Method for detecting preparation success of artificial antigens

Abstract

The invention discloses a method for estimating preparation success of artificial antigens. The method comprises the following steps: a) weighing artificial antigens and carrier protein of the same weight, adding solvent 1 to 10 times the weight for dissolution, applying ultrasound for 1 minute, performing centrifugation at 12,000 rpm, taking supernatant and obtaining an artificial antigen test sample and a carrier protein test sample respectively; b) using an instrument combining ultra performance liquid chromatography and quadrupole-rod electro-spray mass spectrometry to detect the test samples respectively to obtain ultra performance liquid chromatograms and quadrupole-rod electro-spray mass spectrograms respectively; and c) comparing the differences between the ultra performance liquid chromatograms of the artificial antigens and the carrier protein, as well as the corresponding mass spectrograms thereof to determine whether the preparation of the artificial antigens is successful. The method has the advantages of simple sample pretreatment, strong universality, no special requirement for the physical-chemical characteristics of hapten, simple instrument operation conditions, suitability for large-scale popularization and application, and the like.

Description

technical field The invention relates to a method for identifying an artificial antigen, in particular to a method for detecting whether the preparation of the artificial antigen is successful by using ultra-high performance liquid chromatography and quadrupole electrospray mass spectrometry. Background technique Convenient, concise, rapid and sensitive analytical techniques and detection methods are important guarantees for food safety and environmental monitoring. The irrational use or even abuse of pesticides and veterinary drugs has led to excessive drug residues in edible agricultural products, which has become one of the outstanding problems in food safety. The development of reliable, sensitive, fast and practical rapid analysis and detection technologies is undoubtedly the key to monitoring and controlling residues and ensuring people's health. And an important technical means to avoid international trade disputes. As a rapid detection technology, immunoassay (IA), ...

AI Technical Summary

Problems solved by technology

Electrophoresis technology is a technology that uses ions in solution to migrate under a certain electric field to achieve separation. Electrophoresis is widely used in the identification of biological macromolecules; in the identification of artificial antigens, the electrophoresis method currently mainly used is SDS. - Polyacrylamide electrophoresis and SDS-page electrophoresis are used to determine whether the synthesis of the artificial antigen is successful by comparing the position of the bands that appear on the gel with the artificial antigen and its carrier protein; more precisely, it is based on the artificial antigen The molecular weight difference between the carrier protein and the carrier protein is determined; the disadvantage is that if the carrier-coupled hapten is less, the molecular weight of the artificial antigen will not increase significantly, and the difference in the position of the band on the agglutination using the SDS-page method is not obvious; literature It is reported that only when the artificial antigen prepared by coupling the hapten with large molecular weight (>800Da) and the carrier protein with small molecular weight (<20000Da) can be identified by this method, it has a good identification effect; matrix-assisted laser desorption time-of-flight mass spectrometry The method (Qi Xiaohua et al. Rapid determination of the coupling ratio of artificial antigen by biological mass spectrometry. Inspection and Quarantine Science, 2007 Supplement) is the most effective identification method of artificial antigen at present. If the matrix is ​​not properly selected, the repeatability of the test results will be poor; at the same time, the laser desorption time-of-flight mass spectrometer is expensive and the operating conditions are relatively harsh, so it is not suitable for large-scale application

Due to the complex structure of the artificial antigen, it is connected to specific amino acid residues of carrier proteins such as bovine serum albumin, human serum albumin, and ovalbumin by specific groups such as amino groups and carboxyl groups on small molecules through specific reactions. However, the prepared ones have many binding sites between small molecules and proteins, and the analysis is difficult. At present, there is still a lack of a set of universal analysis and detection methods.

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