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Bovine lactoferrin quantitative detection kit and application thereof

A technology for quantitative detection of bovine lactoferrin, which is applied in the field of quantitative detection kits for bovine lactoferrin, can solve the problems of inability to perform accurate quantification, tedious and time-consuming promotion and application, poor stability of capillary electrophoresis, and the like.

Active Publication Date: 2013-08-28
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, a large number of dairy products and formula foods claiming to add bovine lactoferrin have appeared on the market. Due to complex raw materials and different processing techniques, the product quality is uneven, but no accurate and effective quantitative detection method has been established. The main reason is Lack of fast and effective methods for accurate quantitative detection
[0003] At present, the detection methods for bovine lactoferrin at home and abroad are still mainly based on SDS-PAGE method and enzyme-linked immunochemical method. The former is a semi-quantitative method, which cannot be accurately quantified. Due to the cumbersome and time-consuming operation, it is not conducive to popularization and application in routine detection. ; and although the latter has good detection sensitivity and specificity, the detection cost is high, and false positives are prone to occur, and it cannot accurately and effectively detect lactoferrin partially destroyed due to production and processing
In recent years, some researchers have tried to use the HPLC-UV detection method, but due to poor resolution, it cannot be used for the detection of complex matrices, especially foods with low lactoferrin content; Li Jia et al. established a capillary electrophoresis-UV detector method For the determination of bovine lactoferrin in infant formula, the sample is dissolved and extracted by acetic acid solution and then centrifuged and filtered. The separation is realized according to the migration of different protein components in the capillary under high-voltage electric field. The detection is carried out under the wavelength of 214nm ultraviolet light, but the capillary The electrophoresis method has poor stability, is difficult to reproduce, and is limited to the quantitative detection of non-denatured bovine lactoferrin in the sample, but cannot fully reflect the content of lactoferrin in food, and thus cannot fully evaluate the quality and nutritional value of the product
After inquiry, no internal standard quantitative method for the determination of bovine lactoferrin in milk and dairy products has been found so far using the isotope internal standard peptide dilution method combined with HPLC-ESI-MS / MS

Method used

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  • Bovine lactoferrin quantitative detection kit and application thereof
  • Bovine lactoferrin quantitative detection kit and application thereof
  • Bovine lactoferrin quantitative detection kit and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0044] 1. Design and determination of the isotope-specific peptide of the present invention:

[0045] According to the amino acid sequence of bovine lactoferrin-specific peptide determined by experiments, fully considering its related physical and chemical properties, interference and ionization efficiency in the process of mass spectrometry detection, and taking into account the cost and feasibility of actual production and application, the introduction of stable isotope-labeled Valerin amino acid (V * ) and isoleucine (I * ) designed and synthesized the isotope-specific peptide of bovine lactoferrin-specific peptide, whose amino acid sequence is LRPV * AAEI * YGTK.

[0046] Elements such as retention time, linearity and matrix effect can reflect the chromatographic behavior and mass spectrometric ionization efficiency of measured substance to a certain extent, in order to verify the isotope-specific peptide and bovine lactoferrin specific peptide designed in the present ...

Embodiment 2

[0051] Embodiment 2: kit preparation and instructions for use

[0052] 1. Reagent preparation:

[0053] 1. Preparation of bovine lactoferrin-specific peptide standard stock solution: Accurately pipette 5mL of ultrapure water, add standard substance 1 tube (the tube contains bovine lactoferrin-specific peptide that has been accurately weighed in advance), ultrasonically dissolve (30s) , the resulting solution is 500 μM bovine lactoferrin-specific peptide standard stock solution;

[0054]2. Preparation of isotope-specific peptide standard stock solution: Accurately pipette 5mL of ultrapure water, add it into 2 tubes of standard substance (the tube contains pre-accurately weighed isotope-labeled bovine lactoferrin-specific peptide), and ultrasonically dissolve (30s) , the resulting solution is the 500 μM isotope-specific peptide standard stock solution;

[0055] 3. Preparation of isotope internal standard standard stock solution: Accurately pipette 5mL of ultrapure water, add i...

Embodiment 3

[0068] Sample type: Quantitative determination of commercially available infant formula milk powder.

[0069] Weigh 0.5g of the sample into a 100mL volumetric flask, add warm water to dissolve, wait until it cools to room temperature, add water to the mark, accurately draw 500μL, add 80μL 5μM isotope internal standard and 420μL NH 4 HCO 3 Mix the solution (50mM), add 10μL DTT solution (500mM), react at a constant temperature of 50°C for 30min, take it out and cool to room temperature, add 30μL IAA solution (500mM), let stand in the dark for 30min, add 10μL CaCl 2 Solution (100mM) and 40μL 200μg / mL trypsin solution, 37 ℃ constant temperature overnight enzymolysis, take out the next day and add 10μL pure formic acid, let stand at room temperature for 30min, finally add 900μL water to dilute the enzymolysis solution to 2mL, take the obtained sample according to The foregoing method steps were tested and analyzed, and the result was calculated by the internal standard method. The...

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Abstract

The invention provides a bovine lactoferrin quantitative detection kit. The bovine lactoferrin quantitative detection kit mainly comprises a bovine lactoferrin specific peptidase (amino acid sequence being as follows: LRPVAAEIYGTK), an isotope- labeled ovine lactoferrin specific peptidase (amino acid sequence being as follows: LRPV*AAEI*YGTK, wherein I* and L* are carbon-nitrogen isotope-labeled amino acid) and an isotope- labeled ovine lactoferrin internal standard (amino acid sequence being as follows: GRDPYKLRPV*AAEI*YGTKESPQTHY, wherein I* and L* are carbon-nitrogen isotope-labeled amino acid). During a using process, the bovine lactoferrin quantitative detection kit has the advantages of being simple in sample pre-treatment, high in sensitivity, strong in specificity, low in detection cost and the like, is suitable for rapid detection of large-scale samples, and can be used for satisfying accurate quantification of the bovine lactoferrin in various dairy products.

Description

(1) Technical field [0001] The invention relates to a bovine lactoferrin quantitative detection kit and application thereof. (2) Background technology [0002] The main body of bovine lactoferrin (Bovine Lactoferrin) molecule is an iron-binding single-chain glycoprotein composed of 689 amino acid residues, which contains two iron-binding sites and has reversible iron-binding properties. release of iron ions. Generally, the difference in glycosylation and iron binding rate will cause the relative molecular weight of lactoferrin in different states and sources to be different, which is generally between 75-82.6kDa. The amino acid composition of bovine lactoferrin and lactoferrin in human milk is relatively similar, about 70% of the amino acid sequence is identical, and the iron binding capacity of the two is not much different, 93% and 95%, respectively. As a new type of natural nutritional protein, lactoferrin contains a variety of amino acids required by the human body and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88
Inventor 任一平张京顺赖世云蔡增轩徐小民
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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