CN110724192B - Hybridoma cell strain secreting serpentine monoclonal antibody and application thereof - Google Patents

Abstract

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 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% is 1.57-22.6 ng/mL, and the IC is₅₀The 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.

Description

Hybridoma cell strain secreting serpentine monoclonal antibody and application thereof

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)₅₀A value of 5.97 ng/mL) can be used for establishing an immunological detection method of the serpentine and detecting the serpentine residue in the food.

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; one month is left between the first immunization and the boosting immunization, 21 days is left between the boosting immunization, and 18-21 days is left between the boosting immunization and the impact immunization; the first immunization dose is 80 mug/mouse, the boosting immunization dose is 40 mug/mouse, and the sprint immunization dose is 20 mug/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 hybridoma cell strain OVA15 with the preservation number of CGMCC No. 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 Na₂CO₃ 1.59 g，NaHCO₃2.93 g, respectively dissolved inMixing a small amount of double distilled water, adding the double distilled water to about 800mL, uniformly mixing, adjusting the pH value to 9.6, adding the double distilled water to a constant volume of 1000mL, and storing at 4 ℃ for later use;

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 selection of the optimal working point, the serpentin standards were diluted to 8 concentrations (0, 0.27, 0.82, 2.47, 7.41, 22.22, 66.67, 200 ng/mL), followed by ic-ELISA protocol and finally mapped with originPro 8.5, as shown in FIG. 1. Obtaining DAS standard inhibition curve and calculating IC₅₀。

Example 1: synthesis of serpentine hapten

The synthesis route is as follows:

。

the small serpentine molecule has no immunogenicity, and can not stimulate a mouse to generate immune response so as to generate an antibody, so that the serpentine needs to be coupled to protein by a protein connection technology to obtain the immunogenicity; active groups commonly used in the protein coupling technology comprise amino, carboxyl, hydroxyl, sulfydryl and the like, carboxyl is derived from DAS molecules, and a spacer arm is lengthened, so that small molecules can be fully exposed after being coupled with proteins, and the immune effect is improved.

1mg DAS was dissolved in 300. mu.L pyridine, then 7.2mg succinic anhydride and a catalytic amount of 3.4mg DMAP were added. The reaction is stirred for 5h at 50 ℃ in the dark. The reaction was then quenched by the addition of 50. mu.L of ultrapure water and blown dry with nitrogen. Extracting the residue with 1mL chloroform and 1mL water at a ratio of 1: 1 for three times, collecting organic substances, and sequentially drying with nitrogen to obtain derivative HS-DAS.

Example 2: synthesis of serpentine complete antigen

Dissolving the serpentine hapten (HA-DAS) in N, N-dimethylformamide solution, adding 3 times of serpentine substance amount of N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), and reacting for 6-8h (called solution A) under magnetic stirring at room temperature. Adding 2000 times of S-streptocin hemocyanin (KLH) into phosphate buffer solution to obtain solution B (called 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.

Example 3: synthesis of serpentine capsulgen

1mg DAS was dissolved in 200. mu. L N, N-dimethylformamide, 8mg N, N-Carbonyldiimidazole (CDI) was added and magnetic stirring was performed at 37 ℃ for 2 h. The reaction solution was added dropwise to 1mL of 0.05M sodium carbonate buffer (bovine serum albumin BSA 2 mg), stirred at room temperature overnight, dialyzed against 0.01M PBS for three days, and stored at-20 ℃. DAS-CDI-BSA acts as the coating antigen.

Example 4: preparation of hybridoma cell strain secreting serpentine rhzomorph monoclonal antibody

1. Obtaining immunity of animals

Mixing DAS complete antigen and equivalent Freund adjuvant, emulsifying, and performing neck and back subcutaneous multipoint injection immunization (except puncture immunization) on BALB/c mice; complete Freund adjuvant is used for the first immunization, and the dosage is 80. mu.g/vaccine; incomplete Freund's adjuvant is used for multiple times of boosting immunization, and the dosage is reduced by half to be 40 mu g/mouse; the thorny immunity does not use an adjuvant, the thorny immunity is directly diluted by normal saline and then injected into the abdominal cavity, and the dosage is reduced by half to obtain 20 mu g/mouse; one month is separated between the first immunization and the second boosting immunization, 21 days are separated between the multiple boosting immunizations, and 18-21 days are separated between the sprint immunization and the last boosting immunization; the immune effect of the mouse is observed by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), namely the titer and inhibition of the mouse serum are detected;

2. cell fusion

After three days of the spurting immunization, cell fusion is carried out according to a conventional PEG (polyethylene glycol, molecular weight is 4000) method, and the specific steps are as follows:

a. taking eyeballs and blood, immediately putting the mice into 75% alcohol for disinfection after the mice are killed by a cervical vertebra dislocation method, soaking for about 5min, taking out the spleen of the mice by aseptic operation, properly grinding the spleen by using a rubber head of an injector, passing through a 200-mesh cell screen to obtain a spleen cell suspension, collecting, centrifuging (1200 rpm, 8 min), washing the spleen cells for three times by using an RPMI-1640 culture medium, diluting the spleen cells to a certain volume after the last centrifugation, and counting for later use;

b. collecting SP2/0 cells: 7-10 days before fusion, SP2/0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) at 5% CO₂In the incubator, SP2/0 tumor cell number is required to reach (1-4) x 10 before fusion⁷Ensuring SP2/0 tumor cells to be in logarithmic growth phase before fusion, collecting the tumor cells during fusion, suspending the tumor cells in RPMI-1640 basic culture solution, and counting the cells;

c. the fusion process is 7 min: 1min, 1mL of PEG 4000 was added to the cells dropwise from slow to fast; standing for 2 min; dropping 1mL of RPMI-1640 culture medium within 1min at 3min and 4 min; dropping 2mL of RPMI-1640 culture medium within 1min at 5min and 6 min; at 7min, 1mL of RPMI-1640 culture medium is added dropwise every 10 s; then carrying out warm bath at 37 ℃ for 5 min; centrifuging (800 rpm, 8 min), discarding supernatant, resuspending in RPMI-1640 screening medium containing 20% fetal calf serum, 2% 50 × HAT, adding to 96-well cell plate at 200 μ L/well, standing at 37 deg.C and 5% CO₂Culturing in an incubator;

3. cell screening and cell line establishment

Performing RPMI-1640 screening culture medium half-exchange on fused cells on the 3 rd day of cell fusion, performing total-exchange with RPMI-1640 transitional culture medium containing 20% fetal calf serum and 1% 100 XHT on the 5 th day, and taking cell supernatant on the 7 th day for screening;

the screening is divided into two steps: firstly, screening out positive cell holes by using an ic-ELISA method, secondly, selecting serpentine as a standard substance, and determining the inhibition effect of positive cells by using the ic-ELISA method;

selecting a cell hole with good inhibition on a serpentine standard substance, carrying out subcloning by adopting a limiting dilution method, and carrying out detection by using the same method after seven days;

and carrying out subcloning for three times according to the method to finally obtain the serpentine monoclonal antibody cell strain.

Example 5: preparation and identification of serpentine monoclonal antibody

Taking BALB/c mice 8-10 weeks old, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 10⁶Serpentine hybridoma cells, ascites fluid was collected from the seventh day, and antibody purification was performed on the ascites fluid by the octanoic acid-saturated ammonium sulfate method.

Under the condition of partial acid, the caprylic acid can precipitate other hybrid proteins except IgG immunoglobulin in the ascites, then the centrifugation is carried out, and the precipitate is discarded; then, the IgG type monoclonal antibody was precipitated with an ammonium sulfate solution of the same saturation, centrifuged, the supernatant was discarded, and the supernatant was dissolved in a 0.01M PBS solution (pH 7.4), dialyzed and desalted to finally obtain a purified monoclonal antibody, which was stored at-20 ℃.

Using an indirect competition ELISA, a graph was made using originPro 8.5, and the results are shown in FIG. 1. Obtaining a DAS standard inhibition curve, R thereof² = 0.99946，Y = 0.207+1.631/[1+(x/5.967)^1.039]The detection limit (the addition amount corresponding to the inhibition rate of 20-80%) is 1.57-22.6 ng/mL, IC₅₀The value is 5.97ng/mL, the crossing rate of the analog of the serpentine is less than 1%, which indicates that the analog of the serpentine has good sensitivity to the serpentine and can be used for immunoassay detection of the serpentine.

(Cross-over Rate = (IC of Serpentine)₅₀IC of/analogue₅₀）×100%）

Example 6: application of serpentine rhzomorph monoclonal antibody

The monoclonal antibody prepared from the hybridoma cell strain through in-vivo ascites is applied to an ELISA addition recovery test of DAS, and the method comprises the following specific steps:

(1) coating a 96-well enzyme label plate with coating antigen with the concentration of 0.3 mu g/mL diluted by Carbonate Buffer Solution (CBS), coating 100 mu L of the enzyme label plate in each well at 37 ℃ for 2h, washing the plate with PBST washing liquor for three times, wherein 200 mu L of the washing liquor in each well is used for 3min, and drying by beating;

(2) sealing with CBS containing 0.2% gelatin, sealing at 37 deg.C for 2 hr with 200 μ L per well, washing the plate with PBST lotion for three times, each time with 200 μ L per well, each time for 3min, and drying;

(3) preparing 0, 0.27, 0.82, 2.47, 7.41, 22.22, 66.67 and 200 mu g/L of DAS standard solution by using Phosphate Buffered Saline (PBS), respectively adding the standard solution and the extract of a sample to be detected into a closed enzyme label plate, wherein each hole is 50 mu L, repeating 3 holes for each sample, adding 50 mu L of anti-DAS monoclonal antibody diluted by 1:32000 into each hole, reacting at 37 ℃ for 0.5h, and then washing and drying the plate;

(4) adding 100 μ L of HRP-labeled goat anti-mouse IgG secondary antibody diluted with PBS containing 0.1% gelatin at a ratio of 1:3000 into each well, reacting at 37 ℃ for 0.5h, washing and drying;

(5) adding 100 μ L of TMB developing solution into each well, developing at 37 deg.C for 15min, and adding 50 μ L of 2M H into each well₂SO₄Stopping solution, measuring the light absorption value at 450 nm;

(6) adding and recovering and sample pretreatment:

rice was selected as the test sample.

Washing and cutting the sample to be tested, weighing three samples (2 g each), and adding DAS standard (according to antibody linear range and IC) of 100ppb, 250ppb and 500ppb into the sample₅₀Setting the adding concentration), adding 20mL of 25% ethanol-PBS extracting solution for extraction, violently shaking for 1h, centrifuging, and taking supernatant for dilution by 5 times. (i.e., a 50-fold dilution altogether to reduce the effect of the sample matrix).

The additive recovery test was performed by indirect competitive ELISA with 99%, 104%, and 110% recovery, respectively.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (8)

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 hybridoma cell line OVA15 according to claim 1, wherein: the preparation method of the serpentine complete antigen for preparing the hybridoma cell strain OVA15 comprises 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.

7. The hybridoma cell line OVA15 according to claim 6, wherein the structural formula of the serpentine hapten is as follows:

。

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

。

Images