Immunological detection method for content of casein phosphopeptides in infant formula milk powder

Abstract

The invention relates to the technical field of biological and immunological detection, in particular to an immunological detection method for the content of casein phosphopeptides in infant formula milk powder, which comprises the following steps: S1, preparing a mouse anti-casein phosphopeptide monoclonal antibody 6A6; s2, marking a monoclonal antibody 6A6 by adopting horse radish peroxidase (HRP); s3, detecting the working dilution ratio of the HRP-6A6 by adopting a direct ELISA (enzyme-linked immunosorbent assay) method; and S4, drawing a standard curve by adopting a competitive ELISA method, and fitting the sample content. According to the invention, the method for detecting the CPP content in the infant milk powder by adopting a competitive ELISA method overcomes the defects of complex operation, expensive required instruments and equipment and inaccurate detection, once hybridoma cells secreting the monoclonal antibody aiming at the casein phosphopeptides are screened, the antibody can be continuously copied and produced, and the detection time is shortened. The antibody can be used after being labeled with horse radish peroxidase, and the method does not need large instruments or operation of professionals, and can simply, conveniently and quickly realize high-throughput detection.

Description

technical field [0001] The invention relates to the technical field of biological and immunological detection, in particular to an immunological detection method for the content of casein phosphopeptide in formula milk powder for infants and young children. Background technique [0002] Casein phosphopeptide, referred to as CPP, is a biologically active peptide containing 1-6 phosphoserine residues obtained by hydrolyzing, separating and purifying bovine casein with one or more proteases, and has the ability to promote calcium, iron, zinc and other mineral elements CPP has been widely used in calcium-fortified dairy products and nutrient supplements in Japan, Southeast Asia, Europe and other places. China has now widely used CPP as a food additive in infant milk powder and calcium supplement food, and stipulates that the content of casein phosphopeptide in infant formula milk powder shall be ≤3.0g / kg. The detection methods of CPP at home and abroad mainly include barium-etha...

AI Technical Summary

Problems solved by technology

[0002] Casein phosphopeptide, referred to as CPP, is a biologically active peptide containing 1-6 phosphoserine residues obtained by hydrolyzing, separating and purifying bovine casein with one or more proteases, and has the ability to promote calcium, iron, zinc and other mineral elements CPP has been widely used in calcium-fortified dairy products and nutrient supplements in Japan, Southeast Asia, Europe and other places. China has now widely used CPP as a food additive in infant milk powder and calcium supplement food, and stipulates that the content of casein phosphopeptide in infant formula milk powder shall be ≤3.0g / kg. The detection methods of CPP at home and abroad mainly include barium-ethanol precipitation method, sodium lauryl sulfate-polymer Acrylamide gel electrophoresis, high performance liquid chromatography, etc. Among them, barium-ethanol precipitation method and isoelectric focusing electrophoresis are only suitable for the content determination of raw CPP products, and cannot be used for the determination of CPP content in terminal foods; in recent years, there are also Reports on the determination of CPP content by high-performance liquid chromatography and high-performance liquid chromatography-tandem mass spectrometry, the HPLC method has a strong target and good accuracy, but its versatility is not good, and the equipment is expensive, requiring professionals to operate. , China has not yet established an authoritative and unified detection method, and there are no corresponding national standards and industry standards for the detection of CPP in food. Most of the above-mentioned detection methods have the disadvantages of complicated operation, expensive equipment and inaccurate detection.

[0003] Chinese Patent No. CN103760246B discloses a liquid-phase detection method for casein phosphopeptide in milk. The steps include: adding casein phosphopeptide to the milk to be tested to make a 10-100 ppm casein phosphopeptide dispersion solution, and performing cation exchange after deproteinization Resin column decalcification, the eluate obtained after decalcification is passed through a cation exchange resin column for impurity removal and enrichment, the collected liquid is vacuum rotary evaporated at a temperature of 60-70°C and a speed of 50-60rpm, and is used as a liquid after passing through a membrane. Phase detection samples; then carry out liquid phase detection, and calculate the concentration of casein phosphopeptide added in milk by the peak area method according to the external standard method. The purification step is carried out to realize the separation of casein phosphopeptides, which ensures the quantitative accuracy, effectively removes the interference of impurities such as other proteins and lipids, improves the sensitivity of the final detection, and is beneficial to the detection of samples with relatively low content of casein phosphopeptides , but this kind of invention is too rough for the comprehensiveness and subdivision of the detection results, which has a certain impact on the effectiveness of the detection results and needs to be improved

Images