Anti-beta-conglycinin monoclonal antibody and application thereof

A conglycinin and monoclonal antibody technology, applied in the field of monoclonal antibodies, can solve problems such as growth performance decline, death, immune dysfunction, etc., and achieve the effect of low detection limit and wide detection range

Active Publication Date: 2013-10-02
北京龙科方舟生物工程技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Adding soybean meal as a protein source in young animal feed will lead to a series of adverse reactions such as diarrhea, reduced gr

Method used

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  • Anti-beta-conglycinin monoclonal antibody and application thereof
  • Anti-beta-conglycinin monoclonal antibody and application thereof
  • Anti-beta-conglycinin monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1, the acquisition of hybridoma cell line and the preparation of anti-β-conglycinin monoclonal antibody

[0049] 1. Obtaining of hybridoma cell lines

[0050] 1. Animal immunity

[0051] Dissolve β-conglycinin (ie, the immunogen) in 0.5ml of normal saline, add an equal volume of adjuvant to make an emulsifier, and immunize female BALB / c mice (body weight 18-22g). Serum titers were measured 7-10 days after the third immunization. See Table 1 for the immunization steps.

[0052] Table 1. Animal immunization steps

[0053]

[0054] 2. Cell fusion and cloning

[0055] Blood was collected from the tail of the mouse after the third immunization in step 1 to determine the antibody titer, and the mouse with the highest serum titer was selected, and the splenocytes were taken and fused with SP2 / 0 myeloma cells; the cells secreting monoclonal antibodies were screened by limiting dilution method Hybridoma cell line; the stable secreted anti-β-conglycinin monoclo...

Embodiment 2

[0090]Example 2, Competitive ELISA method for detecting β-conglycinin and its special kit

[0091] 1. Checkerboard method to screen the optimal antigen coating concentration and antibody working concentration

[0092] 1. Coating antigen: take β-conglycinin, prepare a solution with a concentration of 1000ng / mL with coating buffer, and then perform 2-fold serial dilution to obtain a concentration of β-conglycinin in order of 1000ng / mL , 500ng / mL, 250ng / mL, 125ng / mL, 62.5ng / mL, 31.2ng / mL, 15.6ng / mL, 7.8ng / mL, 3.9ng / mL, 1.95ng / mL, 0.98ng / mL, 0.49ng 12 kinds of coating solutions per mL; add these 12 kinds of coating solutions in the amount of 0.1ml / well to the columns 1-12 of the microtiter plate in turn, one coating solution for each column, and coat at 4°C for 16h ; Discard liquid.

[0093] 2. Blocking: Add 200 μL / well of blocking solution, block in a 37°C wet box for 1 hour; discard the liquid, wash with washing solution 3 times for 30 seconds each time, and pat dry.

[0094]...

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Abstract

The invention discloses an anti-beta-conglycinin monoclonal antibody and an application of the monoclonal antibody. The monoclonal antibody is secreted by a hybridoma cell strain 2C4CGMCC No.7109; experiments prove that the affinity constant of the beta-conglycinin monoclonal antibody reaches 6.2*10<11>L/mol, the beta-conglycinin monoclonal antibody is applied to detecting beta-conglycinin by means of a competitive ELISA (Enzyme Linked Immunosorbent Assay) for antibody detection, and the detection limit is 0.1ng/ml-8.1ng/mol. The beta-conglycinin monoclonal antibody, a detection method using the monoclonal antibody and a kit with the monoclonal antibody provided by the invention have the advantages of low detection limit, wide detection range, high efficiency, and accuracy, and can be widely applied to the qualitative and quantitative detection of the beta-conglycinin in soybean and a soybean product.

Description

technical field [0001] The invention relates to a monoclonal antibody against β-conglycinin and its application. Background technique [0002] Soybean is rich in protein, and because of its appropriate proportion of essential amino acids (such as lysine, threonine and tryptophan), it is a high-quality vegetable protein source for livestock and poultry. However, the allergic reaction of animals caused by the soybean antigen protein contained in soybean has attracted more and more attention. Adding soybean meal as a protein source in young animal feed will lead to a series of adverse reactions such as diarrhea, reduced growth performance, intestinal mucosal cell proliferation, and immune dysfunction in piglets and calves, and even death in severe cases. [0003] β-conglycinin is the main allergenic protein in soybean, accounting for 10.0%-12.7% of the total protein of soybean seeds, accounting for 30.0% of the total globulin, and its molecular weight is 140-180kDa, which is a...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/16G01N33/68G01N33/577B01J20/24B01J20/281C07K14/415C07K1/14C07K1/22
Inventor 谯仕彦付巍魏寒冰李悦张学何涛
Owner 北京龙科方舟生物工程技术有限公司
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