阅读说明：本技术 氯丹半抗原、人工抗原和抗体及其制备方法和应用 (Chlordan hapten, artificial antigen and antibody, and preparation method and application thereof ) 是由 崔海峰 何方洋 崔廷婷 王兆芹 屈秀玲 崔娜 宋灏 万宇平 于 2020-06-04 设计创作，主要内容包括：本发明公开了氯丹半抗原、人工抗原和抗体及其制备方法和应用,本发明提供的氯丹半抗原既最大程度保留了氯丹的特征结构,使得氯丹半抗原的免疫原性明显增强,又具有可以与载体蛋白发生偶联的羧基；用氯丹半抗原与载体蛋白偶联后得到的氯丹人工抗原去免疫动物,更有利于刺激动物免疫应答产生特异性更强、灵敏度更高的抗体,经检测氯丹抗体的灵敏度可达0.1μg/L,与其他环戊二烯类有机氯农药的交叉反应率低,为后续建立氯丹的各种免疫分析方法提供了基础。(The invention discloses chlordane hapten, artificial antigen and antibody, and a preparation method and application thereof, wherein the chlordane hapten provided by the invention not only retains the characteristic structure of chlordane to the maximum extent, so that the immunogenicity of the chlordane hapten is obviously enhanced, but also has carboxyl which can be coupled with carrier protein; the chlordane artificial antigen obtained by coupling the chlordane hapten and the carrier protein is used for immunizing animals, so that the immune response of the animals can be stimulated to generate antibodies with stronger specificity and higher sensitivity, the sensitivity of the chlordane antibody can reach 0.1 mu g/L through detection, the cross reaction rate with other cyclopentadiene organochlorine pesticides is low, and a foundation is provided for the subsequent establishment of various immunoassay methods of the chlordane.)

1. A chlordane hapten, characterized by the following structural formula:

2. the method for producing a chlordane hapten according to claim 1, which is obtained by an affinity substitution reaction of chlordane oxide with aminobutyric acid.

3. The method for preparing the chlordane hapten as claimed in claim 2, which comprises the following steps: dissolving the chlordane oxide in methanol; dissolving aminobutyric acid in water, adding the dissolved aminobutyric acid into a methanol solution of chlordane oxide, and fully stirring; dissolving potassium carbonate in water, adding the potassium carbonate into the reaction solution, stirring the mixture for 5 hours at 55 ℃, stopping the reaction, adding hydrochloric acid to adjust the pH value to 6, and then separating and purifying the mixture to obtain chlordane hapten; wherein the molar ratio of the chlordane oxide to the aminobutyric acid to the potassium carbonate is 1:2: 3.

4. The method for preparing the chlordane hapten according to claim 2, which comprises the following steps: dissolving 0.423g of chlordane oxide in 100mL of methanol; dissolving 0.206g of aminobutyric acid in 5mL of water, adding the dissolved aminobutyric acid into a methanol solution of chlordane oxide, and fully stirring; dissolving 0.43g of potassium carbonate in 5mL of water, adding the dissolved potassium carbonate into the reaction solution, stirring the mixture at 55 ℃ for 5 hours, stopping the reaction, adding 6mol/L hydrochloric acid to adjust the pH value to 6, performing rotary evaporation and concentration to remove methanol, adding 50mL of water and 100mL of chloroform, extracting the mixture for three times, combining organic phases, evaporating the mixture to dryness to obtain a yellow oily substance, adding 12mL of absolute ethyl alcohol, and recrystallizing to obtain the chlordane hapten.

5. An artificial antigen of chlordane, which is a conjugate obtained by coupling a carrier protein and a chlordane hapten as claimed in claim 1.

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

7. The method for producing a chlordane artificial antigen as claimed in claim 5 or 6, wherein a carrier protein is coupled to a carboxyl group of the chlordane hapten as claimed in claim 1 by the carbodiimide method.

8. A chlordane antibody, which is obtained by immunizing an animal with the chlordane artificial antigen of claim 5, and which specifically reacts with chlordane.

9. Use of the chlordane antibody of claim 8 in the detection of chlordane residues.

Technical Field

The invention belongs to the field of food safety detection. More particularly, the invention relates to chlordane hapten, artificial antigen and antibody, and preparation methods and applications thereof.

Background

Chlordane (Chlordane) is a broad-spectrum organochlorine insecticide, also known as octachloro-hexachloro-methylene indene, and is commonly used for killing underground pests, such as mole cricket, cutworm, straw pests and the like, and has remarkable effect of controlling termites. Chlordane has contact and stomach toxicity effects on insects, has no phytotoxicity on plants under insecticidal concentration, and is toxic to mammals. Chlordane toxicity is mainly reflected in the aspects of causing disorder of a biological endocrine system, destroying a reproductive and immune system, inducing cancer and neurological diseases and the like. It is a serious environmental hazard and can permeate into the groundwater system through the surface soil, causing pollution to the water, soil and atmosphere. Therefore, it has been banned due to its high toxicity and long residue. The national standard GB 2763-2019 'maximum pesticide residue limit in food safety national standard food' specifies the residue limit of chlordane, 0.02mg/kg of vegetables, fruits, nuts and eggs and 0.002mg/kg of raw milk.

At present, analytical methods such as gas chromatography, gas chromatography-mass spectrometry, liquid chromatography tandem mass spectrometry and the like are mainly adopted for detecting chlordane at home and abroad, and the method has the defects of complicated sample pretreatment, long detection time, expensive instruments and the like, so the method cannot be widely applied in China and does not meet the requirements of on-site detection on accurate detection and screening of a large number of samples at low cost in a short time. The immunological detection and analysis technology has the advantages of high sensitivity, high specificity, rapidness, simple and convenient operation and the like, is widely applied to the field of drug residue detection, and has many advantages compared with detection methods such as instruments and the like. Therefore, the immunoassay provides a new analysis and detection method for the research of the chlordane residue.

When an immunological detection method is established and the detection method is applied to detecting the residual quantity of chlordane, the key technology is that an antibody with strong specificity and high sensitivity can be obtained, and the aim is to realize the aim under the precondition that a proper chlordane hapten is synthesized and prepared. However, at present, no relevant report aiming at the chlordane hapten exists in China.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the hapten which can furthest reserve the characteristic structure of chlordane and has a connecting arm with a certain length and the preparation method of the hapten; the artificial antigen prepared by the hapten and the antibody with high detection sensitivity and strong specificity; and the use of such haptens.

In order to achieve the object of the present invention, in a first aspect, the present invention provides a chlordane hapten, which has the following structural formula:

the chlordane hapten provided by the invention introduces carboxyl active groups on a chlordane molecular structure, so that the chlordane hapten can be coupled with carrier protein to obtain an artificial antigen for immunization; the chlordane hapten reserves all characteristic groups of chlordane, changes the original structural characteristics of the chlordane to the minimum, and is coupled with carrier protein to highlight the unique molecular structure of the chlordane, thereby laying a foundation for generating an antibody with stronger specificity and higher sensitivity by subsequent stimulation of animal immune response. The chlordane hapten of the invention makes up the blank of the domestic chlordane immunological detection method technical field, and lays a foundation for the further development of the chlordane immunological detection method.

In a second aspect, the present invention provides a method for preparing the aforementioned chlordane hapten, which is obtained by performing an affinity substitution reaction between chlordane oxide and aminobutyric acid.

Further, the preparation method comprises the following steps: dissolving the chlordane oxide in methanol; dissolving aminobutyric acid in water, adding the dissolved aminobutyric acid into a methanol solution of chlordane oxide, and fully stirring; dissolving potassium carbonate in water, adding the potassium carbonate into the reaction solution, stirring the mixture for 5 hours at 55 ℃, stopping the reaction, adding hydrochloric acid to adjust the pH value to 6, and then separating and purifying the mixture to obtain chlordane hapten; wherein the molar ratio of the chlordane oxide to the aminobutyric acid to the potassium carbonate is 1:2: 3.

Further, the preparation method comprises the following specific steps: dissolving 0.423g of chlordane oxide in 100mL of methanol; dissolving 0.206g of aminobutyric acid in 5mL of water, adding the dissolved aminobutyric acid into a methanol solution of chlordane oxide, and fully stirring; dissolving 0.43g of potassium carbonate in 5mL of water, adding the dissolved potassium carbonate into the reaction solution, stirring the mixture at 55 ℃ for 5 hours, stopping the reaction, adding 6mol/L hydrochloric acid to adjust the pH value to 6, performing rotary evaporation and concentration to remove methanol, adding 50mL of water and 100mL of chloroform, extracting the mixture for three times, combining organic phases, evaporating the mixture to dryness to obtain a yellow oily substance, adding 12mL of absolute ethyl alcohol, and recrystallizing to obtain the chlordane hapten.

According to the invention, the preparation method of the chlordane hapten is reasonably designed according to the structural characteristics of chlordane, the used raw materials are easy to obtain, the reaction operation is simpler, the reaction conditions are easy to control, and the purity and the yield of the prepared chlordane hapten are higher.

In a third aspect, the invention provides a chlordane artificial antigen which is a conjugate obtained by coupling a carrier protein and the chlordane hapten. The chlordane artificial antigen can be used as immunogen and also can be used as coating antigen.

Further, the carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin; bovine serum albumin and ovalbumin are preferred.

More specifically, the following are: chlordane hapten-Bovine Serum Albumin (BSA) forming immunogen; clathrating antigen formed by Chlorella hapten-Ovalbumin (OVA).

The chlordane hapten molecule only has immunoreactivity and is not immunogenic. Therefore, in order to endow the chlordane hapten molecules with immunogenicity, the chlordane hapten molecules are coupled and combined with proper carrier protein molecules, so that the chlordane artificial antigen with immunoreactivity and immunogenicity is generated.

In a fourth aspect, the present invention provides a method for preparing the artificial antigen of chlordane, wherein a carrier protein is coupled to a carboxyl group of the chlordane hapten by using a carbodiimide method.

In a fifth aspect, the invention provides a chlordane antibody, which is obtained by immunizing animals with the chlordane artificial antigen and can generate specific immunoreaction with chlordane.

Further, the chlordane antibody is a monoclonal antibody or a polyclonal antibody. In addition, the chlordane antibody can be prepared by a method conventional in the art.

In a specific embodiment, the chlordane antibody is a murine monoclonal antibody specific for a chlordane artificial antigen of the chlordane hapten described above.

The chlordane antibody obtained by adopting the chlordane artificial antigen has better titer, specificity and affinity, and has low cross reaction rate with other cyclopentadiene organochlorine pesticides.

In a sixth aspect, the invention provides an application of the chlordane antibody in detecting chlordane residues.

The invention induces immune animals to generate antibodies through the chlordane artificial antigen, thereby being used in chlordane immunodetection analysis.

The chlordane immunoassay comprises but is not limited to a chlordane ELISA kit, a chlordane colloidal gold test strip and a chlordane time-resolved fluorescence test strip.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the chlordane hapten provided by the invention not only retains the characteristic structure of chlordane to the greatest extent, so that the immunogenicity of the chlordane hapten is obviously enhanced, but also has carboxyl which can be coupled with carrier protein; the chlordane artificial antigen obtained by coupling the chlordane hapten and the carrier protein is used for immunizing animals, so that the immune response of the animals can be stimulated to generate antibodies with stronger specificity and higher sensitivity, and a foundation is provided for the subsequent establishment of various immunoassay methods of the chlordane.

The method for preparing the chlordane hapten has the advantages of easily available raw materials, simple reaction operation, easily controlled reaction conditions and high purity and yield of the prepared chlordane hapten.

The chlordane antibody obtained by adopting the chlordane artificial antigen has better titer, specificity and affinity, the sensitivity can reach 0.1 mu g/L, and the cross reaction rate with other cyclopentadiene organochlorine pesticides is low.

Drawings

FIG. 1 is a synthetic route of the chlordane hapten of the invention

Detailed Description

The present invention will be described in further detail with reference to specific examples, which are only preferred embodiments of the present invention and are not intended to limit the present invention.