CN107012128B

CN107012128B - Hybridoma cell strain secreting monoclonal antibody against aflatoxin B1 and application thereof

Info

Publication number: CN107012128B
Application number: CN201710231196.9A
Authority: CN
Inventors: 万宇平; 洪小栩; 杨昌松; 袁媛; 何方洋; 崔廷婷; 朱亮亮; 南铁贵
Original assignee: Beijing Kwinbon Biotechnology Co Ltd
Current assignee: Beijing Kwinbon Biotechnology Co Ltd
Priority date: 2017-04-10
Filing date: 2017-04-10
Publication date: 2020-04-28
Anticipated expiration: 2037-04-10
Also published as: CN107012128A

Abstract

The invention provides a hybridoma cell strain F-2-4 secreting monoclonal antibody against aflatoxin B1, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 13805. The monoclonal antibody of anti-aflatoxin B1 secreted by the cell strain has high sensitivity, good specificity and good anti-interference performance, and has 50% inhibition concentration IC of aflatoxin B1₅₀The cross reaction rate of the aflatoxin B/L is 0.141 mug/L, 9 percent with AFB2, 19 percent with AFG1 and less than 1 percent with AFG2 and AFM1, 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 aflatoxin B1 in the traditional Chinese medicine, and has practical application value.

Description

Hybridoma cell strain secreting monoclonal antibody against aflatoxin B1 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-aflatoxin B1 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.

Aflatoxins (Aflatoxins, abbreviated as AFs) are mainly a class of toxic secondary metabolites with biological activity produced by aspergillus flavus a. flavus and aspergillus parasiticus a. parasiticus, and 17 AFs-related derivatives including aflatoxin B1(AFB1), B2(AFB2), G1(AFG1) and G2(AFG2) have been proved at present, wherein AFB1 is the most toxic, so that the cancer research Institute (IARC) of the World Health Organization (WHO) in 1993 classifies the Aflatoxins as class i carcinogens, and the other three Aflatoxins, AFB2, AFG1 and AFG2, are classified as class 2B carcinogens. In view of the extremely strong toxic effect of AFs and the great harmfulness to human and animals, severe limit standards are made by countries and related organizations in the world. The pharmacopoeia of the people's republic of China (2015 edition) requires that AFB1 detection must be carried out on part of Chinese medicinal materials such as polygala tenuifolia, Chinese date, nutmeg, semen cassiae, malt, dried orange peel, rangooncreeper fruit, platycladi seed, boat-fruited sterculia seed, lotus seed, peach seed, betel nut, spina date seed and coix seed, and the AFB1 is regulated not to exceed 5 mug/kg.

At present, the detection method of AFB1 in traditional Chinese medicines is mainly an instrumental analysis method and comprises thin-layer chromatography, high performance liquid chromatography, liquid chromatography-mass spectrometry combined method and the like. The thin-layer chromatography is convenient to detect, but has high detection limit and cannot quantify well; high performance liquid chromatography, liquid chromatography-mass spectrometry combined method and the like are accurate in detection and low in detection limit, but complex sample pretreatment process, special instruments and professional personnel training are required. These congenital deficiencies of instrumental analysis methods have greatly limited their 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 invention aims to provide a hybridoma cell strain secreting monoclonal antibody against aflatoxin B1, the monoclonal antibody prepared by the cell strain has good specificity and high detection sensitivity to AFB1, has good anti-interference performance to mycotoxin, pesticides, heavy metals, auxiliary materials used in the processing process, contaminating bacteria and the like possibly existing in traditional Chinese medicines, and lays a foundation for establishing an immunological detection method for AFB1 in the traditional Chinese medicines.

The technical scheme of the invention is that a hybridoma cell strain secreting monoclonal antibody against aflatoxin B1 is named as hybridoma cell strain F-2-4 of monoclonal antibody against aflatoxin B1, which is preserved in China general microbiological culture Collection center (CGMCC), wherein the preservation number is CGMCC No.13805, and the preservation date is 2017, 03.08 days.

The anti-aflatoxin B1 monoclonal antibody is produced by secreting an aflatoxin B1 monoclonal antibody hybridoma cell strain F-2-4 with the preservation number of CGMCC No.13805, and the anti-aflatoxin B1 monoclonal antibody is applied to the analysis and detection of aflatoxin B1 in traditional Chinese medicines.

The invention provides a basic preparation process of an F-2-4 cell strain, which comprises the following steps:

(1) synthesis and identification of AFB1 hapten: adding 100mg of aflatoxin B1 and 5mL of N, N-dimethylformamide into a 25mL round-bottom flask, dissolving by mechanical stirring, slowly dropwise adding 147.3mg of phosphorus oxychloride at 0 ℃, heating to 40 ℃, reacting for 2h under magnetic stirring, heating to 80 ℃, continuing to react for 3h under stirring, after the reaction is finished, naturally cooling, slowly pouring into ice water, adding a w (NaOH) -10% aqueous solution to adjust the pH value of the solution to 7-8, extracting with ethyl acetate for three times, combining organic phases, drying and evaporating anhydrous sodium sulfate to dryness. Recrystallizing with anhydrous ethanol to obtain yellow solid aldehyde aflatoxin B1103 mg, which is AFB1 hapten, with yield of 95.37%, and identifying result by nuclear magnetic resonance hydrogen spectrum;

(2) complete antigen synthesis: respectively coupling AFB1 hapten with Bovine Serum Albumin (BSA) and Ovalbumin (OVA) to obtain an immunizing antigen (AFB1-BSA) and a coating antigen (AFB 1-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 AFB 1-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 AFB1 by using an indirect ELISA method, determining the inhibition effect of the cell strains by using an indirect competition ELISA method, screening positive cell strains which are best inhibited, carrying out three times of subcloning, and finally obtaining a hybridoma cell strain F-2-4;

(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.13805 of each mouse into the abdominal cavity after 10 days, collecting ascites from the seventh day, purifying the ascites by an octanoic acid-ammonium sulfate method, analyzing the purification effect by an SDS-PAGE gel electrophoresis method, measuring the titer, subtype, cross reactivity and anti-interference performance of the obtained monoclonal antibody, and storing at-20 ℃;

(6) the application of the antibody is as follows: the monoclonal antibody of anti-aflatoxin B1 secreted by the hybridoma cell strain F-2-4 is used for preparing an aflatoxin B1 enzyme linked immunosorbent assay kit for analytical detection of aflatoxin B1 in traditional Chinese medicines.

The invention has the beneficial effects that:

(1) the hybridoma cell strain F-2-4 provided by the invention can be used for preparing a high-titer anti-aflatoxin B1 monoclonal antibody, and the titer measured by an ELISA method for anti-aflatoxin B1 mouse ascites antibody is not less than 20000.

(2) The present invention providesThe anti-aflatoxin B1 monoclonal antibody has high sensitivity, good specificity and good anti-interference performance, and has 50 percent of inhibition concentration IC of aflatoxin B1₅₀0.141 mug/L, 9 percent of cross reaction rate with AFB2, 19 percent of cross reaction rate with AFG1 and less than 1 percent of cross reaction rate with AFG2 and AFM1, 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-aflatoxin B1 monoclonal antibody provided by the invention can be applied to determination of aflatoxin B1 content in traditional Chinese medicines.

Biological material sample preservation: a hybridoma cell strain secreting monoclonal antibody against aflatoxin B1, which is preserved in China general microbiological culture Collection center (CGMCC) for short, and has the address: the collection date of No. 3 Xilu Beijing, Chaoyang, and the institute of microbiology of Chinese academy of sciences is 2017, 03 and 08 days, and the collection number is CGMCC No. 13805.

Drawings

FIG. 1A synthetic route to aflatoxin B1 hapten

FIG. 2H-NMR spectrum of aflatoxin B1 hapten

FIG. 3 is a SDS-PAGE result of the anti-aflatoxin B1 monoclonal antibody

FIG. 4 Aflatoxin B1 competition ELISA standard curve diagram

FIG. 5 Aflatoxin B1 competition ELISA Logit/Log standard curve

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 strain F-2-4

1. Hapten synthesis and identification

The synthetic route of AFB1 hapten is shown in FIG. 1.

Adding 100mg of aflatoxin B1 and 5mL of N, N-dimethylformamide into a 25mL round-bottom flask, dissolving by mechanical stirring, slowly dropwise adding 147.3mg of phosphorus oxychloride at 0 ℃, heating to 40 ℃, reacting for 2h under magnetic stirring, heating to 80 ℃, continuing to react for 3h under stirring, after the reaction is finished, naturally cooling, slowly pouring into ice water, adding a w (NaOH) -10% aqueous solution to adjust the pH value of the solution to 7-8, extracting with ethyl acetate for three times, combining organic phases, drying and evaporating anhydrous sodium sulfate to dryness. And recrystallizing with absolute ethyl alcohol to obtain yellow solid aldehyde aflatoxin B1103 mg, namely AFB1 hapten, with the yield of 95.37%.

The hapten is identified by nuclear magnetic resonance hydrogen spectrum, and the result is shown in figure 2.¹H-NMR(CDCl₃,300MHz)δ：10.36(1H,s,-CHO)，6.78(1H,s,-CH-)，6.22(1H,d,C＝C)，4.82(1H,dd,C＝C)，4.31(1H,dd,-CH-)，3.83(3H,s,-OCH₃)，2.98(2H,dd,CH₂)，2.07(2H,t,CH₂). In the atlas, the aldehyde group hydrogen resonance absorption peak with chemical shift delta of 10.36 is introduced, and the existence of the hydrogen absorption peak except the characteristic hydrogen absorption peak of the original drug proves that the spacer arm coupling is successful, and the AFB1 hapten has a correct structure.

2. Complete antigen synthesis

Immune antigen synthesis-coupling of AFB1 hapten to BSA

Dissolving 12mg of AFB1 hapten in 0.5mL of N, N-dimethylformamide to obtain solution A; dissolving 50mg BSA in acetate buffer solution with pH of 5.40.05mol/L to obtain solution B; dropwise adding the solution A into the solution B, stirring at 4 ℃ overnight, adding 5mg of sodium borohydride, continuously stirring for 2h, dialyzing with 0.05mol/L phosphate buffer solution for three days, changing the solution 3 times every day, packaging, and storing at-20 ℃ for later use.

Synthesis of coating antigen-coupling of AFB1 hapten and OVA

Dissolving 6mg of AFB1 hapten in 0.5mL of dimethyl sulfoxide to obtain solution A; dissolving 50mg of OVA in acetate buffer solution with pH5.40.05mol/L to obtain solution B; dropwise adding the solution A into the solution B, stirring at 4 ℃ overnight, adding 3mg of sodium borohydride, continuously stirring for 2h, dialyzing with 0.05mol/L phosphate buffer solution for three days, changing the solution 3 times every day, packaging, and storing at-20 ℃ for later use.

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 AFB 1-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 coating antigen (AFB1-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 hole, 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 a 50mL 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 a 50mL 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, and in the second step, AFB1, AFB2, AFG1, AFG2 and AFM1 are selected as standard substances, and the inhibition effect of the positive cells is determined by indirect competition ELISA. And selecting a well which has better inhibition on AFB1 standard products and has no inhibition on AFB2, AFG1, AFG2 and AFM1 standard products, performing subcloning by adopting a limiting dilution method, and detecting by using the same method. Repeating the steps for three times to obtain the cell strain F-2-4 capable of stably secreting the anti-aflatoxin B1 monoclonal antibody. The cell strain has been deposited in China general microbiological culture Collection center (CGMCC for short, the address: the institute of microbiology, China academy of sciences, Japan, Kogyo-Yangyuan road, Beijing) on 2017, 03.08, with the preservation number of CGMCC No. 13805.

Example 2: preparation, purification and characteristic identification of anti-aflatoxin B1 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.13805 in logarithmic growth phase after 10 days with RPMI-1640 basic culture medium, counting with blood counting plate and microscope to obtain 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.13805 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 effect by SDS-PAGE gel electrophoresis, and the result is shown in FIG. 3, and the size of the target protein band was consistent with that expected after the antibody was subjected to denaturing SDS-PAGE electrophoresis. The antibody dissociates into two subunits, a large subunit (heavy chain, 50kD) and a small subunit (light chain, 25kD), indicating good purity.

3. Antibody titer determination

The detection is carried out by adopting an indirect ELISA method, and the specific steps are as follows:

(1) diluting the coating antigen (AFB1-OVA) with 0.05mol/L of carbonate buffer solution with pH9.6 at a ratio of 1:1000, 1:2000, 1:4000 and 1:8000, coating an enzyme label plate with 100 μ L of each hole, incubating for 2h at 37 ℃, throwing off the coating solution, washing for 1 time by PBST, and patting to dry;

(3) diluting monoclonal antibodies by PBS (phosphate buffer solution) in a gradient of 1:5000, 1:10000, 1:20000, 1:40000 and 1:80000, simultaneously diluting enzyme-labeled secondary antibodies by 1000 times, mixing the diluted antibodies with the enzyme-labeled secondary antibodies by 10:1, adding 100 mu L of the diluted antibodies into each hole, incubating in a incubator at 25 ℃ for 45min, repeating each concentration, washing by PBST (Poly-p-phenylene benzobisoxazole) for 3-5 times at intervals of 30s, and beating to dry;

(5) substrate addition per well50 mul of color liquid A and B respectively, reacting at 25 deg.C in dark place for 15min, adding 50 mul of 2mol/L H into each well₂SO₄Terminating the reaction by the solution;

(6) OD value at 450nm was measured by microplate reader, and the results are shown in Table 2.

TABLE 2 results of potency assay

As is clear from Table 2, when the titers were measured by the checkerboard method at antibody concentrations having OD values greater than 1.0, the titers of 5 batches of antibodies were 1:80000, 1:40000, 1:20000 (batch 1), 1:80000, 1:40000, 1:20000 (batch 2), 1:80000, 1:40000 (batch 3), 1:80000, 1:40000 (batch 4), 1:40000, 1:20000 (batch 5), respectively, and it was found that the titers of the anti-aflatoxin B1 monoclonal antibodies were not less than 20000.

4. Antibody subtype identification

The subtype of the anti-aflatoxin B1 monoclonal antibody secreted by the hybridoma cell strain F-2-4 is identified to be IgG by using a 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 serial diluted aflatoxin standard working solution (the concentrations are 0, 0.05, 0.15, 0.45 and 1.35 mu g/L respectively) into each well, then adding 50 mu L of monoclonal antibody 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) drawing a standard curve by taking the logarithmic value of the concentration of the aflatoxin as an abscissa and taking the percent absorbance value (the percentage of the OD value of each concentration standard substance to the OD value of a hole without the standard substance) as an ordinate. Mass concentration (IC) at 50% percent absorbance value of each curve₅₀) The cross-reactivity was calculated as follows, and the results are shown in Table 3, and the smaller the cross-reactivity, the higher the antibody specificity.

In the formula:

S_i-cross-reactivity,%;

IC of y-AFB 1 standard working fluid curve₅₀A value;

z-IC of standard working solution curve for aflatoxins other than AFB1₅₀The value is obtained.

TABLE 3 Cross-reaction assay results

As can be seen from Table 3, the anti-aflatoxin B1 monoclonal antibody has less cross reaction and better specificity to other types of aflatoxins.

6. Determination of anti-interference of antibody

The standard curve of aflatoxin B1 was plotted according to the cross-reactivity measurement procedure of 5, and the following interfering substances were diluted with standard buffer solutions to the addition values listed in the tables for analysis, and the cross-reactivity ratio (recovery value/addition value × 100%) was calculated from the OD value corresponding to the drug development in combination with the recovery value analyzed from the standard curve, and the results are shown in table 4.

TABLE 4 anti-interference data for other substances commonly found in Chinese medicine

As can be seen from Table 4, there is no interference from mycotoxin, pesticide, heavy metal, adjuvant used in processing, and contaminating bacteria possibly existing in the Chinese medicinal materials on the properties of the anti-aflatoxin B1 monoclonal antibody.

Example 3: application of anti-aflatoxin B1 monoclonal antibody

The monoclonal antibody of anti-aflatoxin B1 secreted by the hybridoma cell strain F-2-4 is used for preparing an aflatoxin B1 enzyme linked immunosorbent assay kit for analytical detection of aflatoxin B1 in traditional Chinese medicines.

1. Composition of enzyme linked immunosorbent assay kit

(1) An ELISA plate coated with the anti-aflatoxin B1 monoclonal antibody;

(2) enzyme-labeled aflatoxin B1 antigen: the coating antigen described in example 1 is prepared by coupling with horseradish peroxidase (HRP), and diluted to the optimal working concentration by enzyme-labeled antigen diluent;

(3) aflatoxin B1 standard: the concentrations of the standard solution are respectively 0 mug/L, 0.05 mug/L, 0.15 mug/L, 0.45 mug/L and 1.35 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;

(6) washing liquid: each 1L of the washing solution was prepared as follows: mixing 10mL of Tween-20, 5g of sodium azide and 990mL of phosphate buffer; wherein the concentration of the phosphate buffer solution is 0.02mol/L, and the pH value is 7.4;

(7) compounding the solution: each 1L of the redissolution is prepared according to the following method: dissolving 12g of casein 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 7.4.

2. Preparation of kit Components

(1) Preparation of ELISA plate coated with anti-aflatoxin B1 monoclonal antibody

Diluting the anti-aflatoxin B1 monoclonal antibody 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 by 100 mu L, throwing off the coating solution, washing for 1 time by using a washing solution for 30s each time, patting to dry, adding 150 mu L of confining liquid into each hole, incubating for 2h at 37 ℃, pouring off 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 aflatoxin B1 antigen

Taking 15mg of the coating antigen described in the embodiment 1, adding 300 mu L N, dissolving in N-dimethylformamide, adding 10mg of carbodiimide and 10mg of N-hydroxysuccinimide, and carrying out an activation reaction for 0.5 h; dissolving horseradish peroxidase 20mg in 0.5mL of 0.05mol/L CB buffer solution with pH of 9.0, adding 2mL of water, adding 300 μ L of the above activating solution, adding 1mol/L sodium hydroxide 30 μ L, and reacting at room temperature for 4 h; dialyzing with 0.02mol/L PBS for 2 days, changing the solution 4 times per day, and taking out to obtain 2.8mL of dialysate; adding BSA protective protein and five-ten-thousandth preservative, diluting with enzyme-labeled antigen diluent to the optimal working concentration, and subpackaging and storing.

Enzyme-labeled antigen diluent: each 1L of enzyme-labeled antigen diluent is prepared according to the following method: dissolving 8g of bovine serum albumin 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.

3. Kit detection method

(1) Sample pretreatment

Weighing 1.0g of pulverized Chinese medicinal sample into a 50mL centrifuge tube, adding 10mL methanol, shaking for 5min, and centrifuging at 3000g room temperature (20-25 deg.C/68-77 ℉) for 5 min; drying in 2-10 mL clean centrifuge tube under 20-30 deg.C (68-86 ℉) water bath nitrogen flow; adding 2mL deionized water, vortexing for 1min, adding 6mL chloroform, shaking for 5min, and centrifuging at 3000g room temperature (20-25 deg.C/68-77 ℉) for 5 min; removing the upper aqueous phase, and blow-drying the lower 3mL layer in a water bath under nitrogen flow at 20-30 deg.C (68-86 ℉); adding 1mL n-hexane, vortexing for 2min, adding 1mL redissolved solution, vortexing for 3min, and centrifuging at 3000g room temperature (20-25 deg.C/68-77 ℉) for 5 min; the upper organic phase was removed and the lower layer was taken for analysis.

(2) Detection with a kit

Adding 50 mu L/hole of AFB1 standard solution/sample solution and 50 mu L/hole of enzyme-labeled AFB1 antigen into a plate hole, slightly oscillating and uniformly mixing, placing the plate in a dark environment at 25 ℃ for reaction for 45min by using a cover plate film cover plate, pouring out liquid in the hole, adding 250 mu L of washing solution 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 15 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 after reading out the corresponding concentration of the sample from the standard curve, calculating the content of aflatoxin B1 in the sample according to the following formula:

X＝C×n

in the formula: x is the content of aflatoxin B1 in the sample, mu g/kg;

c-concentration of aflatoxin B1 in the sample, μ g/L, 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, 0.05, 0.15, 0.45 and 1.35. mu.g/L, respectively, and the OD values measured are shown in Table 5. 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. 4). By taking logitW as an ordinate and taking a logarithm of the concentration of the standard solution as an abscissa, and converting the attached figure 4, the logitW and the logarithm of the concentration of the aflatoxin B1 show a good linear relation (shown in figure 5) in the concentration range of 0.05 mu g/L-1.35 mu g/L, and a regression equation Y is obtained, wherein the regression equation is-2.393X-2.327, and R is²＝0.994。

TABLE 5 OD value measurement results of the standard solutions

(2) Method detection limit and quantitation limit

Referring to the verification guiding principle of the drug quality standard analysis method of ' pharmacopoeia of the people ' S republic of China ' 2015 edition 9101, the LOD limit of the method is 3.3 delta/S, the LOQ limit of the method is 10 delta/S, and delta in the formula: determining the standard deviation of the blank value; s: slope of the standard curve.

From the above (1), since δ is 0.023 and S is 2.393, the detection limit LOD and the quantitation limit LOQ were determined to be 0.032 μ g/L and 0.096 μ g/L, respectively.

(3) Limit of sample detection

The method is characterized in that 20 parts of 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 from different sources are respectively used for measurement, the detection limit is determined by adding 3 times of standard deviation to the measurement average value, and the result is shown in Table 6.

TABLE 6 sample detection limit results

Sample (I)	Detection limit/(ng/kg)	Sample (I)	Detection limit/(ng/kg)	Sample (I)	Detection limit/(ng/kg)
						Malt	285.7	Peach kernel	592.9	Wild jujube seed	437.2
Semen Sterculiae Lychnophorae	382.3	Bombyx Batryticatus	217.5	Centipede (Scolopendra)	339.8
						Coix seed	159.9	Buthus martensi karsch	388.6	Leech	174.7
Lotus seed	383.5	Fructus quisqualis	291.5	Dried orange peel	235.5
						Root of Polygala	267.8	Cassia seed	371.0	Chinese date	397.0
Betel nut	420.3	Earthworm	337.1	Arborvitae seed	355.8

(4) Accuracy (recovery) and precision (repeatability)

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 which do not contain AFB1 are used as blank sample matrixes, three concentration levels of addition recovery tests are carried out, three batches of kits are respectively used for measuring, the sample addition recovery rate and the variation coefficients in batches and in batches are calculated, and the results are shown in Table 7.

TABLE 7 sample accuracy and precision results

The results show that the adding recovery rate of 18 substrates of 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 is 60-120 percent, and the variation coefficient in batch and between batches is less than 15 percent.

(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 AFB1 addition recovery rate of the kit are 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 monoclonal antibody against aflatoxin B1, named as hybridoma cell strain F-2-4 of monoclonal antibody against aflatoxin B1, has been deposited in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.13805 and the preservation date of 2017, 03 and 08 days, and is characterized in that: the hybridoma cell strain is obtained by immunizing a mouse and screening after obtaining an immune antigen through coupling AFB1 hapten and bovine serum albumin; the synthesis method of the AFB1 hapten comprises the following steps: adding 100mg of aflatoxin B1 and 5mL of N, N-dimethylformamide into a 25mL round-bottom flask, mechanically stirring for dissolving, slowly dropwise adding 147.3mg of phosphorus oxychloride at 0 ℃, heating to 40 ℃, reacting for 2h under magnetic stirring, heating to 80 ℃, continuing stirring for reacting for 3h, after the reaction is finished, naturally cooling, slowly pouring into ice water, adding an aqueous solution with 10% of NaOH by mass fraction to adjust the pH of the solution to be = 7-8, extracting for three times with 30mL of ethyl acetate, combining organic phases, drying and evaporating anhydrous sodium sulfate, and recrystallizing with anhydrous ethanol to obtain yellow solid aldehyde aflatoxin B1103 mg, namely AFB1 hapten.

2. An anti-aflatoxin B1 monoclonal antibody, which is characterized in that: it is produced by the aflatoxin B1 monoclonal antibody hybridoma cell strain F-2-4 with the preservation number of CGMCC No.13805 as claimed in claim 1.

3. The use of the monoclonal antibody against aflatoxin B1 of claim 2, which is characterized in that: is used for analyzing and detecting aflatoxin B1 in traditional Chinese medicines.