IMMUNOADSORBENT FOR PURIFYING FIVE KINDS OF MYCOTOXINS INCLUDING FUMONISIN B1 AND AFLATOXIN B1, AND COMPLEX AFFINITY COLUMN

Abstract

The invention relates to an immunoadsorbent for purifying five kinds of mycotoxins including fumonisin B.sub.1 and aflatoxin B.sub.1, and a complex affinity column. The immunoadsorbent comprises a solid-phase support, and an anti-fumonisin B.sub.1 monoclonal antibody, an anti-aflatoxin B.sub.1 monoclonal antibody, an anti-ochratoxin A monoclonal antibody, an anti-zearalenone monoclonal antibody and an anti-sterigmatocystin monoclonal antibody which are coupled to the solid-phase support, wherein the anti-fumonisin B.sub.1 monoclonal antibody is secreted by a hybridoma cell strain Fm7A11, and the hybridoma cell strain Fm7A11 has been preserved at China Center for Type Culture Collection, Wuhan University, Wuhan, China on Mar. 29, 2016 with the preservation number of CCTCC No. C201636. The complex affinity column can be used for purification and detection of a fumonisin B.sub.1 sample, an aflatoxin B.sub.1 sample, an ochratoxin A sample, a zearalenone sample and a sterigmatocystin sample at the same time.

Claims

1. An immunoadsorbent for purifying fumonisin B.sub.1, aflatoxin B.sub.1, ochratoxin A, zearalenone and sterigmatocystin, comprising a solid-phase support, and an anti-fumonisin B.sub.1 monoclonal antibody, an anti-aflatoxin B.sub.1 monoclonal antibody, an anti-ochratoxin A monoclonal antibody, an anti-zearalenone monoclonal antibody and an anti-sterigmatocystin monoclonal antibody which are coupled to the solid-phase support, wherein the anti-fumonisin B.sub.1 monoclonal antibody is secreted by a hybridoma cell strain Fm7A11, and the hybridoma cell strain Fm7A11 has been preserved at China Center for Type Culture Collection, Wuhan University, Wuhan, China on Mar. 29, 2016 with the preservation number of CCTCC No. C201636.

2. The immunoadsorbent according to claim 1, wherein the solid-phase support is a sepharose.

3. A complex affinity column loaded with the immunoadsorbent according to claim for purifying fumonisin B.sub.1, aflatoxin B.sub.1, ochratoxin A, zearalenone and sterigmatocystin.

4. A preparation method of the complex affinity column according to claim 3, comprising the following steps: (a) preparation of matrix a CNBr-activated sepharose matrix powder is subjected to washing by HCl at pH 2-3 to remove impurities to obtain a CNBr-activated sepharose matrix; (b) ligand coupling each of the anti-fumonisin B.sub.1 monoclonal antibody, the anti-aflatoxin B.sub.1 monoclonal antibody, the anti-ochratoxin A monoclonal antibody, the anti-zearalenone monoclonal antibody and the anti-sterigmatocystin monoclonal antibody for coupling are dissolved in a coupling buffer to obtain a monoclonal antibody solution, the CNBr-activated sepharose matrix in the step (a) is rapidly transferred to the monoclonal antibody solution to obtain a mixture, and the mixture is thoroughly mixed for 2-4 h at room temperature (20-25 C.); (c) ligand blocking to block all remaining active sites; (d) removal of uncoupled ligands after the ligand blocking in the step (c); and (e) column packing.

5. The preparation method according to claim 4, wherein concentration of the HCl for the washing in the step (a) is 1 mmol/L and the washing time is 15 min.

6. The preparation method according to claim 4, wherein the coupling buffer in the step (b) is 0.2 mol/L NaHCO.sub.3, pH 8.3, and concentration of each of the monoclonal antibody is 10-15 mg/mL.

7. The preparation method according to claim 4, wherein the ligand blocking of the step (c) is conducted by transferring the treated CNBr-activated sepharose matrix in the step (b) to a 0.1 mol/L Tris-HCl buffer, and then keep it standing for 2-4 h at room temperature.

8. The preparation method according to claim 4, wherein the step (d) is conducted by washing the treated CNBr-activated sepharose matrix in the step (c) with a buffer of pH 4 and a buffer of pH 8 in sequence for at least 3 cycles, wherein the buffer of pH 4 is a 0.1 mol/L acetate/sodium acetate buffer with pH of 4, and the buffer of pH 8 is a 0.1 mol/L Tris-HCl buffer with pH of 8.

9. The preparation method according to claim 4, wherein the step (e) is conducted by washing with 0.01% NaN.sub.3-PBS with 5 times volume of the CNBr-activated sepharose matrix, storing with the 0.01% NaN.sub.3-PBS, and then packing the column.

10. A method for detecting content of fumonisin B.sub.1, aflatoxin B.sub.1, ochratoxin A, zearalenone and sterigmatocystin with the complex affinity column according to claim 3, wherein when a sample containing fumonisin B.sub.1, aflatoxin, ochratoxin A, zearalenone and sterigmatocystin passes through the complex affinity column, the immunoadsorbent specifically adsorbs fumonisin B.sub.1, aflatoxin, ochratoxin A, zearalenone and sterigmatocystin, other impurities are flowed out from the complex affinity column, followed by elution of the complex affinity column with chromatography-grade methanol to obtain an eluate, and the eluate, namely the purified and concentrated sample is collected and subjected to detection by a high performance liquid chromatography-mass spectrometer, thereby obtaining the content of fumonisin B.sub.1, aflatoxin B.sub.1, ochratoxin A, zearalenone and sterigmatocystin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 shows the mass spectrogram-chromatograms of fumonisin B.sub.1 (FB.sub.1), aflatoxin B.sub.1 (AFB.sub.1), ochratoxin A (OTA), zearalenone (ZEN) and sterigmatocystin (ST) in a sample; and from top to bottom the sequence is sterigmatocystin, zearalenone, ochratoxin A, fumonisin B.sub.1, aflatoxin B.sub.1.

DESCRIPTION OF THE EMBODIMENTS

Embodiment 1

Example 1: Acquisition of an Anti-Aflatoxin B.SUB.1 .Monoclonal Antibody

[0045] An aflatoxin general monoclonal antibody is secreted by a hybridoma cell strain 3G1 with the preservation number of CCTCC No. C201014, and is prepared in advance according to the method reported in the patent number ZL201210117614.9. The preparation method comprises the steps of injecting the obtained hybridoma cell strain 3G1 into a BALB/c mouse previously treated with a Freund's incomplete adjuvant, collecting the ascites of the mouse and conducting purification to obtain the anti-aflatoxin B.sub.1 monoclonal antibody. The purification method is an octanoic acid-ammonium sulfate method. Specifically, the ascites of the mouse is filtered through double-layer filter paper, the filtered ascites is centrifuged at 4 C. and 12000 r/min for more than 15 min, a supernatant is drawn, the supernatant is mixed with an acetate buffer with 4 times volume, n-octanoic acid is slowly added while stirring, the volume of n-octanoic acid required by per ml of ascites is 30-35 L, the mixture is mixed at room temperature for 30-60 min and stands at 4 C. for more than 2 h, then centrifugation is conducted at 4 C. and 12000 r/min for more than 30 min, a precipitate is discarded, a resulting supernatant is filtered through double-layer filter paper, a phosphate buffer with 1/10 filtrate volume and with a molar concentration of 0.1 mol/L and a pH of 7.4 is added, the pH of the mixed liquid is adjusted with 2 mol/L sodium hydroxide solution to 7.4, precooling is conducted at 4 C., ammonium sulfate is slowly added till the final concentration of ammonium sulfate reaches 0.277 g/mL, the mixture stands at 4 C. for more than 2 h, then centrifugation is conducted at 4 C. and 12000 r/min for more than 30 min, a supernatant is discarded, a resulting precipitate is resuspended with 0.01 mol/L phosphate buffer of 1/10 original volume of ascites, placed in a dialysis bag and dialyzed with pure water, the well-dialyzed protein solution is frozen in a 70 C. freezer and then lyophilized with a freezing vacuum dryer, lyophilized powder is collected, thus obtaining the purified anti-aflatoxin B.sub.1 monoclonal antibody, and the antibody is stored in a 20 C. refrigerator for use; and

[0046] the acetate buffer is obtained by adding water to 0.29 g of sodium acetate and 0.141 mL of acetic acid to 100 mL; and the 0.1 mol/L phosphate buffer is obtained by adding water to 0.8 g of sodium chloride, 0.29 g of sodium phosphate dibasic dodecahydrate, 0.02 g of potassium chloride and 0.02 g of potassium dihydrogen phosphate to 100 mL.

Example 2: Acquisition of Anti-Ochratoxin A Monoclonal Antibody

[0047] An anti-ochratoxin A monoclonal antibody is secreted by a hybridoma cell strain 1H2 with the preservation number of CCTCC No. C201329, and is prepared in advance according to the method reported in the patent number 201310115921.8. The preparation method comprises the steps of injecting the hybridoma cell strain 1H2 into the abdomen of a BALB/c mouse previously treated with a Freund's incomplete adjuvant, collecting the ascites of the mouse and conducting purification to obtain the anti-ochratoxin A monoclonal antibody. The purification method is an octanoic acid-ammonium sulfate method. Specifically, the ascites of the mouse is filtered through double-layer filter paper, the filtered ascites is centrifuged at 4 C. and 12000 r/min for more than 15 min, a supernatant is drawn, the supernatant is mixed with an acetate buffer with 4 times volume, n-octanoic acid is slowly added while stirring, the volume of n-octanoic acid required by per ml of ascites is 30-35 L, the mixture is mixed at room temperature for 30-60 min and stands at 4 C. for more than 2 h, then centrifugation is conducted at 4 C. and 12000 r/min for more than 30 min, a precipitate is discarded, a resulting supernatant is filtered through double-layer filter paper, a phosphate buffer with 1/10 filtrate volume and with a molar concentration of 0.1 mol/L and a pH of 7.4 is added, the pH of the mixed liquid is adjusted with 2 mol/L sodium hydroxide solution to 7.4, precooling is conducted at 4 C., ammonium sulfate is slowly added till the final concentration of ammonium sulfate reaches 0.277 g/mL, the mixture stands at 4 C. for more than 2 h, then centrifugation is conducted at 4 C. and 12000 r/min for more than 30 min, a supernatant is discarded, a resulting precipitate is resuspended with 0.01 mol/L phosphate buffer of 1/10 original volume of ascites with a pH of 7.4, placed in a dialysis bag and dialyzed with pure water, the well-dialyzed protein solution is frozen in a 70 C. freezer and then lyophilized with a freezing vacuum dryer, lyophilized powder is collected, thus obtaining the purified anti-ochratoxin A monoclonal antibody, and the antibody is stored in a 20 C. refrigerator for use; and

[0048] the acetate buffer is obtained by adding water to 0.29 g of sodium acetate and 0.141 mL of acetic acid to 100 mL; the 0.01 mol/L phosphate buffer is obtained by adding water to 0.8 g of sodium chloride, 0.29 g of sodium phosphate dibasic dodecahydrate, 0.02 g of potassium chloride and 0.02 g of potassium dihydrogen phosphate to 100 mL; and the 0.1 mol/L phosphate buffer is obtained by adding water to 8 g of sodium chloride, 2.9 g of sodium phosphate dibasic dodecahydrate, 0.2 g of potassium chloride and 0.2 g of potassium dihydrogen phosphate to 100 mL.

Example 3: Acquisition of Anti-Zearalenone Monoclonal Antibody

[0049] An anti-zearalenone monoclonal antibody is secreted by a hybridoma cell strain 2D3 with the preservation number of CCTCC No. C201328, and is prepared in advance according to the method reported in the patent number 201310115825.3. The preparation method comprises the steps of injecting the hybridoma cell strain 2D3 into the abdomen of a BALB/c mouse previously treated with a Freund's incomplete adjuvant, collecting the ascites of the mouse and conducting purification to obtain the anti-zearalenone monoclonal antibody. The purification method is an octanoic acid-ammonium sulfate method. Specifically, the ascites of the mouse is filtered through double-layer filter paper, the filtered ascites is centrifuged at 4 C. and 12000 r/min for more than 15 min, a supernatant is drawn, the supernatant is mixed with an acetate buffer with 4 times volume, n-octanoic acid is slowly added while stirring, the volume of n-octanoic acid required by per ml of ascites is 30-35 L, the mixture is mixed at room temperature for 30-60 min and stands at 4 C. for more than 2 h, then centrifugation is conducted at 4 C. and 12000 r/min for more than 30 min, a precipitate is discarded, a resulting supernatant is filtered through double-layer filter paper, a phosphate buffer with 1/10 filtrate volume and with a molar concentration of 0.1 mol/L and a pH of 7.4 is added, the pH of the mixed liquid is adjusted with 2 mol/L sodium hydroxide solution to 7.4, precooling is conducted at 4 C., ammonium sulfate is slowly added till the final concentration of ammonium sulfate reaches 0.277 g/mL, the mixture stands at 4 C. for more than 2 h, then centrifugation is conducted at 4 C. and 12000 r/min for more than 30 min, a supernatant is discarded, a resulting precipitate is resuspended with 0.01 mol/L phosphate buffer of 1/10 original volume of ascites with a pH of 7.4, placed in a dialysis bag and dialyzed with pure water, the well-dialyzed protein solution is frozen in a 70 C. freezer and then lyophilized with a freezing vacuum dryer, lyophilized powder is collected, thus obtaining the purified anti-zearalenone monoclonal antibody, and the antibody is stored in a 20 C. refrigerator for use; and

[0050] the acetate buffer is obtained by adding water to 0.29 g of sodium acetate and 0.141 mL of acetic acid to 100 mL; the 0.01 mol/L phosphate buffer is obtained by adding water to 0.8 g of sodium chloride, 0.29 g of sodium phosphate dibasic dodecahydrate, 0.02 g of potassium chloride and 0.02 g of potassium dihydrogen phosphate to 100 mL; and the 0.1 mol/L phosphate buffer is obtained by adding water to 8 g of sodium chloride, 2.9 g of sodium phosphate dibasic dodecahydrate, 0.2 g of potassium chloride and 0.2 g of potassium dihydrogen phosphate to 100 mL.

Example 4: Acquisition of Anti-Sterigmatocystin Monoclonal Antibody

[0051] An anti-sterigmatocystin monoclonal antibody is secreted by a hybridoma cell strain ST03 with the preservation number of CCTCC No. C2013187, and is prepared in advance according to the method reported in the patent number 201410115952.8. The preparation method comprises the steps of injecting the obtained hybridoma cell strain ST03 into the abdomen of a BALB/c mouse previously treated with a Freund's incomplete adjuvant, collecting the ascites of the mouse and conducting purification to obtain the anti-sterigmatocystin monoclonal antibody. The purification method is an octanoic acid-ammonium sulfate purification method. Specifically, the ascites of the mouse is filtered through double-layer filter paper, the filtered ascites is centrifuged at 4 C. and 12000 r/min for more than 15 min, a supernatant is drawn, the supernatant is mixed with an acetate buffer with 4 times volume, n-octanoic acid is slowly added while stirring, the volume of n-octanoic acid required by per ml of ascites is 33 L, the mixture is mixed at room temperature for 30-60 min and stands at 4 C. for more than 2 h, then centrifugation is conducted at 4 C. and 12000 r/min for more than 30 min, a precipitate is discarded, a resulting supernatant is filtered through double-layer filter paper, a phosphate buffer with 1/10 filtrate volume and with a molar concentration of 0.1 mol/L and a pH of 7.4 is added, the pH of the mixed liquid is adjusted with 2 mol/L sodium hydroxide solution to 7.4, precooling is conducted at 4 C., ammonium sulfate is slowly added till the final concentration of ammonium sulfate reaches 0.277 g/mL, the mixture stands at 4 C. for more than 2 h, then centrifugation is conducted at 4 C. and 12000 r/min for more than 30 min, a supernatant is discarded, a resulting precipitate is resuspended with 0.01 mol/L phosphate buffer of 1/10 original volume of ascites, placed in a dialysis bag and dialyzed with pure water, the well-dialyzed protein solution is frozen in a 70 C. freezer and then lyophilized with a freezing vacuum dryer, lyophilized powder is collected, thus obtaining the purified anti-sterigmatocystin monoclonal antibody, and the antibody is stored in a 20 C. refrigerator for use; and

[0052] the acetate buffer is obtained by adding water to 0.29 g of sodium acetate and 0.141 mL of acetic acid to 100 mL; and the 0.1 mol/L phosphate buffer is obtained by adding water to 0.8 g of sodium chloride, 0.29 g of sodium phosphate dibasic dodecahydrate, 0.02 g of potassium chloride and 0.02 g of potassium dihydrogen phosphate to 100 mL.

Example 5 Acquisition of Anti-Fumonisin B.SUB.1 .Monoclonal Antibody

[0053] Screening of Hybridoma Cell Strain Fm7A11

[0054] 1. Antigen Synthesis and Animal Immunization

[0055] Commercially available fumonisin B.sub.1 standards are purchased for complete antigen synthesis as follows: 2 mg of FB.sub.1 standard powder and 2 mg of EDC are dissolved in 500 L of a 0.01 mol/L PBS solution respectively to obtain an EDC solution and an FB.sub.1 solution, and 4 mg/mL (0.01 mol/L PBS solution) EDC solution is added dropwise to the solubilized FB.sub.1 solution and gently agitated for 10 min at room temperature. 5 mg/mL of (0.01 mol/L PBS solution) BSA solution is added dropwise to the mixed liquid, and the mixture is stirred at room temperature and reacts for 4 h. Dialysis is conducted for 3 days. Finally, conventional UV scanning identification is conducted, and an identification result shows that FB.sub.1-BSA complete antigen preparation succeeds.

[0056] Six 6-week-old BALB/c mice are purchased and immunized with a fumonisin complete antigen FB.sub.1-BSA synthesized in the laboratory. During the first time of immunization, the fumonisin complete antigen and an equal volume of Freund's complete adjuvant are emulsified and then injected subcutaneously into multiple points of the nape of the mice. The second time of immunization is carried out three weeks later. A Freund's incomplete adjuvant and an equal volume of fumonisin complete antigen are emulsified and then injected subcutaneously into multiple points of the nape of the mice. The third time of immunization and the fourth time of immunization are conducted two weeks after the last time of immunization separately, in the same immunization manner as the second time of immunization. The same dose is adopted for four times of immunization, 100 g per mouse only. On the 7th day after the third time of immunization, blood is collected from the caudal vein of the mice and serum is separated. The serum titer of the mice is monitored through indirect ELISA, the sensitivity of the serum of the mice is measured through indirect competitive ELISA, a mouse corresponding to the serum with higher titer and sensitivity is selected for the final booster immunization, and the immune dose is 2 times the previous dose.

[0057] 2. Cell Fusion

[0058] Three days after the final booster immunization, cell fusion is carried out with a conventional method using 50% (by weight) polyethylene glycol, namely PEG (molecular weight being 1450) as a fusion agent. Specifically, under an aseptic condition, the mice to be fused are killed by breaking the neck, spleen cells are isolated and mixed with murine myeloma cells SP2/0 at a ratio of 5:1, and the mixed cells are washed with an RPMI-1640 basal medium and centrifuged at 1200 rpm for 5 min. A supernatant is discarded, draining is conducted, 1 mL of PEG is added for fusion of 1 min, the RPMI-1640 basal medium is slowly added, centrifugation is conducted, a supernatant is discarded, a precipitation is fusion cells, resuspension is conducted with 20 mL of complete medium, suspended cells are added to 80 mL of semi-solid medium, and the mixture is added to a 6-well cell culture plate after uniform mixing at 2 mL/well, and then placed in a 37 C. CO.sub.2 incubator for culture.

[0059] The cell complete medium containing 1% HAT contains 20% (by volume) of fetal calf serum, 75% (by volume) of RPMI-1640 basal medium, 1% (by weight) of L-glutamine, 1% (by volume) of HEPES, 1% (by volume) of double antibody (10,000 units per milliliter of penicillin and 10,000 micrograms per milliliter of streptomycin), 2% (by volume) of growth factor (HFCS) and 1% (by weight) of hypoxanthine-aminopterin-thymidine (HAT) and methylcellulose, the above components are purchased from Sigma-Aldrich Co. Ltd.

[0060] 3. Cell Strain Screening and Cloning

[0061] Two to three weeks after cell fusion, when cell colonies grow to be visible, clones are picked out from the culture medium with a micropipette and transferred to a 96-well cell culture plate to be cultured with HAT liquid, and a culture supernatant is drawn for testing when the cells grow to the position of of the well bottom. A two-step screening method is adopted. Indirect ELISA is adopted in the first step to screen out positive wells resistant to fumonisin but not resistant to carrier protein BSA; indirect competitive ELISA is adopted in the second step to detect the positive wells screened out in the first step, fumonisin B.sub.1 is used as a competitive antigen, wells with higher absorbance and sensitivity are chosen (higher absorbance means a higher final measured value for a well with a competitive antigen of 0, namely a positive control well, and higher sensitivity means a smaller competitive antigen concentration, namely IC.sub.50 value when the suppression rate is 50%), subcloning is conducted with a limiting dilution method, then the same two-step method is adopted for detection after the subcloning, and subcloning is conducted in the same manner for 4-5 times to obtain a hybridoma cell strain Fm7A11.

[0062] 4. Anti-Fumonisin B.sub.1 Monoclonal Antibody Hybridoma Cell Strain Fm7A11 Antibody Variable Region Sequence Determination

[0063] (1) Total RNA extraction: total RNA capable of producing the hybridoma cell strain Fm7A11 is extracted by using a total RNA extraction kit from TIANGEN according to the instruction manual;

[0064] (2) Synthesis of cDNA: a first strand of cDNA is synthesized by reverse transcription according to a SuperScript-2II reverse transcriptase instruction manual with the total RNA obtained in the step 1 as a template and oligo(dT)15 as a primer, and the primer oligo(dT)15 is purchased from Invitrogen; and

[0065] (3) Cloning of variable region genes by PCR: primers are designed according to the conserved sites of mouse antibody gene sequences in GENBANK, to amplify the antibody heavy chain and light chain variable region genes with cDNA as a template. PCR procedures: 94 C. 30 s, 58 C. 45 s, 72 C. 1 min, amplification of 30 cycles, final extension for 10 min at 72 C. After PCR products are subjected to electrophoretic separation by 1% (by weight) sepharose, DNA fragments are purified and recovered with a kit and ligated into a support pMD18-T to be transformed into escherichia coli DH5 competent cells, and positive clones are picked out and sent to Shanghai Sunny Biotechnology Co., Ltd. for sequencing. The sequences of the primers are as follows: the sequences of the primers of the heavy chain variable region are 5-CAG GTS MAR CTG MAG GAG TCW G-3 (22mer) and 5-CAG GGG CCA GTG GAT AGA CAG ATG GGG G-3 (28mer), wherein S, M, R and W are merging bases, M=A/C, R=A/G, S=G/C, W=A/T, and the sequences of the primers of the light chain variable region are 5-GAC ATC AAG ATG ACC CAG TCT CCA-3 (24mer) and 5-CCG TTT TAT TTC CAG CTT GGT CCC-3 (24mer).

[0066] The obtained gene sequence results: the length of the coding gene sequence of the heavy chain variable region is 379 bp, the sequence is shown in SEQ ID NO: 1, it is deduced from the obtained gene sequence that the heavy chain variable region coded by the gene sequence is composed of 126 amino acids, and the sequence is shown in SEQ ID NO: 3; and the length of the coding gene sequence of the light chain variable region is 348 bp, the sequence is shown in SEQ ID NO: 2, it is deduced from the obtained gene sequence that the light chain variable region coded by the gene sequence is composed of 116 amino acids, and the sequence is shown in SEQ ID NO: 4.

[0067] 5. Preparation and Purification, Subtypes and Characterization of Anti-Fumonisin B.sub.1 Monoclonal Antibody

[0068] The anti-fumonisin B.sub.1 monoclonal antibody hybridoma cell strain Fm7A11 obtained in Example 1 is injected into a BALB/c mouse previously treated with a Freund's incomplete adjuvant, the ascites of the mouse is collected, and the antibody is purified with an octanoic acid-ammonium sulfate method. Specifically, the ascites of the mouse is filtered through double-layer filter paper, the filtered ascites is centrifuged at 4 C. and 12000 r/min for more than 15 min, a supernatant is drawn, the supernatant is mixed with an acetate buffer with 4 times volume, n-octanoic acid is slowly added while stirring, the volume of n-octanoic acid required by per ml of ascites is 30-35 L, and the mixture is mixed at room temperature for 30-60 min and stands at 4 C. for more than 2 h. Then centrifugation is conducted at 4 C. and 12000 r/min for more than 30 min, a precipitate is discarded, a resulting supernatant is filtered through double-layer filter paper, a phosphate buffer with 1/10 filtrate volume and with a molar concentration of 0.1 mol/L and a pH of 7.4 is added, the pH of the mixed liquid is adjusted with 2 mol/L sodium hydroxide solution to 7.4, ammonium sulfate is slowly added in an ice bath till the final concentration of ammonium sulfate reaches 0.277 g/mL, the mixture stands at 4 C. for more than 2 h, then centrifugation is conducted at 4 C. and 12000 r/min for more than 30 min, a supernatant is discarded, a resulting precipitate is resuspended with 0.01 mol/L phosphate buffer of 1/10 original volume of ascites with a pH of 7.4, placed in a dialysis bag and dialyzed with 0.01 mol/L PBS for two days, then dialysis is conducted with PB for two days, a protein solution in the dialysis bag is taken out and centrifuged, a supernatant is collected, a precipitate is discarded, and then the product is pre-frozen at 70 C. and then lyophilized in a lyophilizer. Lyophilized powder is collected, thus obtaining the purified anti-fumonisin B.sub.1 monoclonal antibody;

[0069] the acetate buffer is obtained by adding water to 0.29 g of sodium acetate and 0.141 mL of acetic acid to 100 mL; the 0.01 mol/L phosphate buffer is obtained by adding water to 0.8 g of sodium chloride, 0.29 g of sodium phosphate dibasic dodecahydrate, 0.02 g of potassium chloride and 0.02 g of potassium dihydrogen phosphate to 100 mL; and the 0.1 mol/L phosphate buffer is obtained by adding water to 8 g of sodium chloride, 2.9 g of sodium phosphate dibasic dodecahydrate, 0.2 g of potassium chloride and 0.2 g of potassium dihydrogen phosphate to 100 mL.

[0070] The subtype of the anti-fumonisin B.sub.1 monoclonal antibody secreted by the hybridoma cell strain Fm7A11 is identified as IgG2b by using a commercially available subtype identification kit.

[0071] The titer of the antibody obtained by purifying the ascites of the mouse is measured to be 3.210.sup.5 through conventional non-competitive enzyme-linked immunosorbent assay (ELISA), that is to say, a solution measurement result is positive when the antibody is diluted by 3.210.sup.5 times. The sensitivity to fumonisin B.sub.1 is measured to be 0.32 ng/mL through conventional indirect competitive ELISA. The cross-reactivities to fumonisins B.sub.2 and B.sub.3 are 4.3% and 12.8%. Cross-reactivities with aflatoxin, zearalenone, T-2 toxin, ochratoxin and vomitoxin are all less than 0.1%.

Embodiment 2

[0072] Preparation of Fumonisin B.sub.1, Aflatoxin B.sub.1, Ochratoxin A, Zearalenone and Sterigmatocystin Complex Immunoaffinity Column

[0073] 1. Preparation of Matrix

[0074] 1 g of sepharose lyophilized matrix powder required (3.5 mL of swelling matrix of final volume can be obtained from per gram of lyophilized matrix powder) is weighed and the sepharose lyophilized matrix powder is dissolved in 1 mmol/L HCl. The matrix will swell immediately and is then placed in a sintered glass filter to be washed with 1 mmol/L HCl for 15 min.

[0075] 2. Ligand (Antibody) Coupling

[0076] a. The anti-fumonisin B.sub.1 monoclonal antibody, the anti-aflatoxin B.sub.1 monoclonal antibody, the anti-ochratoxin A monoclonal antibody, the anti-zearalenone monoclonal antibody and the anti-sterigmatocystin monoclonal antibody for coupling are dissolved by using a coupling buffer of 0.2 mol/L NaHCO.sub.3 with a pH of 8.3, wherein the concentration of each antibody is 12.5 mg/mL, and the dissolved antibodies are placed in an ice bath for temporary storage. The antibody-containing coupling buffer is added to a capped container capable of being completely sealed. CNBr activated sepharose is rapidly transferred to the antibody solution. The mixture is thoroughly mixed for 2-4 h at the room temperature (20-25 C.).

[0077] b. Calculation of the coupling rate: centrifugation is conducted at 2,000 rpm till sepharose is centrifuged to the bottom of a tube, a supernatant is transferred to a new centrifuge tube, and the protein content of the supernatant is measured. The coupling rate is calculated to be 98.5% (indicating that coupling is successful). The sepharose centrifuged to the bottom of the tube is taken out and washed with the coupling buffer to remove excess ligands.

[0078] c. Block: the matrix is transferred to a 0.1 mol/L Tris-HCl buffer. The matrix in the 0.1 mol/L Tris-HCl buffer stands for 2-4 h at room temperature to block all remaining active sites.

[0079] d. In order to remove uncoupled unwanted ligands after coupling, the matrix is washed with buffers with a pH of 4 and a pH of 8, namely, 0.1 mol/L acetate/sodium acetate buffer and 0.1 mol/L Tris-HCl buffer in sequence for at least 3 cycles, wherein the usage amount of each buffer is at least 5 times the volume of the matrix. Each washing cycle comprises the steps of conducting washing with the 0.1 mol/L acetate/sodium acetate buffer first and then with the 0.1 mol/L Tris-HCl buffer.

[0080] e. Washing is conducted with 5 times the colloidal volume of 0.01% NaN.sub.3-PBS and storage is conducted with 0.01% NaN.sub.3-PBS.

[0081] 3. Column loading: a slurry is prepared by combining with and using the buffer, and 75% of sedimenting matrix and 25% of phosphate buffer (pH 7.0) are mixed; the slurry is poured into the column continuously; a glass rod leaning against the inner wall of the column is used for column packing, which will help reduce the generation of air bubbles; after column packing, an opening in the lower end of the affinity column is closed and a top part of the affinity column is taken down; the remaining portion of the affinity column is carefully filled with a PBS buffer with a pH of 7.0 to form an upward meniscus at the top end of the affinity column; a top sieve plate is inserted into the affinity column at a certain angle to ensure that there is no air under the sieve plate; the sieve plate is locked at a proper position of the surface of the matrix, an opening in the bottom of the affinity column is opened, 0.01% NaN.sub.3-PBS for sterile filtration with 5 times column bed volume passes through the column, and storage is conducted with 0.01% NaN.sub.3-PBS, thereby completing loading and balancing of the fumonisin B.sub.1, aflatoxin B.sub.1, ochratoxin A, zearalenone and sterigmatocystin affinity column so that the affinity column can be directly used.

Embodiment 3: Detection of Fumonisin B.SUB.1., Aflatoxin B.SUB.1., Ochratoxin A, Zearalenone and Sterigmatocystin in Rice

[0082] 1.0 Detection of Fumonisin B.sub.1, Aflatoxin B.sub.1, Ochratoxin a, Zearalenone and Sterigmatocystin in Rice

[0083] Rice adding recovery experiments: fumonisin B.sub.1 with the concentration gradient of 500 g/kg, 1000 g/kg and 2000 g/kg, and aflatoxin B.sub.1, ochratoxin A, zearalenone and sterigmatocystin with the concentration gradients of 10 g/kg, 20 g/kg and 50 g/kg are added respectively. Five groups of parallel tests are made for each experiment.

[0084] Three Gradients:

[0085] The dose for the first experiment: 500 g/kg of fumonisin B.sub.1, 10 g/kg of aflatoxin B.sub.1, 10 g/kg of ochratoxin A, 10 g/kg of zearalenone and 10 g/kg of sterigmatocystin.

[0086] The dose for the second experiment: 1000 g/kg of fumonisin B.sub.1, 20 g/kg of aflatoxin B.sub.1, 20 g/kg of ochratoxin A, 20 g/kg of zearalenone and 20 g/kg of sterigmatocystin.

[0087] The dose for the third experiment: 2000 g/kg of fumonisin B.sub.1, 50 g/kg of aflatoxin B.sub.1, 50 g/kg of ochratoxin A, 50 g/kg of zearalenone and 50 g/kg of sterigmatocystin.

[0088] Extraction of fumonisin B.sub.1, aflatoxin B.sub.1, ochratoxin A, zearalenone and sterigmatocystin in rice:

[0089] 20.0 g of ground sample (particle size being less than 2 mm) is weighed accurately in a homogenizer, 100 mL of acetonitrile/water/formic acid (80+18+2) is added, and homogenization, high-speed stirring and extraction are conducted for 2 min. Quantitative filter paper filtration: 5.0 mL of filtrate is accurately transferred, 15.0 mL of PBS solution with a PH of 7.0 is added for dilution, and filtration is conducted with glass fiber filter paper for 1-2 times till filtrate becomes clear. The complex immunoaffinity column is attached to the bottom of a 10.0 mL glass syringe. 10.0 mL of sample extract is accurately transferred into the glass syringe, an air pressure pump and the glass syringe are connected, and the pressure is adjusted to enable the solution to slowly pass through the complex immunoaffinity column at a flow rate of about 6 mL/min until 2-3 mL of air passes through the column. The column is eluted twice with 10.0 mL of water, all eluate is discarded, and 2-3 mL of air is made to pass through the column. 1.0 mL of chromatography-grade methanol is accurately added for elution, the flow rate is 1-2 mL/min, and all eluate is collected in a glass test tube for testing.

[0090] 2.0 High Performance Liquid Chromatography-Mass Spectrometry Conditions

[0091] a. Mobile phase: A, 0.05% formic acid/water solution; B, 0.05% formic acid/acetonitrile solution

[0092] b. Gradient elution: 0-3 min, 15%-50% B; 4-5 min, 50%-70% B; 6.5-8 min, 70%-100% B; 8-10 min, 100%-50% B; 10-11 min, 50%-15% B; and 11-15 min, 15% B.

[0093] c. Chromatographic column: C-18 column (column length 50 mm, inner diameter 2.1 mm, packing diameter 1.7 m)

[0094] d. Flow rate: 200 L/min and

[0095] e. The mass spectrometry scanning parameters for various toxins are shown in Table 1.

[0096] 3.0 Quantification

[0097] A standard working solution containing different concentrations of fumonisin B.sub.1, aflatoxin B.sub.1, ochratoxin A, zearalenone and sterigmatocystin is drawn by a sample injector to be injected into a high performance liquid chromatography-mass spectrometer, high performance liquid chromatogram-mass spectrograms of various toxins and the standard solution peak area of each toxin are obtained under the conditions separately, a standard curve of each toxin is drawn, and an external standard method is used to calculate the content of each toxin.

[0098] 4.0 Results

[0099] The results of the adding standard recovery for rice are between 85%-105% with RSD less than 10%. The results show that the method completely meets the analysis requirement for detecting fumonisin B.sub.1, aflatoxin B.sub.1, ochratoxin A, zearalenone and sterigmatocystin in rice. The results are shown in Table 1-Table 5 respectively.

TABLE-US-00002 TABLE 1 Recovery results of fumonisin B.sub.1 in rice Spiked concentration of fumonisin B.sub.1 Recovery Recovery Recovery Recovery Recovery g/kg rate 1 % rate 2 % rate 3 % rate 4 % rate 5 % RSD % 500 104.1 99.3 101.3 99.5 96.7 2.7 1000 86.8 92.3 86.4 87.5 98.3 5.6 2000 93.5 104.6 86.7 97.7 92.5 7.0

TABLE-US-00003 TABLE 2 Recovery results of aflatoxin B.sub.1 in rice Spiked concentration of aflatoxin B.sub.1 Recovery Recovery Recovery Recovery Recovery g/kg rate 1 % rate 2 % rate 3 % rate 4 % rate 5 % RSD % 10 98.6 90.9 91.5 86.7 100.5 6.1 20 103.4 93.3 87.6 87.6 90.6 7.1 50 87.6 101.1 98.4 93.5 102.4 6.3

TABLE-US-00004 TABLE 3 Recovery results of ochratoxin A in rice Spiked concentration of ochratoxin A Recovery Recovery Recovery Recovery Recovery g/kg rate 1 % rate 2 % rate 3 % rate 4 % rate 5 % RSD % 10 92.5 91.2 87.7 103.1 98.8 6.5 20 88.4 91.1 100.9 92.3 86.9 5.9 50 95.6 103.4 96.8 102.2 98.4 3.4

TABLE-US-00005 TABLE 4 Recovery results of zearalenone in rice Spiked concentration of zearalenone Recovery Recovery Recovery Recovery Recovery g/kg rate 1 % rate 2 % rate 3 % rate 4 % rate 5 % RSD % 10 88.8 90.6 95.5 97.6 102.7 5.9 20 90.4 93.8 85.6 94.4 100.8 6.0 50 103.6 100.7 89.7 88.8 98.6 6.9

TABLE-US-00006 TABLE 5 Recovery results of sterigmatocystin in rice Spiked concentration of sterigmatocystin Recovery Recovery Recovery Recovery Recovery g/kg rate 1 % rate 2 % rate 3 % rate 4 % rate 5 % RSD % 10 100.8 100.2 92.7 99.6 95.4 3.6 20 93.3 99.9 101.6 99.7 103.5 3.9 50 89.7 94.4 96.6 102.5 93.2 5.0

Embodiment 4: Detection of Fumonisin B.SUB.1., Aflatoxin B.SUB.1., Ochratoxin A, Zearalenone and Sterigmatocystin in Edible Oil

[0100] 1.0 Detection of Fumonisin B.sub.1, Aflatoxin B.sub.1, Ochratoxin A, Zearalenone and Sterigmatocystin in Edible Oil

[0101] Edible oil adding standard recovery experiments: fumonisin B.sub.1 with the concentration gradient of 500 g/kg, 1000 g/kg and 2000 g/kg, and aflatoxin B.sub.1, ochratoxin A, zearalenone and sterigmatocystin with the concentration gradients of 10 g/kg, 20 g/kg and 50 g/kg are added respectively. Five groups of parallel tests are made for each experiment.

[0102] Extraction of fumonisin B.sub.1, aflatoxin B.sub.1, ochratoxin A, zearalenone and sterigmatocystin in edible oil:

[0103] vegetable oil liquid sample extraction: 5.0 g of vegetable oil sample is weighed accurately in a 50 mL centrifuge tube, 15.0 mL of 70% aqueous methanol is added, oscillation blending is conducted in a vortex mixer for 2 min, centrifugation is conducted at 5000 r/min for 2 min, 10.0 mL of methanol solution layer is transferred, dilution is conducted with 20.0 mL of water, uniform mixing is conducted in a mixer, and filtration is conducted with glass fiber filter paper till filtrate becomes clear. The complex immunoaffinity column is attached to the bottom of a 10.0 mL glass syringe. 10.0 mL of sample extract is accurately transferred into the glass syringe, an air pressure pump and the glass syringe are connected, and the pressure is adjusted to enable the solution to slowly pass through the complex immunoaffinity column at a flow rate of about 6 mL/min until 2-3 mL of air passes through the column. The column is eluted twice with 10.0 mL of water, all eluate is discarded, and 2-3 mL of air is made to pass through the column. 1.0 mL of chromatography-grade methanol is accurately added for elution, the flow rate is 1-2 mL/min, and all eluate is collected in a glass test tube for testing.

[0104] 2.0 High Performance Liquid Chromatography-Mass Spectrometry Conditions

[0105] a. Mobile phase: A, 0.05% formic acid/water solution; B, 0.05% formic acid/acetonitrile solution

[0106] b. Gradient elution: 0-3 min, 15%-50% B; 4-5 min, 50%-70% B; 6.5-8 min, 70%-100% B; 8-10 min, 100%-50% B; 10-11 min, 50%-15% B; and 11-15 min, 15% B.

[0107] c. Chromatographic column: C-18 column (column length 50 mm, inner diameter 2.1 mm, packing diameter 1.7 m)

[0108] d. Flow rate: 200 L/min and

[0109] e. The mass spectrometry scanning parameters for various toxins are shown in Table 1.

[0110] 3.0 Quantification

[0111] A standard working solution containing different concentrations of fumonisin B.sub.1, aflatoxin B.sub.1, ochratoxin A, zearalenone and sterigmatocystin is drawn by a sample injector to be injected into a high performance liquid chromatography-mass spectrometer, high performance liquid chromatogram-mass spectrograms of various toxins and the standard solution peak area of each toxin are obtained under the conditions separately, a standard curve of each toxin is drawn, and an external standard method is used to calculate the content of each toxin.

[0112] 4.0 Results

[0113] The results of the adding recovery for vegetable oil are between 85%-105% with RSD less than 10%. The results show that the method completely meets the analysis requirement for detecting fumonisin B.sub.1, aflatoxin B.sub.1, ochratoxin A, zearalenone and sterigmatocystin in edible oil. The results are shown in Table 6-Table 10 respectively.

TABLE-US-00007 TABLE 6 Recovery results of fumonisin B.sub.1 in vegetable oil Spiked concentration of fumonisin B.sub.1 Recovery Recovery Recovery Recovery Recovery g/kg rate 1 % rate 2 % rate 3 % rate 4 % rate 5 % RSD % 500 92.3 91.6 101.5 88.3 97.2 5.5 1000 104.1 103.4 92.8 90.8 93.4 6.5 2000 97.5 85.8 97.5 91.7 100.5 6.2

TABLE-US-00008 TABLE 7 Recovery results of aflatoxin B.sub.1 in vegetable oil Spiked concentration of aflatoxin B.sub.1 Recovery Recovery Recovery Recovery Recovery g/kg rate 1 % rate 2 % rate 3 % rate 4 % rate 5 % RSD % 10 90.5 103.2 103.1 89.7 95.3 6.8 20 90.6 102.1 94.8 99.8 89.4 5.8 50 98.1 88.5 101.1 102.5 90.6 6.5

TABLE-US-00009 TABLE 8 Recovery results of ochratoxin A in vegetable oil Spiked concentration of ochratoxin A Recovery Recovery Recovery Recovery Recovery g/kg rate 1 % rate 2 % rate 3 % rate 4 % rate 5 % RSD % 10 87.2 102.5 91.8 90.1 96.9 6.5 20 89.8 94.4 99.7 90.6 89.1 4.8 50 87.7 91.6 98.7 89.3 102.2 6.7

TABLE-US-00010 TABLE 9 Recovery results of zearalenone in vegetable oil Spiked concentration of zearalenone Recovery Recovery Recovery Recovery Recovery g/kg rate 1 % rate 2 % rate 3 % rate 4 % rate 5 % RSD % 10 96.5 103.1 85.8 86.3 102.2 8.8 20 88.4 89.1 91 99.5 102.1 6.7 50 102.5 105.7 101.8 102.2 89.9 6.1

TABLE-US-00011 TABLE 10 Recovery results of sterigmatocystin in vegetable oil Spiked concentration of sterigmatocystin Recovery Recovery Recovery Recovery Recovery g/kg rate 1 % rate 2 % rate 3 % rate 4 % rate 5 % RSD % 10 97.3 96.7 94.9 87.9 86.7 5.4 20 90.5 99.1 103.1 89.7 95.6 5.9 50 94.7 90.5 99.4 103.3 104.4 5.9