阅读说明：本技术 一种6-苄基腺嘌呤半抗原、人工抗原及其在免疫检测中的应用 (6-benzyladenine hapten, artificial antigen and application thereof in immunodetection ) 是由 李斌 梁科 梁巧雯 李韵婷 石松 于 2020-06-05 设计创作，主要内容包括：本发明公开了一种6-苄基腺嘌呤半抗原、人工抗原及其在免疫检测中的应用。本发明通过化合物a和化合物b多步反应得到6-苄基腺嘌呤半抗原,所得半抗原引入的手臂不仅具有活性基团,还完整保留了待测目标物的所有胺基；且使用半抗原制得的人工抗原及单克隆抗体IC50值为1.6μg/L,可快速、便捷地实现6-BA的检测,本发明中制备的荧光定量免疫层析试纸条对6-BA检测灵敏度为2μg/L。(The invention discloses a 6-benzyladenine hapten, an artificial antigen and application thereof in immunodetection. The 6-benzyladenine hapten is obtained through multi-step reaction of the compound a and the compound b, an arm introduced by the hapten not only has an active group, but also completely reserves all amino groups of a target object to be detected; the artificial antigen prepared by using the hapten and the value of the monoclonal antibody IC50 are 1.6 mug/L, the detection of 6-BA can be rapidly and conveniently realized, and the sensitivity of the fluorescent quantitative immunochromatographic test strip prepared by the invention to the detection of 6-BA is 2 mug/L.)

1. A 6-benzyladenine hapten, wherein said 6-benzyladenine hapten is of the formula I:

2. the method for producing a hapten according to claim 1, wherein: 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, further comprising the steps of:

(1) carrying out condensation and reduction reactions on the compound a and the compound b under the condition of toluene, and separating and extracting to obtain a compound c;

(2) and (3) carrying out hydrolysis reaction on the compound c under a strong alkali condition, and extracting, filtering and drying 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 the following structural formula:

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

Wherein the hapten is linked to a protein carrier under activator conditions;

the activators include 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide.

7. A6-benzyladenine monoclonal antibody characterized by: the hapten of claim 1 or the artificial antigen of any one of claims 4 to 5.

8. Use of the 6-benzyladenine monoclonal antibody of claim 7 in an ELISA detection method.

9. The use of the 6-benzyladenine monoclonal antibody of claim 7 in immunochromatography.

10. The 6-benzyladenine fluorescent quantitative immunochromatographic test strip is characterized in that: the reaction membrane in the test strip is coated with the artificial antigen of claim 4 or 5.

Technical Field

The invention belongs to the field of immunodetection, and particularly relates to a 6-benzyladenine hapten, an artificial antigen and application thereof in immunodetection.

Background

6-benzyladenine (6-BA) is the first artificially synthesized cytokinin, is white or white-like crystal, is insoluble in water, slightly soluble in ethanol, stable in acid and alkali, and has effects of inhibiting decomposition of chlorophyll, nucleic acid and protein in plant leaf, keeping green and preventing aging; the amino acid, the auxin, the inorganic salt and the like are transported to the treatment part, and the like, and are widely used in various stages from germination to harvest of agricultural, fruit trees and horticultural crops. As the 6-BA has good drug effect and low price, the 6-BA is taken as a growth regulator and widely applied to the planting of the rootless cultivated bean sprouts.

Excessive 6-BA ingested by a human body can stimulate skin mucosa, cause esophagus and stomach mucosa injury, and cause nausea, vomiting and the like. The original national food and drug administration, department of agriculture, national institutes of health and family planning committee all forbid the use of 6-benzyladenine in the production process of bean sprouts. Therefore, the enhancement of the monitoring of the residual 6-BA is very important for ensuring the food quality and safety.

At present, the method for detecting 6-BA mainly adopts the traditional detection method, such as spectrophotometry, high performance liquid chromatography and combined technology thereof, and the like, although the method can accurately quantify, the method can not realize real on-site rapid detection due to expensive equipment and instruments, long detection time and operation of professional staff; the other detection method is an immunological detection technology, which has the characteristics of high specificity and high selectivity, is very suitable for separating or detecting trace components of complex matrixes, and along with the mutual permeation among disciplines, the detection method is endless, the application range is increasingly expanded, and compared with the traditional detection method, the immunological detection technology has the characteristics of economy, rapidness, low technical key points, simplicity and convenience in operation and the like. However, the key of immunoassay detection technology is the determination of the performance of antigen and antibody, and the key of antigen and antibody is the corresponding hapten, so the structural design of hapten is very important for obtaining the antigen and antibody with excellent performance.

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

Disclosure of Invention

One of the objects of the present invention is to provide a 6-benzyladenine hapten;

another object of the present invention is to provide a method for preparing the above hapten;

it is another object of the present invention to provide an artificial antigen;

another object of the present invention is to provide a method for preparing the above artificial antigen;

another object of the present invention is to provide a 6-benzyladenine monoclonal antibody;

the invention also aims to provide the application of the 6-benzyladenine monoclonal antibody in an ELISA detection method;

the invention also aims to provide the application of the 6-benzyladenine monoclonal antibody in immunochromatography.

The invention also aims to provide a 6-benzyladenine fluorescence quantitative immunochromatographic test strip.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided:

a 6-benzyladenine hapten of the formula I:

in a second aspect of the present invention, there is provided:

the preparation method of the hapten comprises the following steps:

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

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

Further, the preparation method also comprises the following steps:

(1) carrying out condensation and reduction reaction on the compound a and the compound b under the condition of toluene, and separating and extracting to obtain a compound c;

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

In a third aspect of the present invention, there is provided:

an 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.

Further, the protein carrier is any one of bovine serum albumin, ovalbumin, human serum albumin, and hemocyanin.

In a fourth aspect of the present invention, there is provided:

the preparation method of the artificial antigen comprises the following steps:

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

Wherein the hapten is linked to a protein carrier under activator conditions;

such activators include 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide.

In a fifth aspect of the present invention, there is provided:

a6-benzyladenine monoclonal antibody prepared by using the hapten or the artificial antigen.

In a sixth aspect of the present invention, there is provided:

the 6-benzyladenine monoclonal antibody is applied to an ELISA detection method.

In a seventh aspect of the present invention, there is provided:

the 6-benzyladenine monoclonal antibody is applied to immunochromatography.

In an eighth aspect of the present invention, there is provided:

a6-benzyladenine fluorescent quantitative immunochromatographic test strip, wherein the artificial antigen is coated on a reaction membrane in the test strip.

The invention has the beneficial effects that:

1. the arm introduced by the designed 6-BA hapten not only has an active group, but also completely reserves all amidogens of a target object to be detected, so that the electron cloud density of the hapten and the target object to be detected is consistent.

2. The 6-BA artificial antigen and the monoclonal antibody have strong detection specificity when used for ELISA, and the IC50 value is 1.6 mu g/L.

3. The 6-BA artificial antigen and the monoclonal antibody are used for a fluorescent quantitative immunochromatographic technique, the detection of 6-BA can be realized quickly and conveniently, and the sensitivity of the fluorescent quantitative immunochromatographic test strip prepared in the invention to the detection of 6-BA is 2 mu g/L.

Drawings

FIG. 1 is a schematic diagram of the synthesis of the 6-BA hapten of the present invention;

FIG. 2 is an ESI-MS analysis of the 6-BA hapten of the invention;

FIG. 3 is a schematic diagram of the synthesis of the 6-BA artificial antigen of the present invention;

FIG. 4 is a four parameter Logistic plot of OD values for different concentrations of 6-BA standard solutions;

FIG. 5 is a four-parameter Logistic plot of the results of the 6-BA assay with the test strip for fluorescent quantitative detection.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.