with acute onset and dangerous conditions. , early diagnosis is particularly important, because the first antibody that appears in the body after infection is IgM antibody, so the detection of speci...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/42G01N33/68G01N33/577G01N33/569C12R1/91
CPCC07K16/4283G01N33/56983G01N33/577G01N33/6854
Inventor 黄家菊王磊舒川李岚敏何涛龙腾镶
Owner SICHUAN ANKERUI NEW MATERIAL TECH CO LTD
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