Double antibody sandwich method for quantitatively detecting cow milk beta-lactoglobulin

A double-antibody sandwich, lactoglobulin technology, applied in the field of immunoassay, can solve the problems of time-consuming, labor-intensive, detection limitations of incorporating foreign proteins, etc., and achieve a good linear effect.

Inactive Publication Date: 2015-09-23
SHENZHEN UNIV +1
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Problems solved by technology

[0011] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a double-antibody sandwich method for the quantitative detection of milk β-...

Method used

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  • Double antibody sandwich method for quantitatively detecting cow milk beta-lactoglobulin
  • Double antibody sandwich method for quantitatively detecting cow milk beta-lactoglobulin

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Embodiment 1

[0038] 1. Animal immunity

[0039] Take 10 BALB / c mice aged 4-6 weeks, and prepare 1 mL of 0.5 mg·mL -1 The natural βLG protein of bovine milk was fully mixed and emulsified with an equal volume of Freund's complete adjuvant, and injected subcutaneously at three points on the back of the mouse, with an immunization dose of 30 μg for each mouse; after the 14th day, an equal amount of Freund's The antigen emulsified with complete adjuvant was injected subcutaneously in mice at multiple points, and the dose was unchanged; after the 21st day, the third routine immunization was carried out. 30 days later (3 days before the fusion with mouse myeloma NS-1 cells), the last injection of natural antigen was done through the tail vein of the mouse, the dose was halved, and a booster immunization was given once.

[0040] 2. Establishment of indirect ELISA method

[0041] Using natural milk βLG as the coating antigen, by the square array method (longitudinal coating with antigen, accor...

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Abstract

The invention discloses a double antibody sandwich method for quantitatively detecting cow milk beta-lactoglobulin. The method includes the steps that an animal is immunized and hybridoma cells are obtained; by means of the hybridoma cells, monoclonal antibodies are prepared; the epitopes of the monoclonal antibodies are identified, monoclonal antibodies with the known epitopes are obtained, two monoclonal antibodies with different epitopes are selected, one of the two monoclonal antibodies and biotin are reacted, and a labeled biotin antibody and an unlabeled biotin antibody are obtained; the unlabeled biotin antibody serves as a coated antibody, the labeled biotin antibody serves as a detection antibody for antibody pairing, and then the cow milk beta-lactoglobulin is quantitatively detected according to antibody pairing data and the sandwich method. On the basis of the principle of antigen-antibody height specific recognition, the probability that other protein is added for adulteration is avoided. By means of the double antibody sandwich method, the content of the betaLG can be efficiently and quantitatively detected. The double antibody sandwich method is high in sensitivity, good in stability and low in cost.

Description

technical field [0001] The invention relates to the technical field of immune analysis, in particular to a double-antibody sandwich method for quantitatively detecting bovine milk β-lactoglobulin. Background technique [0002] In China, in recent years, as people have become more and more aware of the importance of milk to human health, milk products have become a daily necessity for the people, and the market demand for milk products has also continued to grow. Although the base of the total raw milk production is more than 15.9603 million tons and continues to increase at a rate of more than 10% every year, the dairy industry is still in a state of insufficient milk sources. Driven by huge economic interests, the adulteration of dairy products is also increasing. It's getting serious. Milk adulteration mainly includes: First, synthetic milk is adulterated in raw milk. Synthetic milk is made by mixing urea, cooking oil, detergent, caustic soda, sugar, salt, and skim milk p...

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Application Information

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IPC IPC(8): G01N33/68G01N33/577
CPCG01N33/6803G01N33/577G01N2333/4713
Inventor 吴序栎何宗民刘志刚夏兵兵刘波
Owner SHENZHEN UNIV
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