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Anti-human IgG monoclonal antibody and hybridoma cell strain and application thereof

A hybridoma cell line and monoclonal antibody technology, applied in biochemical equipment and methods, instruments, microorganisms, etc., can solve problems such as sensitivity and specificity to be improved, host cell protein contamination, complex antigen components, etc., to achieve optimal Immunity effect, good specific binding ability, excellent long-term and thermal stability effects

Active Publication Date: 2018-12-25
SICHUAN ANKERUI NEW MATERIAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, although the detection of HCMV IgG antibody can only represent past infection and latent HCMV in the body, and is not an indication of active viral infection, it still has market demand
Most of the currently commercially available reagents for detecting HCMV IgG antibodies use HCMV whole virus lysate as the coated antigen, which has complex antigenic components and the possibility of potential host cell protein contamination, as well as the possibility of combining with antibodies against other viruses of the Herpesviridae family to generate false positive signals. Moreover, the production process of the reagent is cumbersome due to the need to culture the virus
A small number of HCMV IgG antibody detection reagents use HCMV recombinant protein as the coated antigen. This kind of reagent is economical and simple to make, but the stability of the reagent is poor, and the sensitivity and specificity need to be improved. The imported reagents are too expensive

Method used

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  • Anti-human IgG monoclonal antibody and hybridoma cell strain and application thereof
  • Anti-human IgG monoclonal antibody and hybridoma cell strain and application thereof
  • Anti-human IgG monoclonal antibody and hybridoma cell strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] The immunization of embodiment 1 mice

[0062] Human blood-derived IgG antigen (Sichuan Mike Bio-New Materials Technology Co., Ltd., batch number 031624) was diluted to 2.0 mg / ml with normal saline, and mixed with an equal volume of Freund's complete adjuvant (Sigma Company, product number SLBF-9338V) (100 μg / ml). BALB / c mice), emulsified into an oily emulsion with a 1ml syringe, until the oily emulsion dripped into the water does not disperse and then stop the emulsification, the emulsion is subcutaneously administered to the BALB / c mice with a dose of 100 μl / axillary limbs ( Chengdu Dashuo Experimental Animal Center, 4-week-old females, 4) Immunization was enhanced 14 days after the first immunization, human IgG was mixed with an equal volume of Freund's incomplete adjuvant (Sigma Company, product number SLBM9367V) (50 μg / BALB / c mice) after emulsification, the immunization dose was 50 μl / mouse, and the immunization was boosted once every other week, and the tail blo...

Embodiment 2

[0065] The preparation of embodiment 2 hybridoma cell lines

[0066] 2-1 Preparation of feeder cells

[0067] Peritoneal macrophages of normal 12-week-old BALB / c mice were used as feeder cells. One day before the fusion, BALB / c was sacrificed by taking blood from the eyes and pulling the neck, soaking in 0.1% bromogeramine for 1 minute, then soaking in 75% alcohol for 1 minute, lifting the abdominal skin from the hind abdomen with sterile scissors in an ultra-clean bench to expose the peritoneum . Wipe the peritoneum with an alcohol swab to disinfect. Inject 2ml of RPMI1640 culture solution into the peritoneal cavity with a syringe, taking care to avoid penetrating into the intestine. Fix the syringe with the right hand so that the needle remains in the abdominal cavity, and gently massage the abdomen with the alcohol cotton ball in the left hand for 1 minute, and then suck out the injected culture solution. Centrifuge at 1000r / min for 5-10 minutes, discard the supernatant...

Embodiment 3

[0080] The preparation of embodiment 3 monoclonal antibody

[0081] Select healthy BALB / c mice of 12-14 weeks, inject 0.5mL liquid paraffin (Tianjin Kemiou) intraperitoneally into each mouse, and inject 2×10 6 a hybridoma cell. Ascites can be produced 7-10 days after cell inoculation. Observe the occurrence of ascites in mice every day. If the abdomen is obviously enlarged and the skin feels tense when touched with hands, the mice can be killed by pulling the neck, and the ascites can be sucked into the test tube with a dropper. One mouse can obtain 1-5mL ascites. 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 by protein A affinity chromatography. The purity of the antibody (the antibody of the present invention is referred to as human IgG-Ab) detected by SDS-PAGE was greater than 90%.

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Abstract

The invention relates to a hybridoma cell strain and a monoclonal antibody secreted by the same. The antibody can specifically bind with human IgG. The invention further relates a kit comprising the hybridoma cell strain or the monoclonal antibody. The monoclonal antibody is good in antibody purity, repeatability, antibody valence and stability.

Description

technical field [0001] The present invention relates to a monoclonal antibody, in particular to an anti-human IgG monoclonal antibody, a hybridoma cell secreting the monoclonal antibody, and the application of the monoclonal antibody. Background technique [0002] Human cytomegalovirus (HCMV) can be transmitted through oral cavity, reproductive tract, placenta, lactation, blood transfusion or organ transplantation. The notable feature of HCMV infection in the population is the high infection rate. The positive rate of HCMV antibody in normal adults is 76.7%-95.8%. my country is also one of the countries with high prevalence of HCMV infection. [0003] HCMV infection may have no obvious symptoms for the first time, but the virus remains latent in the body for a long time. When the body's immune function is low, the latent virus begins to replicate and proliferate in large quantities, followed by active infection and various clinical symptoms. HCMV infection is one of the im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/42G01N33/577G01N33/569C12R1/91
CPCG01N33/56983G01N33/577C07K16/4216C07K2317/94C07K2317/35C07K2317/33
Inventor 舒川黄家菊李岚敏王磊
Owner SICHUAN ANKERUI NEW MATERIAL TECH CO LTD
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