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Test kit for detecting adulterate reconstituted milk mixed in fresh and raw milk

A technology for detection kits and reconstituted milk, which is applied in the direction of measuring devices, color/spectral characteristic measurement, instruments, etc., can solve the problems of organic reagents, complex instruments and equipment, the inability to carry out basic-level laboratories, and the inability to conduct on-site detection applications, etc., to reduce Difficult operation, low cost and easy operation

Active Publication Date: 2017-12-05
北京纳百生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of these methods are that the required equipment is expensive, the operation is cumbersome and complicated, and it cannot be carried out in grassroots laboratories, especially small and medium-sized dairy processing enterprises, milk stations, etc.
For example, when the high performance liquid chromatography method disclosed in the invention patent 201510788655.4 detects fururine, the organic reagents, instruments and equipment are quite complicated and cannot be used as a rapid method for on-site detection; the liquid chromatography tandem mass spectrometry method disclosed in the invention patent 201510404963.2 also has similar defects

Method used

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  • Test kit for detecting adulterate reconstituted milk mixed in fresh and raw milk
  • Test kit for detecting adulterate reconstituted milk mixed in fresh and raw milk

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Synthesis of 7-ketocholesterol antigen

[0040] Such as figure 1 As shown in A, 7-ketocholesterol (7-kc) has a simple chemical structure and a molecular weight of only 400.64. It has no immunogenicity and needs to be coupled with a carrier protein before it can be used as an antigen. According to its structural characteristics, choose to use the succinic anhydride method to couple with the carrier protein, and the specific operation is as follows:

[0041] Weigh 100 mg of 7-ketocholesterol (sigma, product number: C2394), dissolve it in 20 mL of DMF, add 25 mg of succinic anhydride and 50 mL of anhydrous pyridine, heat to reflux for 24 hours, and monitor the reaction progress by thin-layer chromatography. Finally, the organic solvent was distilled off under reduced pressure to obtain the hapten 7-kc-SA.

[0042] The conjugated antigen can be a common carrier protein, such as hemocyanin, ovalbumin, and serum albumin. According to the actual experimental resu...

Embodiment 2

[0045] Example 2: Preparation and purification of 7-ketocholesterol antibody

[0046] After emulsifying 7-kc-SA and KLH-coupled antigen at the amount of 250 μg / rat with an equal amount of adjuvant, several New Zealand rabbits were immunized by subcutaneous multi-point injection, and the immunization interval was 2 weeks. After 3 times of immunization, direct ELISA The serum titer was determined, and the serum inhibition rate was determined by indirect competition ELISA. The serum with suitable screening sensitivity was used as the antibody for the ELISA detection kit.

[0047] In addition, the prepared conjugated antigen can also be used to immunize Balb / C mice, and the monoclonal antibody that can react with the immunized antigen and has appropriate sensitivity can be obtained by screening according to conventional methods.

[0048] After the prepared antibody was precipitated by saturated ammonium sulfate, it was purified by Protein G affinity chromatography column, and the...

Embodiment 3

[0049] Example 3: Recombinant expression of cholesterol oxide and derivative binding protein (OBP)

[0050] According to the sequence published by Genbank, the OBP protein was cloned and expressed by genetic engineering, and purified for later use. For specific operations, refer to the "Molecular Cloning Experiment Guide" (J. Sambrook et al., Science Press, 2015).

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Abstract

A rapid test kit for detecting adulterate reconstituted milk mixed in fresh and raw milk is characterized in that the kit comprises the following components: 1) a series of standard products of a cholesterol oxidation product; 2) an elisa plate coated with a coating antigen; 3) a specific conjugate solution; 4) an enzyme marker solution; 5) a chromogenic substrate; 6) a stop solution and 7) a wash concentrate; wherein the cholesterol oxidation product is any one of 7-keto-cholesterol, 7-alpha-hydroxycholesterol and 25-alpha-hydroxycholesterol; the coating antigen is a coating antigen obtained by coupling any one of the 7-keto-cholesterol, the 7-alpha-hydroxycholesterol and the 25-alpha-hydroxycholesterol with a carrier protein; a specific conjugate is a corresponding monoclonal or polyclonal antibody of one of the 7-keto-cholesterol, the 7-alpha-hydroxycholesterol and the 25-alpha-hydroxycholesterol, or a protein, capable of specifically binding with one of the 7-keto-cholesterol, the 7-alpha-hydroxycholesterol and the 25-alpha-hydroxycholesterol, such as cholesterol oxide and derivative binding protein (OBP, oxysterol binding protein). The rapid test kit aiming at the current complex situation of dairy product quality supervision can play a significant role in grass-roots inspection and government regulation.

Description

technical field [0001] The invention belongs to the field of dairy product safety detection, and relates to a detection kit for raw milk mixed with reconstituted milk and a detection method thereof. Background technique [0002] Milk and its products have become an important animal-derived food. Especially in recent years, with the improvement of people's living standards and the rapid development of my country's dairy industry, raw milk has been widely accepted as a common food. Raw milk is rich in protein, fat, vitamins and trace elements, etc. It is especially suitable for digestion and absorption by the elderly and children. [0003] In recent years, due to the massive dumping of imported dairy products and the rise in the price of domestic fresh milk, in order to reduce costs, some manufacturers have dissolved and reconstituted milk powder and added it to ordinary fresh milk for sale as pure fresh milk, which has seriously affected Therefore, it is very necessary to e...

Claims

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Application Information

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IPC IPC(8): G01N33/535G01N21/31
CPCG01N21/31G01N33/535
Inventor 杨春江莫勋于在江赵荣茂吴迪马孝斌刘彩娟
Owner 北京纳百生物科技有限公司
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