Kit for quantitatively detecting bovine milk serum albumin in milk or dairy product

A serum albumin, quantitative detection technology, applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of inaccurate quantitative results, high operator requirements, accurate quantitative defects, etc., to achieve simple reagent preparation and ensure reproducibility , The effect of low detection cost

Active Publication Date: 2015-02-11
SHANGHAI IMPACT SCI INSTR
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the most important method for bovine milk serum albumin detection at home and abroad is the ELISA method. This method has high sensitivity and strong specificity, but it has high requirements for operators, poor reproducibility, and inaccurate quantitative results, which can meet the initial screening of samples. , but there are still defects in accurate quantification
After inquiry, no method has been found so far that uses the isotopic internal standard peptide dilution method combined with LC-ESI-MS / MS to determine heat-denatured and non-denatured bovine milk serum albumin in milk and dairy products

Method used

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  • Kit for quantitatively detecting bovine milk serum albumin in milk or dairy product
  • Kit for quantitatively detecting bovine milk serum albumin in milk or dairy product
  • Kit for quantitatively detecting bovine milk serum albumin in milk or dairy product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Preparation and verification of bovine milk serum albumin quantitative detection kit

[0029] A bovine milk serum albumin quantitative detection kit mainly includes bovine milk serum albumin specific peptide, isotope-labeled bovine milk serum albumin specific peptide and isotope-labeled bovine milk serum albumin internal standard standard (attached figure 1 ), the amino acid sequence of the bovine milk serum albumin specific peptide is: LVNELTEFAK; the amino acid sequence of the isotope-labeled bovine milk serum albumin specific peptide is: LV * NEL * TEFAK, where V * and L * It is an amino acid labeled with carbon and nitrogen full isotopes; the amino acid sequence of the isotope-labeled bovine milk serum albumin internal standard is: QCPFDEHVKLV *NEL * TEFAKTCVADESHAGC, where V * and L * It is an amino acid labeled with all isotopes of carbon and nitrogen. The key of the kit of the present invention is to carry out isotope labeling on the specific pe...

Embodiment 2

[0049] Embodiment 2: the preparation of reagent in the kit of the present invention and instructions for use thereof

[0050] 1. Reagent preparation:

[0051] 1. Preparation of bovine milk serum albumin-specific peptide standard stock solution: Accurately pipette 5mL of ultrapure water, add it to standard substance 1 tube (the tube contains bovine milk serum albumin-specific peptide that has been accurately weighed in advance), and ultrasonically dissolve ( 30s), the obtained solution is 100 μmol / L bovine milk serum albumin specific peptide standard stock solution;

[0052] 2. Preparation of isotope-labeled bovine milk serum albumin-specific peptide standard stock solution: Accurately pipette 5mL of ultrapure water and add it to standard substance 2 tubes (this tube contains isotope-labeled bovine milk serum albumin-specific peptide that has been accurately weighed in advance) , ultrasonically dissolved (30s), the obtained solution is 100 μmol / L isotope-specific peptide stand...

Embodiment 3

[0065] Example 3. Commercially available infant formula milk powder

[0066] Weigh 0.5g of the sample into a 100mL volumetric flask, add warm water to dissolve, wait to cool to room temperature, add water to the mark, accurately draw 500μL, add 20μL 5μmol / L isotope internal standard and 480μL 50mmol / L NH 4 HCO 3 solution, add 10μL 500mmol / L DTT solution, react at a constant temperature of 50°C for 30 minutes, take it out and cool to room temperature, add 30μL 500mmol / L IAA solution, let it stand in the dark for 30 minutes, add 10μL 100mmol / L CaCl 2 Solution and 30 μL 200 μg / mL trypsin solution, 37 ℃ constant temperature for overnight enzymolysis, take out the next day and add 10 μL pure formic acid, let stand at room temperature for 1 hour, finally dilute the enzymolysis solution to 2mL, take the obtained sample solution according to the method in Example 2 The detection and analysis were carried out, and the result was calculated by the internal standard method. The content ...

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Abstract

The invention provides a kit for quantitatively detecting bovine milk serum albumin. The kit comprises a bovine serum albumin specific peptide, an isotope labeled bovine milk serum albumin specific peptide, an isotope labeled bovine serum albumin internal standard, a proteolytic reagent and a quality control sample. The amino acid sequence of the bovine serum albumin specific peptide is LVNELTEFAK; the amino acid sequence of the isotope labeled bovine milk serum albumin specific peptide is LV *NEL*TEFAK; and the amino acid sequence of the isotope labeled bovine serum albumin internal standard is QCPFDEHVKLV*NEL*TEFAKTCVADESHAGC, wherein V* and L* are carbon and nitrogen isotope labeled amino acids. The kit can accurately quantitatively detect the bovine milk serum albumin in various milk-containing products by adopting the isotope labeled internal standard synthesized through researches and designing, ensures the result reliability, and also has the advantages of less reagent consumption and low detection cost.

Description

technical field [0001] The invention relates to a detection kit capable of measuring heat-denatured and non-denatured constant or trace amounts of bovine milk serum albumin in milk or dairy products. Background technique [0002] Bovine serum albumin (Bovine serum albuminn, BSA) is one of the main globulins in bovine serum and one of the components of whey protein, also known as the fifth component (Fraction V), referred to as BSA, including 583 amino acid residues, molecular weight of 66.43kDa, isoelectric point of 4.7 in water at 25°C, mainly present in bovine serum, accounting for about 52-62% of the total protein in bovine serum; in addition, a small amount of serum is also contained in milk Albumin, about 1.4% of the total protein in milk. [0003] According to reports, the main physiological role of bovine serum albumin is to maintain the balance of osmotic pressure and pH value in human blood, while Karjalainen (1992) and others found that the intake of bovine serum ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88G01N30/06
Inventor 任一平赖世云张京顺
Owner SHANGHAI IMPACT SCI INSTR
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