Method and kit for quantitative detection of content of human alpha-lactalbumin

A technology for the quantitative detection of lactalbumin, which is applied in the field of analytical chemistry, can solve problems such as the inability to accurately quantitatively detect breast milk protein, and achieve high-accuracy results

Active Publication Date: 2014-03-19
贝因美(杭州)食品研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of standard products and the cross-reactivity of antibodies to other breast milk proteins, ELISA cannot be applied to the accurate quantitative detection of breast milk proteins

Method used

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  • Method and kit for quantitative detection of content of human alpha-lactalbumin
  • Method and kit for quantitative detection of content of human alpha-lactalbumin
  • Method and kit for quantitative detection of content of human alpha-lactalbumin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Consistency of isotope-specific peptides and human α-lactalbumin-specific peptides

[0046] The purpose of introducing the isotope-specific peptide in the present invention is to overcome the matrix effect caused by the extraction reagent and the matrix. In order to verify the consistency of the results of the isotope-specific peptides designed in the present invention on human α-lactalbumin specific peptides under the same matrix, the experimental design compared the isotope-specific peptides to human α-lactalbumin specific peptides under the same liquid chromatography and mass spectrometry conditions. The retention time, the fragmentation mode of the product ion and the linearity.

[0047] Standard series working solutions of specific peptides and isotope-specific peptides in the present invention were respectively prepared, and samples were injected and analyzed under the same chromatographic mass spectrometry conditions to obtain their retention time and ...

Embodiment 2

[0050] Example 2: Comparison of isotope internal standard and human α-lactalbumin enzymatic hydrolysis efficiency

[0051] In order to verify whether the isotopic internal standard in the present invention and human α-lactalbumin have more similar enzymatic hydrolysis efficiency in various matrices, the following experiments were designed:

[0052] Dilute breast milk with ammonium bicarbonate solution at a volume ratio of 1:9, accurately measure 10 μL of diluted breast milk in a reaction tube, and add 0, 5, 20, 50, 200 μL of skim milk solution (1g skim milk Milk powder dissolved in 100mL 50mM ammonium bicarbonate solution) as a matrix, which can simulate various nutrients in breast milk without affecting the detection. Then add ammonium bicarbonate solution to a total volume of 945 μL, add 10 μL isotope internal standard and 10 μL dithiothreitol solution, shake well and react at a constant temperature of 60 °C for 30 min, take it out and cool to room temperature, add 10 μL iod...

Embodiment 3

[0054] Embodiment 3: the preparation of reagent

[0055] 1. Preparation of human α-lactalbumin-specific peptide standard solution: Accurately pipette 1mL of ultrapure water, add it to the tube of human α-lactalbumin-specific peptide that has been accurately weighed in advance, dissolve it by ultrasonic for 30s, and the obtained solution is 1mmol / L of α-lactalbumin specific peptide standard solution;

[0056] 2. Preparation of isotope-specific peptide standard solution: Accurately pipette 1mL of ultrapure water, add it to the tube of isotope-specific peptide that has been accurately weighed in advance, and dissolve it by ultrasonic for 30s, the resulting solution is 20μmol / L isotope-specific peptide standard solution;

[0057] 3. Preparation of the isotope internal standard solution: Accurately pipette 1mL of ultrapure water, add it into the tube of the isotope internal standard that has been accurately weighed in advance, and ultrasonically dissolve it for 30s. The resulting...

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Abstract

The invention relates to the field of analytical chemistry and discloses a method and kit for quantitative detection of content of human alpha-lactalbumin. According to the method disclosed by the invention, a specific polypeptide sequence CELSQLLK obtained after enzymolysis of the human alpha-lactalbumin is utilized to design the sequences of an isotope-labeled specific peptide and an isotope-labeled internal standard substance, a brand new accurate quantitative detection method is provided on the basis of an internal standard method to perform the quantitative detection of the human alpha-lactalbumin, and the accuracy, precision and sensitivity are relatively high.

Description

technical field [0001] The invention relates to the field of analytical chemistry, in particular to a method and a kit for quantitatively detecting the content of human α-lactalbumin. Background technique [0002] As early as the 1980s, the advantages of breastfeeding have been discovered by many academic institutions in the world, and many papers have confirmed its superiority. Breast milk provides babies with balanced protein, fat, carbohydrates, minerals and vitamins, enabling babies to grow up healthily. [0003] In modern society, infants cannot be fully breast-fed due to various reasons, and can only partially or completely replace breast-feeding with cow's milk. The composition of nutrients in cow's milk is very different from that of breast milk. For example, every 100mL of milk contains 3.4g of protein, of which about 80% is casein, and the remaining 20% ​​is whey protein; and these three values ​​in breast milk are 1.1g, 40% and 60% respectively. Casein will coa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/37
Inventor 任一平陈启黄焘赖世云张京顺
Owner 贝因美(杭州)食品研究院有限公司
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