CN107365749B

CN107365749B - Hybridoma cell strain secreting anti-vomitoxin monoclonal antibody and application thereof

Info

Publication number: CN107365749B
Application number: CN201710816445.0A
Authority: CN
Inventors: 万宇平; 袁媛; 吴小胜; 杨昌松; 南铁贵; 何方洋; 洪小栩; 李行
Original assignee: Beijing Kwinbon Biotechnology Co Ltd
Current assignee: Beijing Kwinbon Biotechnology Co Ltd
Priority date: 2017-09-12
Filing date: 2017-09-12
Publication date: 2020-04-28
Anticipated expiration: 2037-09-12
Also published as: CN107365749A

Abstract

The invention provides a hybridoma cell strain F-3-4 secreting an anti-vomitoxin monoclonal antibody, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 14304. The anti-vomitoxin monoclonal antibody secreted by the cell strain has high sensitivity, good specificity and good anti-interference performance, and 50 percent of inhibition concentration IC of the anti-vomitoxin₅₀22.1 mu g/L, has better anti-interference performance on possible mycotoxin, pesticide, heavy metal, auxiliary materials used in the processing process, pollution bacteria and the like in the traditional Chinese medicine, provides conditions for immunodetection of vomitoxin in the traditional Chinese medicine, and has practical application value.

Description

Hybridoma cell strain secreting anti-vomitoxin monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a hybridoma cell strain secreting an anti-vomitoxin monoclonal antibody and application thereof.

Background

The traditional Chinese medicinal materials are likely to pollute fungi due to the influence of the properties of the traditional Chinese medicinal materials and external factors in the processes of production, harvesting, processing, transportation, storage and the like, and further mildew and pollute mycotoxin. The mildew can not only affect the quality of the traditional Chinese medicinal materials and cause the traditional Chinese medicinal materials to lose the original medicinal value and cause huge economic loss, but also can cause serious damage to the liver, the kidney, the nerves, the hematopoietic system and the like of a patient and even possibly induce cancer in the mildew process. Meanwhile, along with the popularization of the health care concept of the traditional Chinese medicine, more and more traditional Chinese medicinal materials with homology of medicine and food are used for daily diet and health care of people, the market demand is large, if the traditional Chinese medicinal materials are polluted by mycotoxin, the traditional Chinese medicinal materials inevitably harm the health of more people, the economic burden of individuals and medical systems is increased, and the harmony and stability of the society are more likely to be further influenced.

Vomitoxin (vomintoxin), also known as Deoxynivalenol (DON), belongs to trichothecene compounds, and is mainly produced by fusarium graminearum (f.graminearum) and fusarium flavum (f.culmorum), and most of the fungi slowly grow in cereal crops in low-temperature and humid environments. Vomitoxin is often coexisted with other mycotoxins, and the highest concentration of vomitoxin separated from grains and feed reaches 92 mg/kg. In 1998, vomitoxin was listed as three carcinogens in the evaluation report published by the international agency for research on cancer (IARC). Because of the serious harm of vomitoxin, the general attention of all countries is drawn.

The analytical determination method of vomitoxin is basically applied to the food field at the present stage, and detection methods such as thin layer chromatography, enzyme linked immunosorbent assay, high performance liquid chromatography and the like are generally adopted. The detection of vomitoxin in traditional Chinese medicines is reported less, and a high performance liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS) determination method for vomitoxin in 4 traditional Chinese medicines such as spina date seeds, fermented soybean, semen coicis, fructus psoraleae and the like is established only by referring to maodan and the like [ LC-MS method for determining vomitoxin in 4 traditional Chinese medicines, chinese pharmacist, 2014, 17 (4): 578-581], which has accurate detection and low detection limit, but requires complicated sample pretreatment process, specialized equipment and specialized personnel training, and is greatly limited in practical application.

The immunoassay method, which is an analysis method for detecting various substances using an antigen-antibody specific binding reaction, can make up all of the above disadvantages, and the key to establishing an immunoassay method for small molecule compounds is the ability to produce antibodies with high affinity and high specificity for small molecule compounds.

Disclosure of Invention

The monoclonal antibody prepared by the cell strain has better specificity and higher detection sensitivity to DON, has better anti-interference performance to mycotoxin, pesticide, heavy metal, auxiliary materials used in the processing process, pollution bacteria and the like possibly existing in the traditional Chinese medicine, and lays a foundation for establishing an immunological detection method for the DON in the traditional Chinese medicine.

The technical scheme of the invention is that a hybridoma cell strain secreting an anti-vomitoxin monoclonal antibody is named as a vomitoxin monoclonal antibody hybridoma cell strain F-3-4, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.14304 and the preservation date of 2017, 06 and 15 days.

The anti-vomitoxin monoclonal antibody is generated by the secretion of a vomitoxin monoclonal antibody hybridoma cell strain F-3-4 with the preservation number of CGMCC No.14304, and is applied to the analysis and detection of vomitoxin in traditional Chinese medicines.

The invention provides a basic preparation process of an F-3-4 cell strain, which comprises the following steps:

(1) synthesis and identification of DON hapten: dissolving vomitoxin 50mg in 5mL of ethanol; taking 29.17mg of p-aminobenzene sulfinic acid, adding 2mL of water for dissolving, adding into the vomitoxin ethanol solution, adding 3mL of 1mol/L diluted hydrochloric acid, and stirring at room temperature for 24 hours; stopping the reaction, adjusting the pH to 7 by using sodium hydroxide, adding 10mL of water, adding 15mL of ethyl acetate, performing shaking extraction, standing for layering, removing a water phase, concentrating and evaporating an organic phase, applying a silica gel column, and eluting and separating dichloromethane/methanol (10/1, V/V) to obtain 60mg of sulfoaminated vomitoxin, namely the DON hapten, wherein the yield is 78.95%, and the result is identified by nuclear magnetic resonance hydrogen spectrum;

(2) complete antigen synthesis: respectively coupling the DON hapten with Bovine Serum Albumin (BSA) and Ovalbumin (OVA) to obtain an immune antigen (DON-BSA) and an envelope antigen (DON-OVA);

(3) animal immunization: taking 10 healthy female Balb/c mice (divided into two groups of A and B, and 5 mice in each group) in 6-8 weeks, emulsifying the mice by Freund complete adjuvant for primary immunization, and injecting the mice subcutaneously at the back of the neck and the back at multiple points, wherein the immunization dose of each mouse is 200 mu g of DON-BSA; then boosting immunity and injecting subcutaneous multi-point on the back of the neck once every two weeks, and emulsifying by Freund incomplete adjuvant; the physiological saline is used for replacing Freund incomplete adjuvant in the last immunization, intraperitoneal injection is adopted, and the injection dosage is the same as the previous times; detecting serum titer by indirect ELISA;

(4) cell fusion and cell line screening: on the third day after the enhancement of the immunity, fusing the spleen cells of the mice and myeloma cells of the mice by a polyethylene glycol (PEG4000) method, culturing by complete culture solution, detecting cell strains secreting DON by using an indirect ELISA method, measuring the inhibition effect of the cell strains by using an indirect competitive ELISA method, screening positive cell strains which are best inhibited, carrying out three times of subcloning, and finally obtaining a hybridoma cell strain F-3-4;

(5) antibody preparation and purification and characterization: injecting liquid paraffin into Balb/c mice for 6-8 weeks, wherein each mouse is 500 mu L; injecting 0.5mL hybridoma cell CGMCC No.14304 per mouse into abdominal cavity after 10 days, collecting ascites from the seventh day, purifying the ascites by octanoic acid-ammonium sulfate method, analyzing purification effect by SDS-PAGE gel electrophoresis method, measuring titer, subtype and cross reactivity of the obtained monoclonal antibody, and storing at-20 deg.C;

(6) the application of the antibody is as follows: the anti-vomitoxin monoclonal antibody secreted by the hybridoma cell strain F-3-4 is used for preparing an vomitoxin enzyme linked immunosorbent assay kit for analyzing and detecting the vomitoxin in the traditional Chinese medicine.

The invention has the beneficial effects that:

(1) the hybridoma cell strain F-3-4 provided by the invention can be used for preparing a high-titer anti-vomitoxin monoclonal antibody, and the titer measured by an ELISA method for anti-vomitoxin mouse ascites antibody is not less than 20000.

(2) The anti-vomitoxin monoclonal antibody provided by the invention has the advantages of high sensitivity, good specificity, good anti-interference performance and 50% inhibition concentration IC (integrated Circuit) on vomitoxin₅₀22.1 mu g/L, and has better anti-interference performance on possible mycotoxin, pesticide, heavy metal, auxiliary materials used in the processing process, pollution bacteria and the like in the traditional Chinese medicine.

(3) The anti-vomitoxin monoclonal antibody provided by the invention can be applied to determination of the content of vomitoxin in traditional Chinese medicines.

Biological material sample preservation: a hybridoma cell strain secreting anti-vomitoxin monoclonal antibody is preserved in China general microbiological culture Collection center (CGMCC), and the address: the collection date of No. 3 Xilu Beijing, Chaoyang, and the institute of microbiology of Chinese academy of sciences, 2017, 06 and 15 days, and the collection number is CGMCC No. 14304.

Drawings

FIG. 1 route diagram for synthesis of emetic toxin haptens

FIG. 2 competitive ELISA standard curve for vomitoxin

FIG. 3A graph of the Loit/Log standard for competitive ELISA for vomitoxin

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 invention is further illustrated by the following examples.

Example 1: screening of hybridoma cell line F-3-4

1. Hapten synthesis and identification

The synthetic route of DON hapten is shown in figure 1.

Dissolving vomitoxin 50mg in 5mL of ethanol; taking 29.17mg of p-aminobenzene sulfinic acid, adding 2mL of water for dissolving, adding into the vomitoxin ethanol solution, adding 3mL of 1mol/L diluted hydrochloric acid, and stirring at room temperature for 24 hours; stopping the reaction, adjusting the pH to 7 by using sodium hydroxide, adding 10mL of water, adding 15mL of ethyl acetate, performing shaking extraction, standing for layering, removing a water phase, concentrating an organic phase, evaporating to dryness, passing through a silica gel column, and eluting and separating by using dichloromethane/methanol (10/1, V/V) to obtain 60mg of sulfoaminated vomitoxin, namely the DON hapten, wherein the yield is 78.95%.

Taking the hapten, performing nuclear magnetic resonance hydrogen spectrum identification,¹H-NMR(CDCl₃,300MHz)δ：7.50(2H,d,ArH)，6.69(2H,d,ArH)，6.27(2H,s,NH₂)，3.93(1H,s,CH)，3.61(1H,ddd,CH)，3.58(1H,s,OH)，3.56(1H,s,CH)，3.25(1H,d,CH)，2.34(2H,s,CH)，2.4(1H,s,CH)，1.71(1H,s,CH)，1.46(2H,s,CH₂)，1.11(3H,s,CH₃). In the spectrum, the resonance absorption peak of aromatic amine hydrogen on a spacer arm is shown as chemical shift delta being 6.27, and benzene on the spacer arm is shown as delta being 7.5 and 6.69And (3) ring hydrogen absorption peaks, wherein the existence of the absorption peaks proves that the spacer arm coupling is successful and the structure of the DON hapten is correct.

2. Complete antigen synthesis

Synthesis of immune antigen-coupling of DON hapten with BSA

Dissolving 17mg of DON hapten in 1mL of ethanol, adding 0.2mL of 1mol/L diluted hydrochloric acid, stirring at 0-5 ℃ for 10min, dissolving 8.4mg of sodium nitrite in 1mL of water, adding the dissolved sodium nitrite into a hapten solution, and stirring at 0-5 ℃ for 1h to obtain a hapten activated solution A; dissolving 50mg of BSA in 0.1mol/L carbonate buffer solution, stirring at 0-5 ℃ for 10min, dropwise adding all the solution A, continuously stirring for 2h, dialyzing and purifying with 0.02mol/L phosphate buffer solution for 3 days, changing the solution 3 times per day, and subpackaging at-20 ℃ for later use.

Synthesis of envelope antigen-coupling of DON hapten with OVA

Dissolving 10mg of DON hapten in 1mL of ethanol, adding 0.2mL of 1mol/L diluted hydrochloric acid, stirring at 0-5 ℃ for 10min, dissolving 4mg of sodium nitrite in 1mL of water, adding the dissolved sodium nitrite into the hapten solution, and stirring at 0-5 ℃ for 1h to obtain hapten activated solution A; dissolving OVA50mg in 0.1mol/L carbonate buffer solution, stirring at 0-5 deg.C for 10min, adding all solution A, stirring for 2 hr, dialyzing and purifying with 0.02mol/L phosphate buffer solution for 3 days, changing solution 3 times per day, and packaging at-20 deg.C.

3. Animal immunization

Taking 10 healthy female Balb/c mice (divided into two groups of A and B, and 5 mice in each group) in 6-8 weeks, emulsifying the mice by Freund complete adjuvant for primary immunization, and injecting the mice subcutaneously at the back of the neck and the back at multiple points, wherein the immunization dose of each mouse is 200 mu g of DON-BSA; then boosting immunity and injecting subcutaneous multi-point on the back of the neck once every two weeks, and emulsifying by Freund incomplete adjuvant; the last immunization uses normal saline to replace Freund's incomplete adjuvant, and is injected in the abdominal cavity, and the injection dosage is the same as the previous times. The specific immunization procedure is shown in table 1.

Table 1 mouse immunization procedure

And (3) for the third time, the fourth time and 7d after the boosting immunization, the tail of the mouse is cut off, blood is taken, and the serum titer of the mouse is measured by an ELISA method, wherein the specific steps are as follows:

(1) diluting the coated antigen (DON-OVA) with 0.05mol/L of carbonate buffer solution with pH9.6 at a ratio of 1:1000, coating an enzyme label plate with 100 μ L of each well, incubating at 37 ℃ for 2h, removing the coating solution, washing with PBST for 1 time, and patting to dry;

(2) adding 150 mu L of sealing liquid into each hole, reacting at 37 ℃ for 2h, then pouring off the sealing liquid, and patting to dry;

(3) adding 50 mu L of antiserum diluted by PBS in each hole, reacting at 25 ℃ for 30min, then removing the reaction liquid, washing 3-5 times by PBST, and patting dry at intervals of 30s each time;

(4) adding 100 mu L/hole of enzyme-labeled secondary antibody (1:1000) diluted by PBS, reacting for 30min at 25 ℃, washing for 3-5 times by PBST, and patting dry at intervals of 30s each time;

(5) adding 50 μ L of substrate developing solution A and B into each well, reacting at 25 deg.C in dark for 15min, adding 50 μ L of 2mol/L H into each well₂SO₄Terminating the reaction by the solution;

(6) measuring OD value with wavelength of 450nm by enzyme-labeling instrument, and determining OD of sample hole₄₅₀The titer of positive sera was determined as a dilution factor close to 1.

4. Cell fusion

(1) Preparing feeder cells: the Balb/c mice of 8-10 weeks old are killed after neck breakage, soaked in 75% alcohol for 5min, immediately placed in an ultra-clean bench with the abdomen facing upwards in a plate or fixed on a dissecting plate. The skin of the abdomen of the mouse is clamped by an ophthalmic forceps, a small opening is cut by scissors, and the peritoneum is not cut to avoid the outflow and pollution of the abdominal cavity fluid. Then blunt dissection was performed up and down with scissors to fully expose the peritoneum. Wiping peritoneum with alcohol cotton ball for sterilization. 5mL of RPMI-1640 basic culture solution was aspirated by a syringe, injected into the abdominal cavity of the mouse, the syringe was gently withdrawn, and the leg and tail of the mouse were shaken several times. The liquid in the abdominal cavity is pumped back by the original syringe and is injected into the centrifuge tube. The operation is repeated for 3-4 times. Centrifuging at 1000r/min for 10min, and discarding the supernatant. Resuspending the cells with 20-50 mL of complete culture medium, adding 100 μ L/well dropwise to the culture plate, and placing in an incubator for later use.

(2) Preparation of splenocytes: 3d after enhancing the immunity, taking an immune Balb/c mouse, collecting blood from an orbit, dislocating and killing the mouse, disinfecting the mouse in 75% alcohol, taking the spleen, removing connective tissues, preparing a spleen cell suspension, transferring the spleen cell suspension into a50 mL centrifuge tube, adding RPMI-1640 to 30mL, centrifuging the spleen cell suspension at 1500-2000 r/min for 5min, removing a supernatant, adding RPMI-1640 to 30mL, and counting the number for later use.

(3) Myeloma cell preparation: taking 3 bottles of myeloma cells with good growth state (the number of living cells is more than 95 percent), completely blowing down the myeloma cells, transferring the myeloma cells into a50 mL centrifuge tube, adding RPMI-1640 to 30mL, centrifuging at 1500-2000 r/min for 5min, discarding the supernatant, adding RPMI-1640 to 30mL, and counting for later use.

(4) Cell mixing: spleen cells and myeloma cells are mixed and centrifuged at 1500-2000 r/min for 5min, wherein the ratio of the spleen cells to the myeloma cells is 8: 1.

(5) Cell fusion: centrifuging the mixed cells, pouring out the supernatant, making the precipitated cell mass into paste, placing the paste in a water bath at 37 ℃, adding 1mL of fusion agent which is polyethylene glycol (PEG)4000 within 1min, acting for 2min, slightly stirring the cells, adding 20mL of serum-free PEG nutrient solution within 4min, centrifuging at 1000r/min for 10min, and discarding the supernatant. The cells were resuspended in 20-50 mL of complete medium, plated on 96-well feeder cells-containing cell culture plates at 100. mu.L per well, and placed in an incubator.

5. Cell line selection

And (3) when the cells grow to 1/2-1/3 of the bottom of the hole, carrying out antibody detection. Screening culture wells with hybridoma cell growth by adopting an ELISA method, wherein the screening comprises two steps: in the first step, positive cell holes are screened by indirect ELISA, in the second step, DON is selected as a standard substance, and inhibition effect determination is carried out on positive cells by indirect competition ELISA. Wells with better inhibition of DON standards were selected, subcloned by limiting dilution, and tested in the same manner. Repeating the steps for three times to obtain the cell strain F-3-4 capable of stably secreting the anti-vomitoxin monoclonal antibody. The cell strain is preserved in China general microbiological culture Collection center (CGMCC for short, the address: the great Tunu way of the sunny region in Beijing, China academy of sciences, the postal code 100101) in 2017, 15.06.15.7.4.A preservation number is CGMCC No. 14304.

Example 2: preparation, purification and characteristic identification of anti-vomitoxin monoclonal antibody

1. Preparation of ascites

Liquid paraffin was injected into Balb/c mice for 6-8 weeks at 500. mu.L/mouse. Collecting hybridoma cell CGMCC No.14304 in logarithmic growth phase after 10 days with RPMI-1640 basic culture medium, counting with blood counting plate and microscope to cell concentration of 1.0 × 10⁶～1.5×10⁶In the size per mL range. Each mouse is injected with 0.5mL hybridoma cell CGMCC No.14304 into abdominal cavity. Note that after one week the abdomen of the mouse was enlarged, ascites was collected in the abdomen of the mouse with a sterile syringe once every one to two days, and this was repeated until the mouse died naturally. Centrifuging at 4 deg.C for 5min at 5000r/min, collecting supernatant, and removing fat and protein membrane floating on the upper layer of abdominal water.

2. Antibody purification

The monoclonal antibody is purified by adopting an octanoic acid-ammonium sulfate method, and the method comprises the following specific steps:

(1) the ascites was thawed by taking out from a freezer at-20 ℃. The ascites fluid is filtered with double-layered filter paper to primarily remove fat flakes, cell debris and other impurities. Centrifuging at 12000r/min for 15min, taking supernatant, and discarding precipitate. Accurately measuring the ascites volume;

(2) magnetically stirring and uniformly mixing 1 part by volume of ascites and 3 parts by volume of acetate buffer solution, and adjusting the pH value to 4.5-4.8 by using 2mol/L HCl;

(3) slowly adding n-octanoic acid under magnetic stirring, adding 33 μ L of n-octanoic acid into 1mL of ascites, magnetically stirring at room temperature for 30min, and standing at 4 deg.C for 2 hr;

(4) centrifuging at 12000r/min for 5min, collecting supernatant, filtering with double-layer filter paper, and collecting filtrate;

(5) the volume of the filtrate was measured, 1/10 volumes of 0.1mol/L PBS pH7.4 were added, and the pH was adjusted to 7.4 with 2mol/L NaOH (recording the volume of NaOH);

(6) precooling the supernatant in an ice bath, adding solid ammonium sulfate to 0.277g/mL while stirring, adding the solid ammonium sulfate within 30min, and standing at 4 ℃ overnight;

(7) centrifuging at 12000r/min for 15min, and discarding the supernatant. The pellet was dissolved in a volume of 0.01mol/L PBS. Dialyzing with PB for two days, then dialyzing with 0.01mol/L PBS for two days, collecting dialysate, centrifuging at 12000r/min for 15min, collecting supernatant, and storing at-20 deg.C;

(8) the purified antibody was analyzed for purification by SDS-PAGE gel electrophoresis.

3. Antibody titer determination

The antibody titer was determined by indirect ELISA method, as described in reference example 1, 3. determination of serum titer of animal immunization. The result shows that the titer of the anti-vomitoxin monoclonal antibody is more than or equal to 20000.

4. Antibody subtype identification

Identification of subtype of anti-vomitoxin monoclonal antibody secreted by hybridoma cell strain F-3-4 as IgG by using commercially available SIGMA subtype identification kit₁And (4) molding.

5. Antibody cross-reactivity assay

The indirect competitive ELISA method is adopted for determination, and the specific steps are as follows:

(3) adding 50 mu L of vomitoxin standard working solution (the concentration is 0, 7.5, 25, 75 and 250 mu g/L respectively) which is serially diluted into each well, then adding 50 mu L of monoclonal antibody which is diluted by PBS (1: 20000), reacting at 25 ℃ for 30min, then pouring off the reaction solution, washing 3-5 times by PBST (PBST), and beating to dry at intervals of 30s each time;

(6) and measuring the OD value of the wavelength at 450nm by using a microplate reader.

And (3) taking the logarithmic value of the concentration of the vomitoxin as an abscissa and taking the percent absorbance value (the percentage of the OD value of the standard substance in each concentration to the OD value of the hole without the standard substance) as an ordinate to draw a standard curve. At the same time, the following interfering substances were diluted with standard buffer solutions to the addition values listed in the tables for analysis, and the cross-reactivity (recovery value/addition value × 100%) was calculated from the OD values corresponding to drug color development and the recovery values analyzed by the standard curve, and the results are shown in table 2.

TABLE 2 anti-interference data for other substances commonly found in Chinese medicine

As can be seen from Table 2, there is no interference with properties of the antithetic toxin monoclonal antibody by mycotoxin, pesticide, heavy metal, adjuvant used in the processing, and contaminating bacteria that may be present in the Chinese medicinal herbs.

Example 3: application of anti-vomitoxin monoclonal antibody

The anti-vomitoxin monoclonal antibody secreted by the hybridoma cell strain F-3-4 is used for preparing an vomitoxin enzyme linked immunosorbent assay kit for analyzing and detecting the vomitoxin in the traditional Chinese medicine.

1. Composition of enzyme linked immunosorbent assay kit

(1) An ELISA plate coated with DON-OVA conjugate;

(2) enzyme-labeled anti-emetic toxin monoclonal antibodies: the anti-vomitoxin monoclonal antibody described in the embodiment 2 is prepared by coupling with Horse Radish Peroxidase (HRP);

(3) vomitoxin standard substance: the concentrations of the standard solution are respectively 0 mug/L, 7.5 mug/L, 25 mug/L, 75 mug/L and 250 mug/L;

(4) substrate color developing solution: the liquid A is a 2% carbamide peroxide aqueous solution, and the liquid B is a 1% tetramethylbenzidine aqueous solution;

(5) stopping liquid: 2mol/L sulfuric acid solution.

2. Preparation of kit Components

(1) Preparation of enzyme label plate coated with DON-OVA conjugate

Diluting DON-OVA conjugate to the optimal working concentration by using a coating buffer solution, coating a 96-hole polystyrene ELISA plate, incubating for 2h at 37 ℃ in each hole with 100 mu L of the ELISA plate, throwing off a coating solution, washing for 1 time by using PBST (Poly-p-phenylene benzobisoxazole) (30 s for each time), patting to dry, adding 150 mu L of a sealing solution into each hole, incubating for 2h at 37 ℃, pouring out liquid in the holes, drying, and performing vacuum sealing and storage by using an aluminum film.

Coating buffer solution: 0.05mol/L carbonate buffer solution with the pH value of 9.6;

sealing liquid: each 1L of the confining liquid is prepared according to the following method: mixing 5mL of horse serum, 1g of sodium azide and 30g of casein, dissolving the mixture by using a phosphate buffer solution, and fixing the volume to 1000 mL; wherein the concentration of the phosphate buffer solution is 0.02mol/L, and the pH value is 7.2.

(2) Preparation of enzyme-labeled anti-vomitoxin monoclonal antibody

The anti-vomitoxin monoclonal antibody and horseradish peroxidase (HRP) are coupled by a modified sodium periodate method. The traditional sodium periodate method requires that the molar concentration ratio of enzyme to antibody in a reaction system is 4:1, and horseradish peroxidase generates a plurality of sites for binding with the antibody under the strong oxidation effect, so that the activated horseradish peroxidase molecules serve as bridges for connecting the molecules, the enzyme activity of an enzyme marker is reduced, and a plurality of polymers are mixed in the prepared conjugate. To solve this problem, we modified the conventional method, namely:

(1) the blocking process of the amino group is omitted, because the amino group capable of generating self amino group connection is practically few;

(2) the molar concentration ratio of horseradish peroxidase to antibody is reduced to 2:1, the improved method is simpler than the traditional method, and the loss of enzyme activity is reduced.

3. Kit detection method

(1) Sample pretreatment

Weighing 5.0g + -0.05 g pulverized Chinese medicinal sample into 50mL polystyrene centrifuge tube, adding 25mL deionized water, shaking vigorously with oscillator for 5min, and centrifuging at room temperature (20-25 deg.C/68-77 ℉) above 3000g for 5 min; after diluting the sample extract with deionized water at a volume ratio of 1:3, 50. mu.L of the sample extract was used for analysis.

(2) Detection with a kit

Adding DON standard solution/sample solution 50 muL/hole and enzyme-labeled anti-vomitoxin monoclonal antibody 50 muL/hole into a plate hole, lightly oscillating and uniformly mixing, covering a plate with a cover plate film, reacting for 15min in a dark environment at 25 ℃, pouring out liquid in the hole, adding 250 muL of deionized water into each hole, pouring out liquid in the hole after 30s, repeatedly washing the plate for 5 times, and drying by using absorbent paper; adding 50 μ L of substrate color developing solution A and B into each well, mixing by gentle oscillation, covering with cover plate, and reacting at 25 deg.C in dark environment for 5 min; adding 50 mu L of stop solution into each hole, slightly oscillating and uniformly mixing, setting an enzyme-labeling instrument at 450nm, and measuring the absorbance value of each hole.

(3) Calculation of results

Calculating the percent absorbance value: calculating the ratio of the absorbance value of the obtained standard solution or sample solution to the absorbance value of the blank solution according to the following formula:

in the formula: w-percent absorbance value,%;

a is the average absorbance value of the standard solution or the sample solution;

A₀average absorbance value of blank solution (standard solution at a concentration of 0. mu.g/L).

Drawing a standard curve: the calculated percent absorbance (%) was plotted on the ordinate and the log of the concentration of the standard solution (log10) was plotted on the abscissa to obtain a standard curve.

Sample result calculation: substituting the percent absorbance value of the sample liquid into a standard curve, and reading out the corresponding concentration of the sample from the standard curve, wherein the content of the vomitoxin in the sample is calculated according to the following formula:

X＝C×n

in the formula: x is the content of vomitoxin in the sample, mu g/kg;

c-concentration of emetic toxin in the sample, μ g/L, as determined from the standard curve;

n-sample dilution factor.

The calculations can also be performed using the data processing software of various microplate readers.

Note: and (4) deducting a blank value from the calculated result, and keeping one decimal number in the obtained result.

4. Evaluation of test Effect

(1) Linear relation

The concentrations of the standard solutions were 0, 7.5, 25, 75 and 250. mu.g/L, respectively, and the OD values measured are shown in Table 3. The standard curve is plotted with the percent absorbance values (W,%) as ordinate and the log of the concentration of the standard solution (log10) as abscissa (see FIG. 2). By taking logitW as an ordinate and taking a logarithm of the concentration of the standard solution as an abscissa, and converting the figure 2, the logitW and the logarithm of the concentration of the vomitoxin show a good linear relationship (see figure 3) in the concentration range of 7.5 to 250 mug/L, and a regression equation Y of-2.789X +3.689, R is obtained²＝0.9989。

TABLE 3 OD value measurement results of the standard solutions

(2) Limit of sample detection

The method is characterized in that blank malt, peach kernel, spina date seed, boat-fruited sterculia seed, stiff silkworm, centipede, coix seed, scorpion, leech, lotus seed, rangooncreeper fruit, dried orange peel, polygala root, cassia seed, Chinese date, betel nut, earthworm and platycladi seed samples of different sources are respectively used for measurement, and the detection limit is determined by adding 3 times of standard deviation to the measurement average value. The result shows that the detection limit of the 18 matrixes is less than 150 mug/kg, so that the detection limit of the kit to the traditional Chinese medicine sample is determined to be 150 mug/kg.

(3) Accuracy (recovery) and precision (repeatability)

Taking malt, peach kernel, spina date seed, sterculia lychnophora, stiff silkworm, centipede, coix seed, scorpion, leech, lotus seed, rangooncreeper fruit, dried orange peel, polygala root, cassia seed, Chinese date, betel nut, earthworm and platycladi seed which do not contain DON as blank sample matrixes, carrying out addition recovery tests of three concentration levels (150 mug/kg, 300 mug/kg and 600 mug/kg), respectively measuring by using three batches of kits, and calculating sample addition recovery rate and intra-batch and inter-batch variation coefficients. The results show that the adding recovery rate of 18 matrixes is 70-110%, and the variation coefficient in batch and between batches is less than 15%.

(4) Shelf life of kit

The storage condition of the kit is 2-8 ℃, and the maximum absorbance value (zero standard), 50% inhibition concentration and DON addition recovery rate of the kit are all within a normal range after 12 months of determination. Considering that abnormal storage conditions appear in the transportation and use processes, the kit is placed at 37 ℃ for 8 days for an accelerated aging test, and as a result, all indexes of the kit completely meet the requirements; considering the occurrence of the freezing condition of the kit, the kit is placed in a refrigerator at the temperature of-20 ℃ for 8 days, and as a result, all indexes of the kit are completely normal. From the above results, it was found that the kit can be stored at 2 to 8 ℃ for at least 12 months.

Claims

1. A hybridoma cell strain secreting anti-vomitoxin monoclonal antibodies is named as vomitoxin monoclonal antibody hybridoma cell strain F-3-4, is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.14304 and the preservation date of 2017, 06 and 15 days, and is characterized in that: the hybridoma cell strain is obtained by immunizing a mouse and screening after immune antigen is obtained by coupling DON hapten and bovine serum albumin; the synthesis method of the DON hapten comprises the following steps: dissolving vomitoxin 50mg in 5mL of ethanol; taking 29.17mg of p-aminobenzene sulfinic acid, adding 2mL of water for dissolving, adding into the vomitoxin ethanol solution, adding 3mL of 1mol/L diluted hydrochloric acid, and stirring at room temperature for 24 hours; stopping the reaction, adjusting the pH to 7 by using sodium hydroxide, adding 10mL of water, adding 15mL of ethyl acetate, performing shaking extraction, standing for layering, removing a water phase, concentrating and evaporating an organic phase, performing silica gel small column chromatography, and performing elution separation by using dichloromethane/methanol with the volume ratio of 10/1 to obtain 60mg of sulfoacid aminated vomitoxin, namely the DON hapten.

2. An anti-emetic toxin monoclonal antibody, characterized in that: it is secreted and produced by the vomitoxin monoclonal antibody hybridoma cell strain F-3-4 with the preservation number of CGMCCNo.14304 in claim 1.

3. The use of an anti-emetic toxin monoclonal antibody as claimed in claim 2, characterized in that: used for analyzing and detecting vomitoxin in the traditional Chinese medicine.