Hybridoma cell strain zqm4B4, anti-aspergillus ochraceus monoclonal antibody generated by hybridoma cell strain zqm4B4 and application of monoclonal antibody

Abstract

The invention relates to a hybridoma cell strain zqm4B4, an anti-aspergillus ochraceus monoclonal antibody generated by the hybridoma cell strain zqm4B4 and application. The hybridoma cell strain zqm4B4 provided by the invention can be used for preparing a high-titer anti-aspergillus ochraceus monoclonal antibody, and the titer measured by an enzyme-linked immunosorbent assay (ELISA) method can reach 6.51 * 10 < 5 >. The anti-aspergillus ochraceus monoclonal antibody provided by the invention is high in sensitivity and good in specificity, the lowest detection limit of the anti-aspergillus ochraceus monoclonal antibody to aspergillus ochraceus is 0.0962 mu g / mL, and the anti-aspergillus ochraceus monoclonal antibody has no cross reaction with other fungi.

Description

Technical Field

[0001] The invention relates to a hybridoma cell strain zqm4B4 and an anti-ochratoaspergillus monoclonal antibody produced by the hybridoma cell strain and application thereof. Background Art

[0002] Aspergillus westerdijkiae is a filamentous fungus belonging to the Aspergillus genus. Its main secondary metabolite, ochratoxin A (OTA), has strong liver and kidney toxicity and potential carcinogenicity and mutagenicity. The International Agency for Research on Cancer (IARC) ranks it second in terms of hazard, second only to aflatoxin (Group 2B). Studies have shown that Aspergillus westerdijkiae is one of the main species causing OTA contamination in agricultural products. Compared with other Aspergillus species, A. westerdijkiae produces the highest concentration of OTA. The presence of A. westerdijkiae and OTA contamination can be detected in grains or feeds such as corn and wheat, as well as coffee, beer, and fruit juice drinks, which seriously endangers human health and livestock safety. In addition to the presence of Aspergillus ochraceus in agricultural products, Aspergillus ochraceus can also be found in air dust and deep sea environments. Therefore, it is of great significance to ensure the health of humans and animals to establish rapid quantitative detection technology for specific and highly sensitive detection of Aspergillus ochraceus and eliminate OTA contamination at the source.

[0003] The existing detection methods for Aspergillus ochraceus are mainly laboratory confirmatory detection technologies such as LAMP (Loop-Mediated Isothermal Amplification) and PCR-DGGE (Denaturing Gradient Gel Electrophoresis). LAMP has the advantages of good specificity and high sensitivity, but it requires strict experimental conditions, expensive instruments and equipment, and professional operators, which is difficult to meet the requirements of rapid on-site detection. PCR-DGGE is a method of amplifying rDNA in samples by PCR using fungus-specific primers, and using DGGE electrophoresis separation method based on the differences in rDNA molecular sequence structure to achieve sensitive detection. PCR-DGG is a convenient tool for analyzing fungal communities, but it is not suitable for accurately describing the species present. Immunological detection methods are based on antigen-antibody specific recognition, and the immune signal is converted into a detectable signal through enzyme, fluorescence and other labeling technologies, thereby qualitatively or quantitatively displaying the content of the target. This type of method has the advantages of simple sample processing, high sensitivity, strong specificity, and short experimental cycle, among which the sensitivity and specificity of the antibody directly determine the accuracy of the immunological detection method. At present, there are few reports on immunological detection technology for the detection methods of Aspergillus ochraceus at home and abroad. Therefore, the research on anti-Aspergillus ochrae monoclonal antibodies can open up new ideas for the rapid detection of Aspergillus ochrae and is of great significance for the realization of immunoquantitative detection of Aspergillus ochrae. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a hybridoma cell line zqm4B4, an anti-Aspergillus westerdijkiae monoclonal antibody produced by the hybridoma cell line, and applications thereof.

[0005] The first aspect of the present invention provides a hybridoma cell line zqm4B4, which was deposited in the China Center for Type Culture Collection (CCTCC) on March 22, 2023. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C202365.

[0006] The second aspect of the present invention provides an anti-ochratoaspergillus monoclonal antibody, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1 in the sequence table; and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 2 in the sequence table. It is secreted and produced by the hybridoma cell line zqm4B4 with a deposit number of CCTCC NO: C202365. The anti-ochratoaspergillus monoclonal antibody provided by the present invention has high sensitivity and good specificity. Its minimum detection limit for ochratoaspergillus is 0.0962 μg / mL, and there is no cross reaction with other fungi such as Aspergillus flavus, Aspergillus parasiticus, Penicillium citrinum, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Fusarium, Aspergillus terreus, Penicillium chrysogenum, Aspergillus tubingensis, Aspergillus piperits, Aspergillus liu, Aspergillus clavatus, etc.

[0007] The third aspect of the present invention provides the use of the above anti-Aspergillus ochrata monoclonal antibody in detecting the content of Aspergillus ochrata.

[0008] A fourth aspect of the present invention provides use of the above anti-Aspergillus ochrata monoclonal antibody in the preparation of a product for detecting Aspergillus ochrata.

[0009] The fifth aspect of the present invention provides an immunoassay product for detecting Aspergillus ochraceus comprising the above-mentioned anti-Aspergillus ochraceus monoclonal antibody.

[0010] According to the above scheme, the product for detecting Aspergillus ochraceus is an ELISA kit for immunoassay of Aspergillus ochraceus or an immunoaffinity column for Aspergillus ochraceus. The ELISA kit for immunoassay of Aspergillus ochraceus, such as the double antibody sandwich immunoassay kit for Aspergillus ochraceus, the marker in the ELISA test kit can be horseradish peroxidase, or other markers with similar functions to horseradish peroxidase, such as colloidal gold labeled probes, time-resolved fluorescent microspheres, up-conversion fluorescent materials, biomimetic catalytic enzymes, etc., and then a series of immunoassay methods are established based on these probe-labeled immune recognition materials for immunoassay of Aspergillus ochraceus; the immunoaffinity column for Aspergillus ochraceus can be used for sample pretreatment in, for example, HPLC detection methods and HPLC-MS / MS detection methods, effectively reducing or even eliminating the matrix effect, and achieving high recovery rate and low matrix effect of Aspergillus ochraceus.

[0011] The present invention provides a variable region sequence of a monoclonal antibody specifically binding to Aspergillus ochraceus, wherein the heavy chain variable region of the monoclonal antibody specifically binding to Aspergillus ochraceus is an amino acid sequence shown in SEQ ID NO: 1 in the sequence table or a conservative mutant obtained by adding, deleting, replacing or modifying conservative mutations of one or more amino acids; the light chain variable region is an amino acid sequence shown in SEQ ID NO: 2 in the sequence table or a conservative mutant obtained by adding, deleting, replacing or modifying conservative mutations of one or more amino acids. The variable region sequence of the monoclonal antibody provided by the present invention can specifically bind to Aspergillus ochraceus.

[0012] The present invention also provides a gene sequence encoding the variable region sequence of the above-mentioned monoclonal antibody specifically binding to Aspergillus ochraceus.

[0013] The present invention further provides a method for preparing an anti-Aspergillus ochraceus monoclonal antibody, comprising the following steps: injecting the hybridoma cell line zqm4B4 obtained above into the abdomen of a BALB / c mouse that has been treated with Freund's incomplete adjuvant in advance, collecting the ascites of the mouse, and obtaining the anti-Aspergillus ochraceus monoclonal antibody after purification. In the present invention, the anti-Aspergillus ochraceus monoclonal antibody can be purified by the octanoic acid-ammonium sulfate method.

[0014] According to the above scheme, the specific operation of the caprylic acid-ammonium sulfate method for purifying antibodies is as follows: filter the mouse ascites with a double-layer filter paper, centrifuge at 4°C, 12000r / min for more than 15min, absorb the supernatant, mix the obtained ascites supernatant with 4 volumes of acetate buffer, slowly add caprylic acid under stirring, the volume of caprylic acid required for each milliliter of ascites is 30-35μL, mix at room temperature for 30-60min, and let stand at 4°C for more than 2h. 12000r / min, centrifuge at 4℃ for more than 30min, discard the precipitate, filter the obtained supernatant with double-layer filter paper, add 1 / 10 volume of the filtrate with a molar concentration of 0.1mol / L and a pH of 7.4 phosphate buffer, adjust the pH of the mixture to 7.4 with 2mol / L sodium hydroxide solution, slowly add ammonium sulfate in an ice bath to a final concentration of 0.277g / mL ammonium sulfate, let stand at 4℃ for more than 2h, then centrifuge at 12000r / min, 4℃ for more than 30min, discard the supernatant, resuspend the obtained precipitate with 1 / 10 volume of the original ascites water volume with a molar concentration of 0.01mol / L and a pH of 7.4 phosphate buffer, put it into a dialysis bag, dialyze it with 0.01mol / L PBS for two days, and then dialyze it with PB for two days, take out the protein solution in the dialysis bag, centrifuge, collect the supernatant, discard the precipitate, pre-freeze it at -70℃, and then put it into a freeze dryer for freeze drying. Collecting the lyophilized powder is the purified anti-Aspergillus ochraceus monoclonal antibody;

[0015] The acetate buffer is obtained by adding water to 0.29g sodium acetate and 0.141mL acetic acid to make the volume to 100mL; the 0.01mol / L phosphate buffer is obtained by adding water to 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, and 0.02g potassium dihydrogen phosphate to make the volume to 100mL; the 0.1mol / L phosphate buffer is obtained by adding water to 8g sodium chloride, 2.9g disodium hydrogen phosphate dodecahydrate, 0.2g potassium chloride, and 0.2g potassium dihydrogen phosphate to make the volume to 100mL.

[0016] Beneficial effects of the present invention:

[0017] (1) The hybridoma cell line zqm4B4 provided by the present invention can be used to prepare high-titer monoclonal antibodies against Aspergillus ochraceus. The antibody titer measured by enzyme-linked immunosorbent assay (ELISA) can reach 6.51×10 5 .

[0018] (2) The anti-Aspergillus ochraceus monoclonal antibody provided by the present invention has high sensitivity and good specificity. The minimum detection limit of Aspergillus ochraceus is 0.0962 μg / mL, and there is no cross reaction with other fungi including Aspergillus flavus, Aspergillus parasiticus, Penicillium citrinum, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Fusarium, Aspergillus terreus, Penicillium chrysogenum, Aspergillus tubingensis, Aspergillus piperits, Aspergillus liufuga, Aspergillus clavatus and the like.

[0019] (3) The anti-Aspergillus ochraceus monoclonal antibody provided by the present invention can be used for the determination of Aspergillus ochraceus content. DETAILED DESCRIPTION

[0020] Example 1: Screening of hybridoma cell line zqm4B4

[0021] The hybridoma cell line zqm4B4 provided by the present invention is obtained by a two-step screening method. The specific steps are: BALB / C mice are immunized with Aspergillus ochraceus strains for 4-6 times, and then a complete antigen AO standard strain with a dose of 1 / 2 times the previous immunization dose is used for booster immunization, cell fusion is performed 3 days later, and after culturing with HAT semi-solid medium for 1-2 weeks, a single cell colony is transferred to a cell culture plate for culture, and when the cells grow to 2 / 3 of the bottom of the well, an indirect non-competitive ELISA method is used for screening, wells with higher absorbance values ​​are selected, and subcloning is performed 4-5 times by a limiting dilution method, and finally the hybridoma cell line zqm4B4 is screened.

[0022] 1. Animal immunization

[0023] The ochratoxin-producing fungal strains in the laboratory were revived by AFPA medium, cultured in Czapek liquid medium, and the culture fluid was collected after filtering the mycelial pellets to prepare the mycelial pellet lysate antigen. It was used as an antigen to immunize 6-week-old female BALB / C mice. In the first immunization, the ochratoxin antigen was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at five points on the back of the mouse's neck. The second immunization was carried out 21 days later, and the ochratoxin antigen was emulsified with an equal volume of Freund's incomplete adjuvant and injected intraperitoneally into the mouse. The third immunization was 2 weeks apart from the second immunization, and the immunization method was the same. The fourth immunization was carried out 3 weeks after the third immunization, and the immunization method was the same as the second immunization, also intraperitoneal injection. The 4 immunization doses were the same, all 200μg per mouse. 8-10 days after each of the first three immunizations, the tail was cut and blood was collected, and the serum was separated. The serum titer of the mouse was detected by indirect ELISA. Eight days after the third immunization, the mice were tail-cut and blood was collected. The mice with sera with relatively high titer and sensitivity were selected for the final booster immunization, with the immunization dose being 1 / 2 times of the previous one.

[0024] 2. Cell Fusion

[0025] Three days after booster immunization, 50% polyethylene glycol PEG (molecular weight 1450) was used as a fusion agent, and cell fusion was performed according to conventional methods. The specific steps were aseptically dislocated the mouse, the spleen was removed, the spleen was crushed with a homogenizer, and the spleen cells were separated with a filter, mixed with mouse myeloma cells SP2 / 0 at a ratio of 5:1-10:1, centrifuged at 1000rpm for 5min, and the mixed cells were resuspended with RPMI-1640 basic culture medium, centrifuged at 1000rpm for 5min, and the supernatant was discarded. 1mL of 50% PEG was added, and the total time was 1 minute. 20mL of RPMI-1640 basic culture medium was added to the adherent cells to resuspend the cells, centrifuged, and the supernatant was discarded. The fused cells at the bottom of the tube were resuspended with 20mL of complete cell culture medium containing 1% HAT, and the suspended cells were added to 80mL of semi-solid culture medium, mixed and added to a 6-well cell culture plate, 1-2mL / well, and placed in a 37°C carbon dioxide incubator for static culture. The complete cell culture medium containing 1% HAT contains 20% (volume percentage) fetal bovine serum, 75% (volume percentage) RPMI-1640 basic culture medium, 1% (weight percentage) L-glutamine, 1% (volume percentage) HEPES, 1% (volume percentage) double antibody (10,000 units per milliliter of penicillin and 10,000 micrograms per milliliter of streptomycin), 1% (volume percentage) growth factor (clone easy) and 1% (weight percentage) hypoxanthine-aminopterinary-thymidine nucleoside, i.e., HAT and methylcellulose, which are purchased from Sigma-Aldrich.

[0026] 3. Screening and cloning of cell lines

[0027] 1-2 weeks after cell fusion, when the cell colonies grow to be visible to the naked eye, use a micropipette to pick out the clones from the culture medium and transfer them to a 96-well cell culture plate for HAT liquid culture. When the cells grow to 2 / 3 of the bottom of the well, aspirate the culture supernatant for detection. The indirect non-competitive ELISA method was used to screen out the positive wells against Aspergillus ochraceus, and the limiting dilution method was used for subcloning. After subcloning, the same method was used for detection. After repeating the subcloning 4-5 times, the hybridoma cell line zqm4B4 was obtained. The hybridoma cell line was deposited in the China Center for Type Culture Collection (CCTCC) on March 22, 2023. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C202365.

[0028] Example 2: Determination of the sequence of the variable region of the antibody of the anti-Aspergillus ochraceus monoclonal antibody hybridoma cell line zqm4B4.

[0029] (1) Extracting total RNA: Using the total RNA extraction kit of Tiangen Company and following the instructions, extract the total RNA that can produce the hybridoma cell line zqm4B4;

[0030] (2) cDNA synthesis: Using the total RNA obtained in step 1 as a template and oligo(dT)15 as a primer, the cDNA was synthesized according to SuperScript TM -2II reverse transcriptase instructions were used for reverse transcription to synthesize the first strand of cDNA; primer oligo(dT)15 was purchased from Invitrogen;

[0031] (3) PCR method for cloning variable region genes: Primers were designed based on the conserved sites of mouse antibody gene sequences in GENBANK, and cDNA was used as a template to amplify antibody heavy chain and light chain variable region genes. The PCR program was: 94°C for 30s, 58°C for 45s, 72°C for 1min, 30 cycles of amplification, and finally 72°C for 10min. After the PCR products were separated by 1% (weight percent) agarose gel electrophoresis, the DNA fragments were purified and recovered using a kit, connected to the vector pMD18-T, transformed into E. coli DH5α competent cells, and the positive clones were picked and sequenced. The sequences of the primers are: the heavy chain variable region primers are 5'-CAG GTS MAR CTGMAG GAG TCW G-3' (22mer) and 5'-CAG GGG CCA GTG GAT AGA CAG ATG GGG G-3' (28mer), where S, M, R and W are merged bases, M = A / C, R = A / G, S = G / C, W = A / T; the light chain variable region primers are 5'-GAC ATCAAG ATG ACC CAG TCT CCA-3' (24mer) and 5'-CCG TTT TAT TTC CAG CTT GGT CCC-3' (24mer).

[0032] The gene sequence results obtained: the amino acid composition of the heavy chain variable region is shown in SEQ ID NO: 1. The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2.

[0033] Example 3: Preparation, purification, subtype and characterization of anti-Aspergillus ochraceus monoclonal antibodies

[0034] The anti-ochratoaspergillus monoclonal antibody hybridoma cell line zqm4B4 obtained in Example 1 was injected into BALB / C mice treated with Freund's incomplete adjuvant in advance, and the ascites of the mice was collected. The antibody was purified by the caprylic acid-ammonium sulfate method. The specific operation was: the mouse ascites was filtered with a double-layer filter paper, centrifuged at 4°C and 12000r / min for more than 15min, the supernatant was aspirated, and the obtained ascites supernatant was mixed with 4 times the volume of acetate buffer, and caprylic acid was slowly added under stirring, the volume of caprylic acid required per milliliter of ascites was 30-35μL, mixed at room temperature for 30-60min, and allowed to stand at 4°C for more than 2h. 12000r / min, centrifuge at 4℃ for more than 30min, discard the precipitate, filter the obtained supernatant with double-layer filter paper, add 1 / 10 volume of the filtrate with a molar concentration of 0.1mol / L and a pH of 7.4 phosphate buffer, adjust the pH of the mixture to 7.4 with 2mol / L sodium hydroxide solution, slowly add ammonium sulfate in an ice bath to a final concentration of 0.277g / mL ammonium sulfate, let stand at 4℃ for more than 2h, then centrifuge at 12000r / min, 4℃ for more than 30min, discard the supernatant, resuspend the obtained precipitate with 1 / 10 volume of the original ascites water volume with a molar concentration of 0.01mol / L and a pH of 7.4 phosphate buffer, put it into a dialysis bag, dialyze it with 0.01mol / L PBS for two days, and then dialyze it with PB for two days, take out the protein solution in the dialysis bag, centrifuge, collect the supernatant, discard the precipitate, pre-freeze it at -70℃, and then put it into a freeze dryer for freeze drying. Collecting the lyophilized powder is the purified anti-Aspergillus ochraceus monoclonal antibody;

[0035] The acetate buffer is obtained by adding water to 0.29g sodium acetate and 0.141mL acetic acid to make the volume to 100mL; the 0.01mol / L phosphate buffer is obtained by adding water to 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, and 0.02g potassium dihydrogen phosphate to make the volume to 100mL; the 0.1mol / L phosphate buffer is obtained by adding water to 8g sodium chloride, 2.9g disodium hydrogen phosphate dodecahydrate, 0.2g potassium chloride, and 0.2g potassium dihydrogen phosphate to make the volume to 100mL.

[0036] The subtype of the anti-Aspergillus ochraceus monoclonal antibody secreted by the hybridoma cell line zqm4B4 was identified as IgG2a using a commercially available subtype identification kit.

[0037] The antibody titer purified from mouse ascites was measured by conventional non-competitive enzyme-linked immunosorbent assay (ELISA) to be 6.51×10 5 , that is, the antibody dilution is 6.51×10 5The result of the solution determination was positive when the concentration was doubled. The minimum detection limit of Aspergillus ochraceus was 0.0962 μg / mL by conventional indirect non-competitive ELISA. The specificity of the antibody can be evaluated by the cross-reaction rate. The zqm4B4 monoclonal antibody was determined by the indirect non-competitive ELISA method. The fungi such as Aspergillus flavus, Aspergillus parasiticus, Penicillium citrinum, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Fusarium, Aspergillus terreus, Penicillium chrysogenum, Aspergillus tubingensis, Aspergillus piperitus, Aspergillus liu, and Aspergillus clavatus were treated with Aspergillus ochraceus under the same conditions. They were then added to the ELISA plate as coating materials and coated overnight at 4°C. Then, they were used for indirect non-competitive ELISA detection of zqm4B4 monoclonal antibody. The OD value at 450 nm was determined by the microplate reader. The results showed that only Aspergillus ochraceus had an OD450 nm>1, and the OD450 nm of other fungi was <0.3. Therefore, the zqm4B4 monoclonal antibody had no cross-reaction with other fungi.

[0038] Example 4: Preparation of double antibody sandwich immunoassay method and kit for Aspergillus ochraceus

[0039] 1. Sample pretreatment:

[0040] Weigh 0.2 g of crushed healthy peanuts and corn, add them to 20 mL of PBST, mix well, and dilute 10 times. Mix the mycelium with the peanut solution and corn solution so that the mycelium accounts for 10% of the total. -2 , 10 -1 , 10, 10 2 , 10 3 μg / mL, used for double antibody sandwich immunoassay method and kit detection.

[0041] 3. Taking the ELISA method as an example, the steps for establishing the standard curve of the immunoassay method are as follows:

[0042] (1) The determined capture antibody monoclonal antibody zqm4B4 was diluted to 1 μg / mL with 0.1 mol / L pH 9.6 carbonate buffer and coated on a 96-well ELISA plate at 4°C overnight.

[0043] (2) Wash the plate three times with PBST, add 300 μL / well of PBS solution containing 5% (W / V) skim milk powder as blocking solution, and react at 37°C for 2 h.

[0044] (3) Wash the plate three times with PBST, and dilute the AO standard strain mycelial pellet lysate three-fold to 11 concentration gradients starting from 0.01 M PBS to 1 mg / mL, 100 μL / well. The blank group plus PBS was added to the ELISA plate and reacted at 37°C for 1 h.

[0045] (4) Wash the plate three times with PBST, add 100 μL / well of rabbit polyclonal antibody against Aspergillus ochraceus (4 μg / mL), and react at 37°C for 1 h.

[0046] (5) Wash the plate nine times with PBST, add 100 μL / well of HRP-labeled goat anti-rabbit antibody (1:5000), and react at 37°C for 1 h.

[0047] (6) Wash the plate 6 times with PBST, add 100 μL / well of TMB colorimetric solution, react at 37°C in the dark for 15 min, and measure the OD450 value with an ELISA reader. The sandwich ELISA standard curve can be fitted by the following formula:

[0048]

[0049] The content of Aspergillus ochraceus in the sample solution was obtained based on the relationship curve between the OD value and the concentration of Aspergillus ochraceus. The detection limits of the established Sandwich-ELISA method for Aspergillus ochraceus in peanut and corn samples were 0.118μg / mL and 0.1285μg / mL, respectively.

[0050] The markers in the above ELISA test kit can also use other markers besides horseradish peroxidase, such as labeled probe colloidal gold, time-resolved fluorescent microspheres, upconversion fluorescent materials, biomimetic catalytic enzymes, etc., and then establish a series of immunoassay methods based on these probe-labeled immune recognition materials for the detection of Aspergillus ochraceus.

[0051] Example 5: Preparation of Aspergillus ochraceus immunoaffinity column

[0052] 1. Matrix Preparation

[0053] Weigh the required 1g CNBr-activated Sepharose lyophilized matrix powder (each gram of lyophilized matrix powder can form a final volume of 3.5mL of swollen matrix) and dissolve it in 1mmol / L HCl. The matrix will swell immediately and then be placed in a sintered glass filter and washed with 1mmol / L HCl for 15min.

[0054] 2. Ligand (antibody) coupling

[0055] a. Dissolve the anti-Aspergillus ochraceus monoclonal antibody to be coupled using coupling buffer 0.2 mol / L NaHCO3 pH 8.3, with an antibody concentration of 15 mg / mL. Store the dissolved antibody in an ice bath. Add the coupling buffer containing the antibody to a fully sealable container with a lid. Quickly transfer the CNBr-activated Sepharose to the antibody solution. Mix thoroughly for 2-4 hours at room temperature (20-25°C).

[0056] b Calculation of coupling rate: Centrifuge at 2000rpm to centrifuge the agarose gel to the bottom of the tube, transfer the supernatant to a new centrifuge tube, and measure the protein content of the supernatant. The coupling rate is calculated to be 98.5%. Take the agarose gel that has been centrifuged to the bottom of the tube and wash it with coupling buffer to remove excess ligand.

[0057] c Blocking: Transfer the matrix to 0.1 mol / L Tris-HCl buffer and let stand for 2-4 hours at room temperature to block all residual active groups.

[0058] d is to remove the excess ligand that is not coupled after coupling, and wash the matrix for 3-5 cycles with buffers of pH 4 and 8, namely 0.1mol / L acetic acid / sodium acetate buffer and 0.1mol / L Tris-HCl buffer, respectively, and the amount of each buffer used is 3-5 times the volume of the matrix. Each washing cycle step: first wash with 0.1mol / L acetic acid / sodium acetate buffer, and then wash with 0.1mol / L Tris-HCl buffer.

[0059] e Wash with 3-5 times the volume of gel in 0.01% NaN3-PBS and store in 0.01% NaN3-PBS.

[0060] 3. Column Packing Prepare the slurry using binding buffer, mixing in a ratio of 75% sedimentation matrix and 25% phosphate buffer (pH 7.0). Pour the slurry into the column in a continuous operation. Using a glass rod leaning against the inner wall of the column to fill the column will help reduce the generation of bubbles. After filling the column, close the opening at the lower end of the affinity column and remove the top part of the affinity column. Carefully, use pH 7.0 PBS buffer to add the remaining part of the affinity column to form an upward curved liquid surface at the top of the affinity column. Insert the top screen into the affinity column at a certain angle to ensure that there is no air under the screen. The sieve plate is locked in an appropriate position on the surface of the matrix, the opening below the affinity column is opened, 3-5 times the volume of the column bed is filtered with sterile 0.01% NaN3-PBS, and the column is preserved with 0.01% NaN3-PBS. At this point, the affinity column has been filled and balanced and can be used directly; for example, the sample pretreatment in the HPLC detection method and the HPLC-MS / MS detection method effectively reduces or even eliminates the matrix effect, thereby achieving a high recovery rate of Aspergillus ochraceus and a low matrix effect.

Claims

1. The hybridoma cell line zqm4B4 was deposited in the China Center for Type Culture Collection (CCTCC) on March 22, 2023. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCCNO: C202365.

2. Anti-Aspergillus ochraceus monoclonal antibody, characterized in that: The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing; the light chain variable region has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.

3. The method for preparing the anti-Aspergillus ochraceus monoclonal antibody according to claim 2, characterized in that: The hybridoma cell line zqm4B4 described in claim 1 is injected into BALB / C mice treated with Freund's incomplete adjuvant in advance, and the ascites of the mice is collected and purified to obtain anti-Aspergillus ochraceus monoclonal antibodies.

4. Use of the anti-Aspergillus ochraceus monoclonal antibody according to claim 2 in detecting the Aspergillus ochraceus content.

5. Use of the anti-Aspergillus ochraceus monoclonal antibody according to claim 2 in the preparation of a product for detecting Aspergillus ochraceus.

6. A product for detecting Aspergillus ochraceus comprising the anti-Aspergillus ochraceus monoclonal antibody according to claim 1.

7. The product for detecting Aspergillus ochraceus according to claim 6, characterized in that: It is an ELISA kit for Aspergillus ochraceus immune detection or an Aspergillus ochraceus immunoaffinity column.

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