阅读说明：本技术 一种磺胺类药物半抗原、人工抗原及其在免疫检测中的应用 (Sulfonamide hapten, artificial antigen and application thereof in immunodetection ) 是由 李斌 王莹莹 邬秀锋 刘远高 苏振贤 黄家怡 于 2019-10-09 设计创作，主要内容包括：本发明公开了一种磺胺类药物半抗原、人工抗原及其在免疫检测中的应用,属于免疫检测领域。半抗原结构中具有两个胺基苯磺酰胺的结构,使得制备而成的人工抗原的空间结构更突出,更有利于呈现磺胺类药物的共有特征基团胺基苯磺酰胺,提高了抗原的免疫原性,将人工抗原结合ELISA检测技术,磺胺二甲嘧啶的IC50值为0.5μg/L；使用本发明制备的磺胺类药物胶体金免疫层析试纸条,可以实行快速、准确的检测。(The invention discloses a sulfonamide hapten, an artificial antigen and application thereof in immunodetection, belonging to the field of immunodetection. The hapten structure has a structure of two aminobenzene sulfonamides, so that the space structure of the prepared artificial antigen is more prominent, the aminobenzene sulfonamides which are common characteristic groups of sulfonamides can be presented more favorably, the immunogenicity of the antigen is improved, the artificial antigen is combined with an ELISA detection technology, and the IC50 value of the sulfadimidine is 0.5 mu g/L; the colloidal gold immunochromatographic test strip for sulfonamides prepared by the invention can realize rapid and accurate detection.)

1. A hapten, the hapten having the formula:

2. a method of preparing a hapten according to claim 1, characterized in that: the method comprises the following steps:

1) reacting the compound a with the compound b to obtain a compound c;

2) hydrolyzing the compound c to obtain the hapten

Wherein the structural formula of the compound a is

The structural formula of the compound b is

The structural formula of the compound c is

3. The method of claim 2, comprising the steps of:

(1) carrying out substitution reaction on the compound a and the compound b under the condition of triethylamine, and obtaining a compound c after extraction and column chromatography purification;

(2) and (3) carrying out hydrolysis reaction on the compound c under a strong alkali condition, and extracting and recrystallizing to obtain the hapten.

4. An artificial antigen obtained by coupling the hapten of claim 1 with a protein carrier, wherein the artificial antigen has a structural formula as follows:

wherein the protein is a protein carrier.

5. The artificial antigen of claim 4, wherein: the protein carrier is any one of bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.

6. A method for preparing the artificial antigen of claim 4 or 5, comprising the steps of:

linking the hapten of claim 1 to a protein carrier to obtain the artificial antigen

7. A monoclonal antibody against sulfonamides, which is characterized in that: the hapten of claim 1 or the artificial antigen of any one of claims 4 to 5.

8. The artificial antigen of claim 4 or 5 and the sulfonamide monoclonal antibody of claim 7 for use in an ELISA detection method.

9. The artificial antigen of claim 4 or 5 and the sulfonamide monoclonal antibody of claim 7 for use in immunochromatography.

10. A colloidal gold immunochromatographic test strip for sulfonamides is characterized in that: the reaction membrane in the test strip is coated with the artificial antigen of claim 4 or 5; further, the preparation method comprises the following steps:

1) preparing a nano gold-labeled sulfonamide monoclonal antibody: mixing the sulfonamide monoclonal antibody of claim 7 and nanogold uniformly under weak base, centrifuging, adding a gold seed protective agent for resuspension, and obtaining the nanogold-labeled sulfonamide monoclonal antibody;

2) preparing a gold pad: diluting the gold-labeled sulfonamide monoclonal antibody by using gold seed diluent, and smearing the gold-labeled sulfonamide monoclonal antibody on a glass fiber membrane to obtain a gold pad of the gold-labeled sulfonamide monoclonal antibody;

3) preparing standard solutions of sulfonamides with series of concentrations by using PBS (phosphate buffer solution), dripping the standard solutions into sample loading holes of test strips, judging the color development depth of a T/C line by naked eyes, judging the color development depth of the T/C line to be negative when the color development of the T line is deeper than that of the C line or the same depth, and judging the color development depth of the T line to be positive when the color development of the T line is shallower than that of the C line; and (3) adding a standard solution into a blank sample, and judging the result, so that the rapid quantitative detection of the sulfonamides in the sample can be realized.

Technical Field

The invention belongs to the field of immunodetection, and particularly relates to a sulfonamide hapten, an artificial antigen and application thereof in immunodetection.

Background

Sulfonamides (SAs) are artificial synthetic therapeutic drugs with sulfanilamide structural characteristics for preventing and treating bacterial infectious diseases. Because of its broad spectrum, convenient and easily available features, sub-therapeutic concentration of the drug is usually used as feed additive to prevent diseases, increase the conversion rate of feed and promote animal growth. However, the unreasonable use and even abuse of human beings easily cause that SAs are remained in animal tissues and then accumulated in the human bodies through food chains and other ways, thus harming human health. The world specifies the Maximum Residual Limits (MRLs) of sulfonamides in animal food. Besides controlling the abuse of SAs from the source, the detection and monitoring of drug residues in animal-derived food are also important measures for ensuring food safety.

At present, methods for detecting sulfonamide residues mainly include microbiological methods, physicochemical analysis methods, immunological methods and the like. The microbiological method is simple and convenient, the cost is low, but the operation is time-consuming, the characteristics such as sensitivity, specificity and the like are poor, the qualitative and quantitative determination are difficult, the requirement of modern residue detection cannot be met, and even some detection results cannot meet the requirement of the maximum residue limit. Physicochemical analysis methods are often used as verification methods for determining drug residues in animal tissues, such as Gas Chromatography (GC), High Performance Liquid Chromatography (HPLC), gas-mass spectrometry (GC-MS), liquid-mass spectrometry (HPLC-MS), and the like, but because equipment and instruments are expensive, the detection time is long, and professional operation is required, the real on-site rapid detection cannot be realized. The immunological detection technology is very suitable for separating or detecting trace components of complex matrixes due to the characteristics of economy, rapidness, low technical key points, simple and convenient operation and the like. The immunoassay detection technology is gradually one of the main methods for rapidly screening and detecting toxic and harmful residues, and a new way is provided for the detection of sulfonamides.

The key of the immunoassay detection technology is the performance determination of the antigen and the antibody, and the key of the antigen and the antibody is based on the hapten of the corresponding medicine. Although the sulfonamides have active groups, the active groups of the drugs are strong recognition groups of the sulfonamides, and the artificial antigens prepared directly have the defects of poor sensitivity and low cross reaction rate, so that the requirements of the existing markets cannot be met.

Therefore, the need to design complete antigens with higher recognition degree is a problem to be solved urgently.

Disclosure of Invention

The invention mainly aims to provide a sulfonamide hapten.

The invention also aims to provide a sulfonamide artificial antigen.

The invention also aims to provide the application of the sulfonamide artificial antigen in immunodetection.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the invention, a hapten is provided, the hapten having the formula:

in a second aspect of the present invention, there is provided a method for preparing the hapten, comprising the steps of:

1) reacting the compound a with the compound b to obtain a compound c;

2) hydrolyzing the compound c to obtain the hapten

Wherein the structural formula of the compound a is

The structural formula of the compound b is

The structural formula of the compound c is

According to an embodiment of the invention, the method comprises the following steps:

(1) carrying out substitution reaction on the compound a and the compound b under the condition of triethylamine, and obtaining a compound c after extraction and column chromatography purification;

(2) and (3) carrying out hydrolysis reaction on the compound c under a strong alkali condition, and extracting and recrystallizing to obtain the hapten.

In a third aspect of the present invention, an artificial antigen is provided, wherein the artificial antigen is obtained by coupling the hapten and a protein carrier, and the structural formula of the artificial antigen is as follows:

wherein the protein is a protein carrier.

According to an embodiment of the invention, the protein carrier is any one of bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.

In a fourth aspect of the present invention, a method for preparing the artificial antigen is provided, which comprises the following steps:

linking the hapten with a protein carrier to obtain the artificial antigen

In the fifth aspect of the invention, a sulfonamide monoclonal antibody is provided, which is prepared by using the hapten or any one of the artificial antigens.

In the sixth aspect of the invention, the artificial antigen and the sulfonamide monoclonal antibody are applied to an ELISA detection method.

In the seventh aspect of the invention, the artificial antigen and the monoclonal antibody of the sulfonamide are applied to immunochromatography.

In an eighth aspect of the present invention, there is provided a sulfonamide colloidal gold immunochromatographic test strip, wherein the reaction membrane of the test strip is coated with the artificial antigen, and in some embodiments, the preparation method comprises the following steps:

1) preparing a nano gold-labeled sulfonamide monoclonal antibody: uniformly mixing the sulfanilamide drug monoclonal antibody and nanogold under weak base, centrifuging, adding a gold seed protective agent for heavy suspension, and obtaining the nanogold-labeled sulfanilamide drug monoclonal antibody;

2) preparing a gold pad: diluting the gold-labeled sulfonamide monoclonal antibody by using gold seed diluent, and smearing the gold-labeled sulfonamide monoclonal antibody on a glass fiber membrane to obtain a gold pad of the gold-labeled sulfonamide monoclonal antibody;

3) preparing standard solutions of sulfonamides with series of concentrations by using PBS (phosphate buffer solution), dripping the standard solutions into sample loading holes of test strips, judging the color development depth of a T/C line by naked eyes, judging the color development depth of the T/C line to be negative when the color development of the T line is deeper than that of the C line or the same depth, and judging the color development depth of the T line to be positive when the color development of the T line is shallower than that of the C line; and (3) adding a standard solution into a blank sample, and judging the result, so that the rapid quantitative detection of the sulfonamides in the sample can be realized.

According to the embodiment of the invention, the gold seed diluent of the colloidal gold immunochromatographic test strip for sulfonamides comprises Tris, bovine serum albumin, thimerosal and sucrose.

The invention has the beneficial effects that:

the hapten prepared by the invention not only retains the common characteristic group amino of sulfonamides, but also has the characteristic structure of two aminobenzene sulfonamides on the molecular structure of one hapten, so that the space structure of the prepared artificial antigen is more prominent, the common characteristic group aminobenzene sulfonamides of sulfonamides can be presented more favorably, the immunogenicity and the sensitivity of the antigen are improved, the artificial antigen is combined with an ELISA detection technology, and the IC50 value of the sulfadimidine is 0.5 mug/L; the colloidal gold immunochromatographic test strip for sulfonamides prepared by the invention is used for carrying out rapid and accurate detection.

Drawings

FIG. 1 is a mass spectrum of sulfonamide hapten prepared by the invention.

Detailed Description

The technical solution of the present invention is clearly and completely illustrated below with reference to the following examples, but is not limited thereto.