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Hybridoma cell line able to secrete anti-bovine immunoglobulin IgG monoclonal antibody and application thereof

A hybridoma cell line and monoclonal antibody technology, which is applied in the field of bovine immunoglobulin IgG immunoassay, can solve the problems of complex operation, cumbersome processing, and difficulty in rapid detection, and achieve high sensitivity and good specificity

Inactive Publication Date: 2014-10-15
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the measurement error of the diffusion diameter, the results are quite different, the accuracy and sensitivity are low, and at the same time, it is labor intensive and time-consuming.
HPLC analysis is accurate and repeatable, but it requires complex instruments and equipment, the sample pretreatment process is cumbersome, the detection process takes a long time, and the requirements for the experimental environment are high, so it is difficult to achieve rapid detection
Enzyme-linked immunoassay has high sensitivity and specificity, and can measure dozens or even hundreds of samples at the same time. It is widely used in foreign research, but the method is complicated, time-consuming and expensive.

Method used

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  • Hybridoma cell line able to secrete anti-bovine immunoglobulin IgG monoclonal antibody and application thereof
  • Hybridoma cell line able to secrete anti-bovine immunoglobulin IgG monoclonal antibody and application thereof
  • Hybridoma cell line able to secrete anti-bovine immunoglobulin IgG monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: the screening of hybridoma cell line 9C6

[0017] 1. Animal immunization

[0018] Four 6-week-old female BALB / c mice were selected and immunized three times with bovine immunoglobulin IgG (manufactured by Sigma-Aldrich, product number I5506), 100 μg / mouse for the first time, with the same amount of complete Freund's adjuvant, fully emulsified, Intraperitoneal injection; 2 weeks later for the second immunization, 100 μg / monkey, the same amount of incomplete Freund's adjuvant, fully emulsified, intraperitoneal injection; 2 weeks later for the third immunization, the operation is the same as the second immunization. Serum titer was measured, and the one with the highest serum titer was selected for booster immunization 3 days before fusion, 100 μg / rat, without adjuvant, and injected intraperitoneally.

[0019] 2. Cell Fusion

[0020] Three days after the booster immunization, mouse splenocytes were collected according to conventional methods, and fused with...

Embodiment 2

[0037] Embodiment 2: the preparation purification, subtype and characteristic identification of anti-bovine immunoglobulin IgG monoclonal antibody

[0038] The anti-bovine immunoglobulin IgG monoclonal antibody hybridoma cell line 9C6 obtained in Example 1 was injected into BALB / c mice treated with Freund's incomplete adjuvant in advance, and the ascites of the mice was collected, centrifuged and defatted, Antibody was purified by Protein A affinity purification method (operate according to the manual). Immediately after purification, dialyze against 0.01M pH7.2 phosphate buffer, adjust the protein concentration to 10mg / mL, and freeze at -70°C for later use;

[0039] The subtype of the anti-bovine immunoglobulin IgG monoclonal antibody secreted by the hybridoma cell line 9C6 was identified as IgG1 with a subtype identification kit.

[0040] The titer of 9C6 mouse ascites antibody measured by conventional indirect ELISA method can reach 5.12×10 5 (antigen coating concentratio...

Embodiment 3

[0041] Example 3 Preparation and Application of Immunochromatography Detection Card or Test Strip

[0042] The anti-bovine immunoglobulin IgG monoclonal antibody that embodiment 2 obtains is used for the preparation of bovine immunoglobulin IgG immunochromatographic detection card, and preparation method comprises the following steps:

[0043] (1) Preparation of antibody

[0044] The anti-bovine immunoglobulin IgG monoclonal antibody secreted by the above-mentioned cell line was selected as the labeled antibody, and the anti-bovine polyclonal antibody derived from rabbit, cow, horse, donkey, etc. was selected as the detection line (T line) coating antibody, sheep Anti-mouse polyclonal antibody was used as the quality control line (C line) to coat the antibody.

[0045] (2) Preparation of detection membrane

[0046] Prepare the T-line coating antibody and C-line coating antibody with T-line coating solution and C-line coating solution to a concentration of 0.5-2.0 mg / mL, respec...

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Abstract

The invention relates to a screening method for a hybridoma cell line able to secrete an anti-bovine immunoglobulin IgG monoclonal antibody for bovine immunoglobulin IgG detection, the hybridoma cell line, the monoclonal antibody secreted thereby and application in the field of bovine immunoglobulin IgG detection. The hybridoma cell is preserved in China Center For Type Culture Collection, Wuhan University at Luojiashan, Wuchang, Wuhan City in Hubei Province, and the preservation number is CCTCC No:2013183. The anti-bovine immunoglobulin IgG monoclonal antibody secreted by the hybridoma cell has the advantages of strong specificity, large affinity and high titer and the like, and can be widely applied in the detection reagent or detection equipment field of bovine immunoglobulin IgG. A bovine immunoglobulin IgG rapid detection card built based on the immunochromatography principle can be used for detection of bovine immunoglobulin IgG in colostrum and its products, bovine blood and other samples, and has significant advantages compared with conventional detection methods in the aspects of specificity, sensitivity and detection efficiency, etc.

Description

technical field [0001] The invention relates to the field of bovine immunoglobulin IgG immunodetection, in particular to a hybridoma cell line 9C6 capable of secreting anti-bovine immunoglobulin IgG monoclonal antibody and an application thereof. Background technique [0002] The bovine immunoglobulin system is similar to other mammals in terms of physical and chemical properties and classification and naming, mainly including IgG, IgA and IgM. IgG is divided into two subclasses, IgG1 and IgG2, of which IgG1 is the main component. IgG molecule is composed of 4 peptide chains, 2 long chains are called heavy chain (heavy chain, H), composed of about 440 amino acid residues, molecular weight is about 50-70kD; 2 short chains are called light chain (light chain) , L), consisting of about 220 amino acids, with a molecular weight of about 22.5kD. The four peptide chains are linked together by interchain disulfide bonds (-S·S-). Bovine immunoglobulin is widely distributed in bovi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/18G01N33/577C12R1/91
Inventor 岳喜庆王丽威林晶武俊瑞陈娜王忠霞乌日娜姜楠丛敏艾志松邢宇李桂宇李云海
Owner SHENYANG AGRI UNIV
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