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Anti-human IgM monoclonal antibody, hybridoma cell strain and application thereof

A hybridoma cell line and monoclonal antibody technology, applied in biochemical equipment and methods, material inspection products, instruments, etc., can solve the problem of systematic evaluation of cross-reactivity, stability, detection sensitivity and specificity, antibody It is not clear whether it is suitable for the preparation of in vitro diagnostic reagents, and the specificity of polyclonal antibodies is low, so as to achieve the effects of excellent long-term and thermal stability, good immune effect, loose storage conditions and operating conditions

Active Publication Date: 2018-07-27
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 2010026758 A1 discloses an anti-human IgM monoclonal antibody. This patent aims to solve the problem of low specificity of polyclonal antibodies, and focuses on 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 in in vitro diagnostic reagents (eg, antibody potency). valence, 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, hybridoma cell strain and application thereof
  • Anti-human IgM monoclonal antibody, hybridoma cell strain and application thereof
  • Anti-human IgM monoclonal antibody, hybridoma cell strain and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0065] The immunization of embodiment 1 mice

[0066] Human IgM extracted from blood (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 A 1ml syringe was emulsified into an oily emulsion until the oily emulsion dripped into water did not disperse and the emulsification could be stopped. The emulsion was subcutaneously administered to BALB / c mice in the armpits of limbs at a dose of 120 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 volume and then emulsified. After immunization once, the tail blood was collected before each immunization, the serum was separated, and the titer was determined by indirect ELISA method. After 5 times of immunization, ...

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 killed by taking 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 aseptic operation in the ultra-clean bench to expose the peritoneum. Inject 4ml of RPMI1640 basal culture solution into the peritoneal cavity with a syringe, then take it out with a dropper, then add 6ml of RPMI1640 basal culture solution to wash repeatedly, recover the washing solution, centrifuge at 1000rpm for 5 minutes to leave the precipitate, and use RPMI1640 culture solution that has been added with 20% newborn bovine serum Resuspend, adjust cell concentration to 2.7×10 5 cells / ml, added to 96-well plate, 100 μl / well, 37°C, 5% CO 2 to cultivate.

[0070] 2-2 Preparat...

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) intraperitoneally into each mouse, and inject 1.2×10 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. Ascitic fluid was saturated and precipitated with ammonium sulfate, and then purified with protein A affinity chromatography column. The purity of this antibody (referred to as IgM-Ab3) was detected by SDS-PAGE to be greater than 90%, and the electrophoresis results were as follows: figure 1...

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Abstract

The invention relates to a hybridoma cell strain and a monoclonal antibody secreted by the hybridoma cell strain. The antibody can be specifically bound with human IgM, and can be used for in-vitro detection of the human IgM, and especially suitable for the early diagnosis of cytomegalovirus infection. The invention further relates to a kit containing the hybridoma cell strains or the monoclonal antibodies.

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 Applications(China)
IPC IPC(8): C12N5/20C07K16/42G01N33/577G01N33/68
CPCC07K16/4283G01N33/577G01N33/6854G01N33/6893G01N2800/26
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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