CN-115772215-B - Artificial composite antigen of sulfonamide and tetracycline antibiotics, broad-spectrum detection antibody and ELISA detection method thereof

CN115772215BCN 115772215 BCN115772215 BCN 115772215BCN-115772215-B

Abstract

The invention provides artificial composite antigens of sulfonamides and tetracycline antibiotics, a broad-spectrum detection antibody and an ELISA detection method thereof, and belongs to the technical field of immunoassay. According to the invention, artificial composite antigens of sulfonamides (sulfonamides, SAs) and tetracyclines (TETRACYCLINES, TCs) antibiotics are constructed, a sulfonamide segment and a tetracyclines segment are introduced into the artificial antigens, and the sulfonamide segment and the tetracyclines segment are connected with carrier proteins through glutaraldehyde connecting arms, so that the artificial composite antigens of the sulfonamides and the tetracyclines antibiotics are obtained. The artificial composite antigen can obtain broad-spectrum antibody with specificity and high titer to SAs and TCs after animal immunization, and can directly detect the total amount of SAs and TCs in a sample. The invention provides an ELISA detection method for total amount of sulfonamide and tetracycline antibiotics, which has good linearity for SAs and TCs antibiotics.

Inventors

LIU XUDONG
ZHANG YUCHAO
WU FUYONG
YU MEI

Assignees

茅台学院

Dates

Publication Date: 20260505
Application Date: 20221117

Claims (4)

1. An artificial composite antigen has a structure shown in a formula I: A formula I; in the formula I, "N-Protein-N" represents a carrier Protein; the preparation method of the artificial composite antigen comprises the following steps: mixing sulfanilamide, glutaraldehyde, carrier protein, an alcohol solvent and a buffer solution, and performing a first coupling reaction to obtain a compound with a structure shown in a formula a; A formula (a); Mixing the compound with the structure shown in the formula a, tetracycline and glutaraldehyde, and performing a second coupling reaction to obtain an artificial composite antigen with the structure shown in the formula I; The molar ratio of the sulfanilamide to the glutaraldehyde to the carrier protein is 33:178:1-1.4; The molar ratio of the compound with the structure shown in the formula a to the tetracycline to the glutaraldehyde is 1:1:5-5.5; the alcohol solvent is methanol, and the buffer solution is PBS buffer solution; the temperature of the first coupling reaction is 22-26 ℃ and the time is 4-5 h; the temperature of the second coupling reaction is 22-26 ℃ and the time is 4-5 h.
2. The artificial composite antigen of claim 1, wherein the carrier protein is bovine serum albumin or egg albumin.
3. The method for preparing artificial composite antigen according to claim 1 or 2, comprising the steps of: mixing sulfanilamide, glutaraldehyde, carrier protein, an alcohol solvent and a buffer solution, and performing a first coupling reaction to obtain a compound with a structure shown in a formula a; A formula (a); Mixing the compound with the structure shown in the formula a, tetracycline and glutaraldehyde, and performing a second coupling reaction to obtain an artificial composite antigen with the structure shown in the formula I; The molar ratio of the sulfanilamide to the glutaraldehyde to the carrier protein is 33:178:1-1.4; The molar ratio of the compound with the structure shown in the formula a to the tetracycline to the glutaraldehyde is 1:1:5-5.5; the alcohol solvent is methanol, and the buffer solution is PBS buffer solution; the temperature of the first coupling reaction is 22-26 ℃ and the time is 4-5 h; the temperature of the second coupling reaction is 22-26 ℃ and the time is 4-5 h.
4. The application of the artificial composite antigen as claimed in claim 1 or 2 or the artificial composite antigen prepared by the preparation method as claimed in claim 3 in preparing sulfonamides and tetracycline antibiotics broad-spectrum detection antibodies; The sulfonamide antibiotics are one or more of sulfanilamide, sulfapyridine, sulfadiazine and sulfathiazole; the tetracycline antibiotics are one or more of tetracycline, aureomycin, oxytetracycline and cyclosporine.

Description

Artificial composite antigen of sulfonamide and tetracycline antibiotics, broad-spectrum detection antibody and ELISA detection method thereof Technical Field The invention relates to the technical field of immunoassay, in particular to an artificial composite antigen of sulfonamides and tetracycline antibiotics, a broad-spectrum detection antibody and an ELISA detection method thereof. Background The antibiotics can possibly enter the environment in each link from production to consumption, and residual antibiotics in the environment can cause the generation of drug resistance genes to change the ecological environment of microorganisms, and meanwhile, the antibiotics can cause unpredictable harm to human bodies due to accumulation of food chains. At present, the pollution of antibiotics to water bodies is increasingly serious, and medical and health, livestock breeding and aquaculture become main pollution sources of antibiotics in environmental water bodies. Although the pollution degree is quite different in all places, the detection frequency is higher, and antibiotics with higher detection concentration in China are sulfonamides, tetracyclines, macrolides, quinolones and chloramphenicol in sequence. Therefore, it is important to construct a rapid and efficient detection method for antibiotics. At present, the detection method of antibiotics in water bodies at home and abroad is mainly an instrument analysis method, and the method has high requirements on instrument conditions, and the pretreatment operation is relatively complex and time-consuming. ELISA utilizes antigen-antibody specific reaction, well makes up the deficiency of the instrumental analysis method, because expensive instrumental equipment is not needed, and the specificity is strong, the sensitivity is high, the operation is simple and convenient and quick, has shown its unique advantage in the rapid detection of screening and a large number of samples on the spot. At present, ELISA detection methods for antibiotics in water bodies are studied, wherein the methods all adopt a carrier protein coupled with a hapten to prepare artificial antigens, and specific polyclonal/monoclonal antibodies are prepared by immunizing animals so as to develop corresponding ELISA detection methods. However, antibiotics are various, only one kind of antibiotics can be measured by an ELISA detection method, if the total amount of a certain kind of antibiotics or a plurality of kinds of antibiotics in a sample is needed to be known, the results are summarized after a plurality of times of measurement, the workload is increased, and the detection time is prolonged. Disclosure of Invention In view of the above, the present invention aims to provide artificial complex antigens of sulfonamides and tetracyclines, broad-spectrum detection antibodies and ELISA detection methods thereof. The artificial composite antigen provided by the invention can be used for preparing a broad-spectrum antibody capable of simultaneously detecting various antibiotics, so that the simultaneous detection of sulfonamides and tetracyclines is realized. In order to achieve the above object, the present invention provides the following technical solutions: The invention provides an artificial composite antigen, which has a structure shown in a formula I: in formula I, "N-Protein-N" represents a carrier Protein. Preferably, the carrier protein is bovine serum albumin or ovalbumin. The invention provides a preparation method of the artificial composite antigen, which comprises the following steps: Mixing sulfanilamide, glutaraldehyde, carrier protein, an alcohol solvent and a buffer solution, and performing a first coupling reaction to obtain a compound with a structure shown in a formula a; And mixing the compound with the structure shown in the formula a, tetracycline and glutaraldehyde, and performing a second coupling reaction to obtain the artificial composite antigen with the structure shown in the formula I. Preferably, the temperature of the first coupling reaction is 22-26 ℃ and the time is 4-5 hours; the temperature of the second coupling reaction is 22-26 ℃ and the time is 4-5 h. The invention provides application of the artificial composite antigen in preparation of a broad-spectrum detection antibody of sulfonamides and tetracycline antibiotics. The invention provides a broad-spectrum detection antibody for sulfonamide and tetracycline antibiotics, which is obtained by immunizing host animals with the artificial composite antigen. The invention provides a kit for detecting sulfonamide and/or tetracycline antibiotics, which comprises a coating antigen, a blocking agent, a broad-spectrum detection antibody for the sulfonamide and the tetracycline antibiotics, an enzyme-labeled secondary antibody, a color developing agent and an enzyme-labeled plate; the coating antigen is the artificial composite antigen; the carrier protein in the coating antigen structure is different from the