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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 systematic evaluation of 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: 2020-08-11
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, 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

[0067] The immunization of embodiment 1 mice

[0068] Dilute the human IgM extracted from the blood source (Sichuan Mike Bio-New Materials Technology Co., Ltd., batch number 150127) to 1.0 mg / ml with normal saline, and mix it with the quick-immune adjuvant QuickAntibody 5w (Beijing Boaolong Immunology Technology Co., Ltd.) , mix well, and give BALB / c mice (Chengdu Dashuo Experimental Animal Center, 6-week-old female, 2 mice) with a dose of 50 μl / limb intramuscular injection, and boost immunity 21 days after the first immunization, method and dosage Same as before, after 35 days, the tail blood was collected, the serum was separated, and the titer was determined by indirect ELISA method, and the titer of all mouse serum was greater than 1:10 6 , which can be used for fusion. Three days before the fusion, human IgM was diluted to 1.0 mg / ml with normal saline, and then mixed with normal saline in equal volume for booster immunization through the tail vein, with a dose of 50 μl p...

Embodiment 2

[0069] The preparation of embodiment 2 hybridoma cell lines

[0070] 2-1 Preparation of feeder cells

[0071] 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 4.5ml of RPMI1640 basal culture solution into the peritoneal cavity with a syringe, then take it out with a dropper, then add 5.5ml RPMI1640 basal culture solution to wash repeatedly, recover the washing solution, centrifuge at 1000rpm for 5 minutes to leave the precipitate, and use RPMI1640 that has added 20% newborn bovine serum The culture medium was resuspended, and the cell concentration was adjusted to 3.2×10 5 cells / ml, added to 96-well plate, 100 μl / well, 37°C, 5% CO 2 to cultivate.

[0072]...

Embodiment 3

[0082] The preparation of embodiment 3 monoclonal antibody

[0083] Choose healthy BALB / c mice of 6-8 weeks, inject 0.5ml liquid paraffin (Tianjin Kemiou) into each mouse intraperitoneally, and inject 1.1×10 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. After the ascitic fluid was saturated and precipitated with ammonium sulfate, it was purified with a protein A affinity chromatography column. The purity of the antibody detected by SDS-PAGE was greater than 90% (denoted as IgM-Ab10). 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 realize the specific combination with the human IgM, can be used forhuman IgM in-vitro detection, and is particularly suitable for being used for early diagnosis of hepatitis B virus. The invention also comprises 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): C12N5/20C07K16/42G01N33/68G01N33/569C12R1/91
CPCC07K16/4283G01N33/56983G01N33/686
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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