Immunodetection kit for detecting adulterated cow milk in camel milk and application thereof

An immunoassay and camel milk technology, which is applied in biological testing, measuring devices, material inspection products, etc., can solve problems such as complicated operation, low sensitivity, and complicated extraction process of genetic material

Inactive Publication Date: 2019-07-16
XINJIANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection methods that can be applied to camel milk mixed with cow milk mainly include: (1) Electrophoresis detection technology: Although this method is easy to operate, its sensitivity is low, and it is not suitable for the detection of a large number of samples
(2) Liquid chromatography analysis: This method has high sensitivity and requires a small amount of sample for detection, which is suitable for the detection of a large number of samples, but this method requires expensive instruments and skilled instrument operators and is complicated to operate, so it is not suitable for on-site inspection
(3) Nucleic acid differential analysis: This method has high sensitivity, but the extraction process of genetic material is relatively complicated

Method used

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  • Immunodetection kit for detecting adulterated cow milk in camel milk and application thereof
  • Immunodetection kit for detecting adulterated cow milk in camel milk and application thereof
  • Immunodetection kit for detecting adulterated cow milk in camel milk and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Comparison of protein composition between cow milk and camel milk

[0071] Camel milk whey, bovine milk whey, camel milk casein, and bovine milk casein were subjected to polyacrylamide gel electrophoresis (SDS-PAGE), Coomassie brilliant blue stained gel and decolorized, and the results were recorded with a gel imager, as shown in the attached figure 1 As shown, it is found that camel whey lacks β-lactoglobulin protein relative to bovine whey, and the content of α-lactalbumin protein in camel whey is significantly higher than bovine whey, so bovine milk β-lactoglobulin and camel milk α-lactoglobulin are selected protein for the preparation of polyclonal antibodies; compared with bovine milk, camel milk casein contains less κ casein, α s1 - Casein has a large molecular weight, so cow's milk κ casein and camel milk α are selected s1 - Casein for the preparation of polyclonal or monoclonal antibodies.

Embodiment 2

[0072] Example 2: Camel milk α-lactalbumin and α s1 - Extraction of casein

[0073] (1) Crude extraction of camel milk α-lactalbumin by ammonium sulfate fractional precipitation

[0074] Take defatted camel milk, adjust the pH to 4.3-4.8, centrifuge at 3000-12000r / min for 10-60min, and repeat the centrifugation several times. The supernatant is subjected to fractional precipitation according to the ammonium sulfate saturation of 70%-100%, and the obtained precipitate is desalted and freeze-dried spare.

[0075] (2) Calcium ion precipitation crude extraction of camel milk α s1 -casein

[0076] Take defatted camel milk, adjust the pH to 10-12, add calcium ions to produce a precipitate, adjust the pH to 6-8, centrifuge at 3000-8000r / min for 10-30min, take the precipitate and dissolve it in 3.3mol / L urea, adjust the pH to 4 ~5, centrifuge at 3000~8000r / min for 10~30min, get the precipitate and dry it for later use.

[0077] (3) Ultrafiltration and centrifugation for coarse se...

Embodiment 3

[0081] Example three: α-lactalbumin, β-lactoglobulin, α s1 - Preparation of polyclonal antibodies against casein and κ-casein

[0082] 1. Bovine milk β-lactoglobulin polyclonal antibody, camel milk α-lactalbumin polyclonal antibody, camel milk α s1 - Preparation of casein polyclonal antibody and milk kappa-casein polyclonal antibody

[0083] In this experiment, bovine milk β-lactoglobulin, bovine milk κ-casein prepared by Sigma Company and self-made camel milk α-lactalbumin, camel milk α s1 - Casein is the immunization antigen, mix Freund's complete adjuvant (initial immunization) or Freund's incomplete adjuvant (boost immunization) to immunize white rabbits, use indirect ELISA technology to detect the titer of polyclonal antibody, and the titer of each protein antibody reaches at least When the ratio of 1:20000 or more is effective, the rabbit is sacrificed, and the serum is collected by collecting blood from the heart, purified, and polyclonal antibody is obtained.

[008...

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Abstract

The invention discloses an immunodetection kit for detecting adulterated cow milk in camel milk and an application thereof, wherein alpha-lactalbumin and alphas1-casein in camel milk are used as characteristic protein of the camel milk, and beta-lactoglobulin and kappa casein in cow milk are used as characteristic protein of the cow milk. After the camel milk characteristic protein is separated and purified, the polyclonal antibody and the monoclonal antibody prepared by the commercialized cow milk characteristic protein and the self-made camel milk characteristic protein can be applied to ELISA detection. The kit mainly adopts an indirect ELISA method to qualitatively or quantitatively detect the content of camel-derived alpha-lactalbumin, alphas1-casein or bovine-derived beta-lactoglobulin and kappa-casein in camel milk, the sample pretreatment process is simple, and after a camel milk sample is repeatedly centrifuged to obtain a milk sample, the content can be directly measured after diluted by diluent, and a large number of samples can be simultaneously detected. The determination method is accurate, rapid and convenient, and has wide practicability and development value in thefield of camel milk adulteration monitoring.

Description

technical field [0001] The invention mainly relates to the technical field of immunoassay, in particular, the invention relates to the technical field of an immunoassay kit for detecting adulterated cow's milk in camel milk. Background technique [0002] With the development of the economy, people's awareness of health care has increased, and camel milk products have gradually entered people's daily life. Scholars at home and abroad have increasingly deepened the research on the nutritional value and efficacy of camel milk, and found that it has certain effects in anti-tumor and diabetes treatment. As an adjuvant treatment, most of the camels come from small-scale breeding by herdsmen, and the output of camel milk is low. There is a possibility of mixing low-priced cow's milk into high-priced camel milk, which will affect the development of camel milk industry. [0003] The common allergen in milk is β-lactoglobulin, which accounts for about 7-12% of fresh milk protein. β-l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/531G01N33/577G01N21/31
CPCG01N21/31G01N33/531G01N33/577G01N33/68
Inventor 杨洁邓露李敏婧王雪岳海涛申彤陈红征
Owner XINJIANG UNIVERSITY
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