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Preparation method and application of cow milk protein and goat milk protein bigeminy detection card

The technology of cow milk protein and goat milk protein is applied in the field of preparation of cow milk protein and goat milk protein dual detection card, which can solve the problems of high homology of casein, no identification of cow milk protein and goat milk protein, etc., and achieves detection operation. Simple, good resistance to matrix interference, strong immunogenic effect

Active Publication Date: 2021-08-27
深圳市计量质量检测研究院(国家高新技术计量站国家数字电子产品质量监督检验中心)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high homology of casein in these two species, there is no quick and convenient method to identify bovine milk protein and goat milk protein

Method used

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  • Preparation method and application of cow milk protein and goat milk protein bigeminy detection card
  • Preparation method and application of cow milk protein and goat milk protein bigeminy detection card
  • Preparation method and application of cow milk protein and goat milk protein bigeminy detection card

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Preparation of target antigen (immunogen).

[0045] According to the predicted results of the epitope of the milk beta-casein antigen, the amino acid sequence of milk β-casein semi-antigens is designed by Discovery Studio2.5 software: EvmgvskvKeama-ASP (SEQ ID NO: 1 ), A polyxine semi-antibiotic pre-activated polyxereonic acid is counted by a polyxereonic acid pre-activated polyxynamic acid. The coupling method is as follows:

[0046] Multi-lysine activation: a polyleidine having a molecular weight of 40 kDa was selected from 10 ml of pure water using a 100 mg molecular weight of 40 kDa, dissolved in 10 ml of pure water. 1 mg benzoic acid, 1 mg NHS, 1 mg EDC, dissolved in 1 ml DMF as a B liquid. The A liquid and B mixture were mixed for 24 hours, and the dialysis pockets were used as 4 kDa dialysis purification. Lyophilized saved.

[0047] Coupling: Carbon diimine, polypeptide, 1 mg of dissolved in 1 ml of dimethyl sulfoxide, gradually added to 1 ml of 0.01 Ml pH 7.4PBS for...

Embodiment 2

[0050] Preparation and purification of antibodies.

[0051] The immunogen prepared by the examples was immunized from 6 weeks of female BALB / C rats, 3 per group. When immunojection was first immunized, 100 μg / mL was 100 μL, and the isometric Forte is fully emulsified, and the abdominal cavity is directly injected. After two weeks of interval, the sampled antigen was taken, and the same method was injected with 100 μl of incomplete adjuvant.

[0052] In the first 1D cell fusion, Kunming mice was sacrificed on the day, soaking in 70% alcohol, sterilizing the body surface; fixed the Kunming rat on the wax, using the big head, cutting the abdominal tablet on the ultra-net table, Improve 5 ml RPMI-1640 complete culture solution (15% fetal bovine serum) was added from GIBICO RPMI-1640 base culture solution), gently blinking with hand, and moved into a sterile straw in a sterile straw into 75ml Hat complete culture solution (by 74.25 ml RPMI-1640 complete culture solution was added t...

Embodiment 3

[0060] Preparation of colloidal gold.

[0061] 1% 1 ml of chlorosolic acid solution was added, add 99 ml of ultrapure water to a final concentration of 0.01% chloroetic acid solution. After heating boiling, 1% sodium citrate was taken from 1.9 ml of a boiling chloric acid solution, Continue to heating to the solution from light yellow to blue and black ultimately changed to bright red, and the color is stable and then heated 5 min, cooling at room temperature, ultrapure water to 100 ml.

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Abstract

The invention provides a preparation method and application of a cow milk protein and goat milk protein bigeminy detection card. The preparation method comprises the following steps: respectively preparing an anti-cow milk protein specific monoclonal antibody and an anti-goat milk protein specific monoclonal antibody, and respectively recording as Anti-CC and Anti-SC; preparing a monoclonal antibody for resisting a common site of cow milk protein and goat milk protein, and recording the monoclonal antibody as Anti-total; labeling colloidal gold with a monoclonal antibody of a common site of anti-cow milk protein and anti-goat milk protein to obtain an antibody colloidal gold marker; and preparing the bigeminy detection card through the antibody colloidal gold marker. The bigeminy detection card comprises a PVC backing, wherein a sample pad, a combination pad, a reaction pad and a water absorption pad are sequentially arranged on the PVC backing, a sample hole is formed in the sample pad of the bigeminy detection card, and a detection line is arranged on the reaction pad. According to the invention, the detection card is good in specificity, high in sensitivity, good in matrix interference resistance effect, good in detection result accuracy and good in repeatability, and cow milk protein and goat milk protein in food can be rapidly identified on site.

Description

Technical field [0001] The present invention relates to the field of dairy detection, and more particularly to the preparation method and application of milk protein and lactarin two detection cards. Background technique [0002] Special dairy such as goat milk is welcomed by more and more consumers from their unique taste, nutritional value or low sensitivity. In 2018, my country's milk production enterprises more than 500, with an annual output value of more than 20 billion yuan. At present, the price of goat milk is more than 4 times that of milk. Some irritable farmers and manufacturers are often incorporated into these high-value dairy in these high-value dairy due to their interests. [0003] Methods for the current identification of milk proteins and lamb proteins mainly include gene methods and protein methods. However, because DNA in the milk protein is mainly derived from breast cells from cattle, and there is no proportional relationship with the content of the milk pr...

Claims

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Application Information

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IPC IPC(8): G01N33/558G01N33/543G01N33/577G01N33/68
CPCG01N33/558G01N33/577G01N33/54313G01N33/68
Inventor 冯荣虎张恒张世伟林霖劳翠瑜郑奕鑫朱成杰王坤吴佳辉王珍妮董珊
Owner 深圳市计量质量检测研究院(国家高新技术计量站国家数字电子产品质量监督检验中心)
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