阅读说明：本技术 一株分泌蛇形菌素单克隆抗体的杂交瘤细胞株及其应用 (Hybridoma cell strain secreting serpentine monoclonal antibody and application thereof ) 是由 匡华 金国浩 胥传来 徐丽广 马伟 刘丽强 吴晓玲 宋珊珊 胡拥明 于 2019-12-12 设计创作，主要内容包括：一株分泌蛇形菌素单克隆抗体的杂交瘤细胞株及其应用,属于食品安全免疫检测领域。本发明分泌蛇形菌素单克隆抗体的杂交瘤细胞株OVA15,已保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC,保藏编号CGMCC No.18515。利用该菌株分泌获得的蛇形菌素单克隆抗体用于检测食品中蛇形菌素残留物。本发明获得的蛇形菌素单克隆抗体,对蛇形菌素有较好的检测灵敏度和特异性；其检测限,即抑制率20%-80%对应的加标量为1.57–22.6ng/mL,IC<Sub>50</Sub>值为5.97ng/mL；对蛇形菌素类似物交叉率小于1%。本发明还提供了一种全新的蛇形菌素完全抗原及其合成思路,采用该完全抗原筛选所获得的蛇形菌素单克隆抗体细胞株可以用于免疫分析检测。(A hybridoma cell strain secreting a serpentine monoclonal antibody and application thereof belong to the field of food safety immunodetection. The hybridoma cell strain OVA15 secreting the serpentine monoclonal antibody has been preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 18515. The serpentine monoclonal antibody secreted by the strain is used for detecting serpentine residues in food. The snake shape obtained by the inventionThe rhzomorph monoclonal antibody has better detection sensitivity and specificity to the serpentin; the detection limit, namely the addition amount corresponding to the inhibition rate of 20-80% is 1.57-22.6 ng/mL, and the IC is 50 The value was 5.97 ng/mL; the crossing rate of the serpentine analog is less than 1 percent. The invention also provides a brand-new snakelike rhzomorph complete antigen and a synthesis thought thereof, and the snakelike rhzomorph monoclonal antibody cell strain obtained by adopting the complete antigen screening can be used for immunoassay detection.)

1. A hybridoma cell strain OVA15 secreting serpentine monoclonal antibody has been deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences microorganism institute No. 3, West Lu No.1 institute of North Chen West Lu, Kyoho, Beijing, and is classified and named as monoclonal cell strain, the preservation date is 2019, 10 and 14 days, and the preservation number is CGMCC No. 18515.

2. A serpentine monoclonal antibody characterized by: it is secreted and produced by hybridoma cell strain OVA15 with the preservation number of CGMCC No.18515 as claimed in claim 1.

3. The use of a serpentine monoclonal antibody as claimed in claim 2, characterized in that: the method is used for detecting the serpentine bacterin residues in food.

4. The use of a serpentine monoclonal antibody according to claim 3, characterized in that: the food is specifically cereal.

5. The use of a serpentine monoclonal antibody according to claim 3, characterized in that: the detection method is to prepare a reagent for detecting the serpentin by an ELISA competition method.

6. The method for producing the hybridoma cell line OVA15 according to claim 1, wherein the method comprises the steps of: immunizing the serpentine to obtain a serpentine complete antigen; mixing and emulsifying the serpentine complete antigen and equivalent Freund's adjuvant, immunizing mouse, and collecting high titer and low IC₅₀The spleen cells of the immunized mice are fused with myeloma cells, and hybridoma cell strains OVA15 are obtained by screening subclones through ELISA detection.

7. The method for preparing hybridoma cell line OVA15 according to claim 6, wherein the serpentine complete antigen is prepared by the following steps:

(1) preparation of serpentine hapten: reacting the serpentine with succinic anhydride to introduce active carboxyl to obtain serpentine hapten;

(2) preparation of a serpentin complete antigen: dissolving a serpentine hapten in an N, N-dimethylformamide solution, adding N-hydroxysuccinimide NHS and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC with 3 times of serpentine substance mass, and magnetically stirring at room temperature for 6-8h to obtain solution A; adding hemocyanin KLH with the mass of 2000 times of snakehead rhzomorph into a phosphate buffer solution to obtain a solution B; and dropwise adding the solution A into the solution B, reacting at room temperature overnight, dialyzing by using a PBS solution, and removing unreacted small molecule hapten to obtain the serpentine complete antigen.

8. The method for preparing hybridoma cell line OVA15 according to claim 7, wherein the structural formula of the serpentine hapten is as follows:

9. the method for preparing hybridoma cell line OVA15 according to claim 6, wherein the structural formula of the serpentine complete antigen is as follows:

Technical Field

The invention relates to a hybridoma cell strain secreting a serpentine monoclonal antibody and application thereof, and belongs to the field of food safety immunodetection.

Background

Snakes (DAS) are highly toxic secondary metabolites produced by fusarium, belong to trichothecene type a toxins, are widely distributed in the nature, and seriously harm human and animal health. Serpentines are natural contaminants of agricultural feed and food worldwide, commonly found in cereals, especially wheat, corn, rice, and have been shown by clinical studies to be highly toxic to various animal species. The main manifestations are anorexia, vomiting, growth inhibition, neurotoxicity, immunotoxicity, genotoxicity and hepatotoxicity. The potential threat of anorexia caused by trichothecene type a toxins is of particular concern from a human and animal health perspective.

Highly sensitive and specific trichostatin assays are of critical importance for assessing the safety of food and animal feed, since they are a serious hazard to human and animal health.

The traditional detection methods of the serpentine are a gas chromatography-mass spectrometry method, a capillary electrophoresis method, a high performance liquid chromatography and a capillary electrophoresis-mass spectrometry method, however, the pretreatment methods are complex and time-consuming, and are not suitable for rapid detection of a large number of samples, and in order to maintain the benefits of consumers, a high-efficiency and rapid detection method for the serpentine is needed to be established.

An enzyme-linked immunosorbent assay (ELISA) is an extremely high-efficiency, sensitive and rapid detection method, has low requirement on the purity of a sample during detection, is simple and convenient to operate, and is suitable for on-site rapid detection of a large number of samples, however, the premise of using the ELISA to detect the serpentine is to obtain a monoclonal antibody with high specificity and high sensitivity to the serpentine, so that a method for preparing the monoclonal antibody with high specificity and high sensitivity to the serpentine is very critical.

The invention tries to prepare the serpentine monoclonal antibody through the hybridoma cell, but in the process of preparing the hybridoma cell strain capable of secreting the serpentine monoclonal antibody, further research is needed on how to prepare the serpentine hapten and the serpentine complete antigen and how to make a mouse generate strong immunity; whether the prepared hybridoma cell strain can secrete the serpentine monoclonal antibody or not needs further verification; the specificity and sensitivity of the secreted serpentine rhzomorph monoclonal antibody also need to be further verified.

Disclosure of Invention

The invention aims to overcome the defects and provides a hybridoma cell strain secreting a snakelike rhzomorph monoclonal antibody and application thereof, wherein the snakelike rhzomorph monoclonal antibody secreted by the hybridoma cell strain has better specificity and detection sensitivity (IC)₅₀Value of 5.97 ng/mL) can be used for establishing an immunological detection method of the serpentine and detectingThe food is tested for serpentine residues.

According to the technical scheme, the hybridoma cell strain OVA15 secreting the serpentine monoclonal antibody is deposited in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms, and is classified and named as a monoclonal cell strain by the microbiological research institute of China academy of sciences No. 3 of West Lu 1 Hospital, North Kyoho, the Beijing city, the date of deposition is 2019, 10 months and 14 days, and the number of deposition is CGMCC No. 18515.

The invention provides a preparation method of a hybridoma cell strain OVA15 secreting a serpentine monoclonal antibody, which comprises the following steps:

(1) preparing a snakelike bacterin hapten and a snakelike bacterin complete antigen, and preparing the obtained snakelike bacterin complete antigen into an antigen-containing Freund adjuvant and an antigen-containing incomplete Freund adjuvant;

(2) injecting the obtained Freund adjuvant containing the antigen into a BALB/c mouse body through back subcutaneous injection for multiple times of immunization, wherein the complete Freund adjuvant containing the antigen is adopted for the first immunization, and the incomplete Freund adjuvant containing the antigen is adopted for the boosting immunization;

(3) collecting blood from the mice on the 7 th day after the 3 rd immunization process, detecting the serum immune titer and the immune suppression capacity of the mice through indirect ELISA, and screening the mice with high content of the serpentine rhzomorph antibody in the serum to obtain immunized mice;

(4) performing last boosting immunization on the screened mice by using incomplete Freund's adjuvant, and performing sprint immunization by intraperitoneal injection, wherein the sprint immunization is performed by using a snakelike rhzomorph complete antigen without Freund's adjuvant;

(5) fusing splenocytes and myeloma cells of BALB/c mice 3 days after the completion of the impact immunization by a polyethylene glycol (PEG 4000) method, culturing the fused cells by an RPMI-1640 culture medium, detecting positive cell pores by using indirect ELISA, further determining the inhibition effect of the positive cell pores by using an indirect competitive ELISA method, carrying out 3 times of subcloning on the positive cell pores with the best inhibition by using a limiting dilution method, and finally screening to obtain a hybridoma cell strain OVA15 capable of secreting the serpentine monoclonal antibody.

The immune process in the steps (2) and (4) comprises 1 first immunity, 4 boosting immunizations and 1 sprint immunity, wherein the interval between the first immunity and the boosting immunizations is one month, the interval between the boosting immunizations is 21 days, the interval between the boosting immunizations and the sprint immunizations is 18 ~ 21 days, the first immunity dose is 80 mu g/mouse, the boosting immunity dose is 40 mu g/mouse, and the sprint immunity dose is 20 mu g/mouse.

The invention provides a hybridoma cell strain OVA15 secreting a serpentine monoclonal antibody, a preparation method of the hybridoma cell strain OVA15 secreting the serpentine monoclonal antibody and application of the hybridoma cell strain OVA15 secreting the serpentine monoclonal antibody in preparation of the serpentine monoclonal antibody.

The invention provides a snakelike rhzomorph hapten which has the following structural formula:

。

the invention provides a preparation method of a snakelike rhzomorph hapten. And extracting the residue with chloroform and water, combining organic phases, and continuously blowing the organic phases with nitrogen to obtain the derivative HS-DAS.

In one embodiment of the invention, 1mg of S.S.sub.S. is dissolved in 300. mu.L of pyridine, 7.2mg of succinic anhydride and 3.4mg of DMAP for catalysis are added, and the reaction is carried out under the condition of 50 ℃ and magnetic stirring in the dark for 5 hours. The reaction was then quenched by the addition of 50. mu.L of water and blown dry with nitrogen. The residue was purified with 1mL of chloroform and 1mL of water 1: 1, extracting for three times, combining organic phases, and continuously blowing the organic phases by using nitrogen to obtain the derivative HS-DAS.

The invention provides application of the serpentine hapten in preparation of serpentine complete antigen, hybridoma cell strains secreting serpentine monoclonal antibodies and serpentine monoclonal antibodies.

The invention provides a serpentine complete antigen, which has the following structural formula:

。

the invention provides a preparation method of the serpentine complete antigen, which is characterized by dissolving a serpentine hapten into an N, N-dimethylformamide solution, adding N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), and carrying out magnetic stirring reaction at room temperature (called liquid A). Adding hemocyanin (KLH) into phosphate buffer solution to obtain solution B (named as solution B), dropwise adding the solution A into the solution B, reacting at room temperature overnight, dialyzing by PBS solution, and removing unreacted small molecule hapten to obtain complete antigen.

In one embodiment of the invention, the method is to dissolve the serpentine hapten in N, N-dimethylformamide solution, add 3 times the mass of serpentine and N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), and magnetically stir the reaction at room temperature for 6-8h (called solution A). Adding 2000 times mass of hemocyanin (KLH) into phosphate buffer to obtain solution B (named as solution B), dropwise adding the solution A into the solution B, reacting at room temperature overnight, dialyzing by PBS solution, and removing unreacted small molecule hapten to obtain the serpentine complete antigen.

In one embodiment of the invention, the Phosphate Buffered Saline (PBS) is present at a concentration of 0.01mol/L, pH of 7.4.

The invention provides application of the serpentine complete antigen or the preparation method of the serpentine complete antigen in preparation of hybridoma cell strains secreting serpentine monoclonal antibodies and serpentine monoclonal antibodies.

In one embodiment of the invention, the serpentine monoclonal antibody is secreted and produced by a hybridoma cell strain OVA15 with the preservation number of CGMCCNo.18515.

The invention provides a preparation method of the serpentine monoclonal antibody, which comprises the steps of taking a BALB/c mouse, injecting paraffin oil into the abdominal cavity, injecting a hybridoma cell strain OVA15 with the preservation number of CGMCC No.18515 into the abdominal cavity, collecting ascites after injection, purifying the ascites, and storing the obtained monoclonal antibody at low temperature.

In one embodiment of the invention, the method is to take BALB/c mice 8-10 weeks old, inject 1mL paraffin oil into the abdominal cavity of each mouse, and inject 1X 10 paraffin oil into the abdominal cavity of each mouse 7 days later⁶Collecting ascites from 7 days, purifying the ascites by an octanoic acid-ammonium sulfate method, and storing the obtained monoclonal antibody at-20 ℃.

The invention provides an application of the serpentine monoclonal antibody or the preparation method of the serpentine monoclonal antibody in recognition of serpentine.

The invention provides a detection kit prepared by applying the hybridoma cell strain secreting the snakelike rhzomorph monoclonal antibody or the snakelike rhzomorph complete antigen or the snakelike rhzomorph monoclonal antibody, and the detection kit is used for detecting snakelike rhzomorph residues in food.

The food is specifically cereal.

The detection method is to prepare a reagent for detecting the serpentin by an ELISA competition method.

The invention has the beneficial effects that: the serpentine monoclonal antibody obtained by the invention has better detection sensitivity and specificity to serpentine; the detection limit, namely the addition amount corresponding to the inhibition rate of 20-80 percent, is 1.57-22.6 ng/mL, and the IC is₅₀The value was 5.97 ng/mL; crossing rate of serpentine analog is less than 1%, and crossing rate = (IC of serpentine)₅₀IC of/analogue₅₀）×100%）。

The invention also provides a brand-new snakelike rhzomorph complete antigen and a synthesis thought thereof, and the snakelike rhzomorph monoclonal antibody cell strain obtained by adopting the complete antigen screening can be used for immunoassay detection.

Biological material sample preservation: a hybridoma cell strain OVA15 secreting serpentine monoclonal antibody has been deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences microorganism institute No. 3, West Lu No.1 institute of North Chen West Lu, Kyoho, Beijing, and is classified and named as monoclonal cell strain, the preservation date is 2019, 10 and 14 days, and the preservation number is CGMCC No. 18515.

Drawings

FIG. 1 is a standard curve of inhibition of a serpentine monoclonal antibody against serpentine.

Detailed Description

The following examples are provided as further illustration of the invention and are not to be construed as limitations or limitations of the invention.

The media involved in the following examples are as follows: PMI-1640 medium.

The reagents involved in the following examples are as follows:

carbonate Buffer (CBS): weighing Na₂CO₃1.59 g，NaHCO₃2.93 g of the mixture is respectively dissolved in a small amount of double distilled water and then mixed, the double distilled water is added to about 800mL and mixed evenly, the pH value is adjusted to 9.6, the double distilled water is added to the volume of 1000mL, and the mixture is stored for later use at 4 ℃;

phosphate Buffered Saline (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH₂PO₄，2.9g Na₂HPO₄·12 H₂Dissolving O in 800mL of pure water, adjusting the pH value to 7.2-7.4 by using NaOH or HCl, and fixing the volume to 1000 mL;

PBST: PBS containing 0.05% tween 20;

TMB color development liquid: solution A: na (Na)₂HPO₄·12H₂18.43g of O, 9.33g of citric acid and pure water to reach the constant volume of 1000 mL; and B, liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. And the volume ratio of the solution B to the solution B is 5: 1 to obtain the TMB color developing solution which is mixed at present.

The detection methods referred to in the following examples are as follows:

the serpentine inhibition rate detection method comprises the following steps: the most suitable antigen and antibody concentrations in the ic-ELISA were selected by a checkerboard assay. Antigen was diluted to 0, 01, 0.03, 0.1 and 0.3. mu.g/mL with coating buffer and antibody was diluted to 0.03, 0.1, 0.3 and 1. mu.g/mL with antibody diluent. After the optimal working point is selected, the serpentine standard is diluted to 8 concentrations (0, 0.27, 0.82, 2.47, 7.41, 22.22, 66.67, 200 ng/mL),the results are shown in FIG. 1, following the ic-ELISA protocol, and finally mapped with originPro 8.5. Obtaining DAS standard inhibition curve and calculating IC₅₀。