Hybridoma cell strain zqm4B4 and the aspergillus niger monoclonal antibody produced thereby and use thereof

Abstract

The present application relates to hybridoma cell strain zqm4B4, and an anti-aspergillus niger monoclonal antibody produced by the hybridoma cell strain and application thereof. The hybridoma cell strain zqm4B4 provided by the present application can be used to prepare an anti-aspergillus niger monoclonal antibody with high titer, and the titer measured by enzyme-linked immunosorbent assay (ELISA) can reach 6.51x10 5 The anti-aspergillus niger monoclonal antibody provided by the present application has high sensitivity and good specificity, and the minimum detection limit for aspergillus niger is 0.0962 μg / mL, and has no cross reaction with other fungi.

Description

Technical Field

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

[0002] Aspergillus westerdijkiae is a filamentous fungus belonging to the Aspergillus genus. Its primary secondary metabolite, ochratoxin A (OTA), is highly toxic to the liver and kidneys, and potentially carcinogenic and mutagenic. The International Agency for Research on Cancer (IARC) ranks ochratoxin A (OTA) second only to aflatoxins in its hazard ranking (Group 2B). Studies have shown that A. ochratoxin A is one of the most common species responsible for OTA contamination in agricultural products, producing the highest concentrations of OTA compared to other Aspergillus species. A. westerdijkiae and OTA contamination have been detected in grains and feeds such as corn and wheat, as well as in coffee, beer, and fruit drinks, posing a serious threat to human health and livestock safety. In addition to being found in agricultural products, A. ochratoxin A is also found in airborne dust and deep-sea environments. Therefore, it is of great significance to ensure the health of humans and animals to study and establish rapid quantitative detection technology to perform specific and highly sensitive detection of Aspergillus ochraceus and eliminate OTA contamination at the source.

[0003] Existing methods for detecting Aspergillus ochraceus primarily rely on laboratory confirmatory techniques such as LAMP (Loop-Mediated Isothermal Amplification) and PCR-DGGE (Denaturing Gradient Gel Electrophoresis). LAMP offers the advantages of high specificity and sensitivity, but requires stringent experimental conditions, expensive equipment, and specialized personnel, making it difficult to meet the requirements for rapid on-site testing. PCR-DGGE uses fungus-specific primers to amplify rDNA in a sample and electrophoretically separates it using DGGE based on sequence structure differences. PCR-DGGE is a convenient tool for analyzing fungal communities, but it is not suitable for accurately characterizing the species present. Immunological detection methods rely on specific antigen-antibody recognition, converting immune signals into detectable signals through enzymes, fluorescence, and other labeling techniques, thereby qualitatively or quantitatively indicating the target's content. These methods offer advantages such as simple sample preparation, high sensitivity, strong specificity, and short experimental cycles. The sensitivity and specificity of the antibody directly determine the accuracy of the immunoassay. Currently, few reports on immunological detection techniques for Aspergillus ochraceus detection have been published domestically or internationally. Therefore, the research on anti-Aspergillus ochra monoclonal antibodies can open up new ideas for the rapid detection of Aspergillus ochra and is of great significance for the realization of quantitative immunoassay detection of Aspergillus ochra. 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. 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 CCTCC NO: C202365.

[0006] A second aspect of the present invention provides an anti-Aspergillus ochrae monoclonal antibody, wherein the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing; and the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing. The antibody is secreted and produced by the hybridoma cell line zqm4B4, deposited with CCTCC NO: C202365. The anti-Aspergillus ochrae monoclonal antibody provided by the present invention has high sensitivity and good specificity. Its minimum detection limit for Aspergillus ochrae is 0.0962 μg / mL, and it exhibits no cross-reactivity with other fungi, including Aspergillus flavus, Aspergillus parasiticus, Penicillium citrinum, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Fusarium spp., Aspergillus terreus, Penicillium chrysogenum, Aspergillus tubingensis, Aspergillus piperita, Aspergillus slatiensis, and Aspergillus clavatus.

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

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

[0009] A 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 ochrae is an ELISA kit for immunoassay detection of Aspergillus ochrae or an immunoaffinity column for Aspergillus ochrae. The ELISA kit for immunoassay detection of Aspergillus ochrae, such as the double-antibody sandwich immunoassay kit for Aspergillus ochrae, can be labeled with horseradish peroxidase. Other markers with similar functions to horseradish peroxidase can also be used, such as colloidal gold labeled probes, time-resolved fluorescent microspheres, upconversion fluorescent materials, biomimetic catalytic enzymes, etc., and then a series of immunoassay methods for immunoassay detection of Aspergillus ochrae can be established based on these probe-labeled immune recognition materials. The immunoaffinity column for Aspergillus ochrae can be used for sample pretreatment in, for example, HPLC detection methods and HPLC-MS / MS detection methods, effectively reducing or even eliminating matrix effects, thereby achieving high recovery rates and low matrix effects for Aspergillus ochrae.

[0011] The present invention provides a variable region sequence of a monoclonal antibody that specifically binds to Aspergillus ochraceus, wherein the heavy chain variable region of the monoclonal antibody that specifically binds to Aspergillus ochraceus is the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, or a conservative mutant thereof obtained by adding, deleting, replacing, or modifying one or more amino acids through conservative mutation; and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, or a conservative mutant thereof obtained by adding, deleting, replacing, or modifying one or more amino acids through conservative mutation. 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 BALB / c mice previously treated with incomplete Freund's adjuvant, collecting the ascites from the mice, and purifying the ascites to obtain the anti-Aspergillus ochraceus monoclonal antibody. In the present invention, the anti-Aspergillus ochraceus monoclonal antibody can be purified using the octanoic acid-ammonium sulfate method.

[0014] According to the above scheme, the specific operation of the octanoic acid-ammonium sulfate method for purifying antibodies is as follows: filter the mouse ascites with double filter paper, centrifuge at 12000r / min at 4°C for more than 15 minutes, aspirate the supernatant, mix the obtained ascites supernatant with 4 volumes of acetate buffer, slowly add octanoic acid with stirring, the volume of octanoic acid required per milliliter of ascites is 30-35μL, mix at room temperature for 30-60 minutes, and let it stand at 4°C for more than 2 hours. 12000r / min, centrifuge at 4℃ for more than 30min, discard the precipitate, filter the obtained supernatant with double filter paper, add 1 / 10 volume of 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 ammonium sulfate of 0.277g / mL, let it stand at 4℃ for more than 2h, then centrifuge at 12000r / min, 4℃ for more than 30min, discard the supernatant, and resuspend the obtained precipitate with 1 / 10 volume of original ascites water with a molar concentration of 0.01mol / L and 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. Remove the protein solution in the dialysis bag, centrifuge, collect the supernatant, discard the precipitate, pre-freeze it at -70℃, and then freeze-dry it in a freeze dryer. Collecting the lyophilized powder is the purified anti-Aspergillus ochraceus monoclonal antibody;

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

[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 ochrae monoclonal antibody provided by the present invention has high sensitivity and good specificity. Its minimum detection limit for Aspergillus ochrae is 0.0962 μg / mL, and it has no cross-reaction with other fungi, including Aspergillus flavus, Aspergillus parasiticus, Penicillium citrinum, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Fusarium spp., Aspergillus terreus, Penicillium chrysogenum, Aspergillus tubingensis, Aspergillus piperitum, Aspergillus liufuga, and Aspergillus clavatus.

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

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

[0021] The hybridoma cell line zqm4B4 provided by the present invention was obtained by a two-step screening method. The specific steps are: BALB / C mice were immunized with Aspergillus ochraceus strains 4-6 times, then boosted with a complete antigen AO standard strain at half the dose of the previous immunization. Cell fusion was performed 3 days later. After 1-2 weeks of culture in HAT semi-solid medium, single cell colonies were transferred to cell culture plates for culture. When the cells grew to 2 / 3 of the bottom of the wells, they were screened using an indirect non-competitive ELISA method. Wells with higher absorbance values ​​were selected. Subcloning was performed 4-5 times using the limiting dilution method. Finally, the hybridoma cell line zqm4B4 was screened.

[0022] 1. Animal immunization

[0023] An ochratoxin-producing fungal strain already in the laboratory was revived using AFPA medium and cultured in Czapek's liquid medium. After filtering the mycelial pellets, the culture medium was collected and used to prepare a pellet lysate antigen. This was then used as an antigen to immunize six-week-old female BALB / C mice. For the first immunization, the Aspergillus ochratoxin antigen was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at five points on the back of the neck. The second immunization took place 21 days later, using an equal volume of the Aspergillus ochratoxin antigen emulsified with Freund's incomplete adjuvant and injected intraperitoneally. The third immunization was performed two weeks after the second, using the same immunization method. The fourth immunization was performed three weeks after the third, also using the same intraperitoneal injection method. The dose for all four immunizations was the same: 200 μg per mouse. Eight to ten days after each of the first three immunizations, blood was collected by tail clipping, and serum was isolated. Serum titers were determined using an indirect ELISA. Eight days after the third immunization, the mice were tail-cut and blood was collected. The mice with serum with relatively high titer and sensitivity were selected for the final booster immunization, and the immunization dose was 1 / 2 times of the previous one.

[0024] 2. Cell Fusion

[0025] Three days after the booster immunization, cell fusion was performed using 50% polyethylene glycol (PEG) (molecular weight 1450) as a fusion agent according to conventional methods. The following steps were performed: mice were sacrificed by cervical dislocation under sterile conditions, the spleens were removed and crushed with a homogenizer, and splenocytes were separated using a filter. The cells were mixed with murine myeloma SP2 / 0 cells at a cell population ratio of 5:1-10:1, centrifuged at 1000 rpm for 5 minutes, and resuspended in RPMI-1640 basal medium. The mixture was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. One mL of 50% PEG was added, and the mixture was incubated for 1 minute. Once adhered, cells were resuspended in 20 mL of RPMI-1640 basal medium, centrifuged, and the supernatant discarded. The fused cells at the bottom of the tube were resuspended in 20 mL of complete cell culture medium containing 1% HAT. The suspended cells were added to 80 mL of semi-solid culture medium, mixed thoroughly, and plated into a 6-well cell culture plate at a rate of 1-2 mL / well. The plates were then incubated in a 37°C CO2 incubator. The complete cell culture medium containing 1% HAT contains 20% (volume percentage) fetal bovine serum, 75% (volume percentage) RPMI-1640 basal culture medium, 1% (weight percentage) L-glutamine, 1% (volume percentage) HEPES, 1% (volume percentage) double antibody (10,000 units per milliliter penicillin and 10,000 micrograms per milliliter streptomycin), 1% (volume percentage) growth factor (clone easy), and 1% (weight percentage) hypoxanthine-aminopterin-thymidine (HAT) and methylcellulose, which were 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, transfer them to a 96-well cell culture plate and culture in HAT liquid. 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 for resistance to 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: Sequence determination of the variable region of the anti-Aspergillus ochraceus monoclonal antibody hybridoma cell line zqm4B4.

[0029] (1) Extraction of total RNA: Total RNA from hybridoma cell line zqm4B4 was extracted using the total RNA extraction kit from Tiangen Company according to the instructions;

[0030] (2) Synthesize cDNA: Use the total RNA obtained in step 1 as a template and oligo(dT)15 as a primer. TM Reverse transcription was performed using the instructions of the 2II reverse transcriptase to synthesize the first-strand cDNA; the primer oligo(dT)15 was purchased from Invitrogen;

[0031] (3) PCR cloning of variable region genes: Primers were designed based on conserved sites in the mouse antibody gene sequence in GENBANK, and cDNA was used as a template to amplify the antibody heavy and light chain variable region genes. The PCR program was as follows: 94°C for 30 seconds, 58°C for 45 seconds, and 72°C for 1 minute, with 30 cycles of amplification and a final extension at 72°C for 10 minutes. The PCR products were separated by electrophoresis on a 1% (weight percent) agarose gel, and the DNA fragments were purified and recovered using a kit. The DNA fragments were ligated into the vector pMD18-T and transformed into competent E. coli DH5α cells. Positive clones were selected and sequenced. The sequences of the primers are: 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; 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 obtained gene sequence results: 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-Aspergillus ochraceus monoclonal antibody hybridoma cell line zqm4B4 obtained in Example 1 was injected into BALB / C mice pre-treated with Freund's incomplete adjuvant, and the ascites of the mice was collected. The antibody was purified using the octanoic acid-ammonium sulfate method. The specific operation was as follows: the mouse ascites was filtered through double filter paper, centrifuged at 12000 r / min at 4°C for more than 15 min, and the supernatant was aspirated. The obtained ascites supernatant was mixed with 4 volumes of acetate buffer, and octanoic acid was slowly added under stirring. The volume of octanoic acid required per milliliter of ascites was 30-35 μL. The mixture was mixed at room temperature for 30-60 min and allowed to stand at 4°C for more than 2 h. 12000r / min, centrifuge at 4℃ for more than 30min, discard the precipitate, filter the obtained supernatant with double filter paper, add 1 / 10 volume of 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 ammonium sulfate of 0.277g / mL, let it stand at 4℃ for more than 2h, then centrifuge at 12000r / min, 4℃ for more than 30min, discard the supernatant, and resuspend the obtained precipitate with 1 / 10 volume of original ascites water with a molar concentration of 0.01mol / L and 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. Remove the protein solution in the dialysis bag, centrifuge, collect the supernatant, discard the precipitate, pre-freeze it at -70℃, and then freeze-dry it in a freeze dryer. Collecting the lyophilized powder is the purified anti-Aspergillus ochraceus monoclonal antibody;

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

[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 titer of the antibody 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 solution tested positive when the concentration was doubled. The minimum detection limit for Aspergillus ochrae was 0.0962 μg / mL using a conventional indirect noncompetitive ELISA. The specificity of the antibody can be evaluated by the cross-reactivity rate. The zqm4B4 monoclonal antibody was tested using an indirect noncompetitive ELISA. Fungi such as Aspergillus flavus, Aspergillus parasiticus, Penicillium citrinum, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Fusarium spp., Aspergillus terreus, Penicillium chrysogenum, Aspergillus tubingensis, Aspergillus piperita, Aspergillus slatifugae, and Aspergillus clavatus were treated under the same conditions as Aspergillus ochrae. The plates were then coated with the zqm4B4 monoclonal antibody overnight at 4°C. The plates were then used in an indirect noncompetitive ELISA assay. The OD values ​​at 450 nm were measured on a microplate reader. Only Aspergillus ochrae showed an OD > 1, while the OD < 0.3 for all other fungi was < 0.3, indicating that the zqm4B4 monoclonal antibody showed no cross-reactivity 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 each 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 amount. -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 1 mg / mL using 0.01 M PBS to 11 concentration gradients, 100 μL / well. The blank group was added with PBS and 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 9 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 six times with PBST, add 100 μL / well of TMB colorimetric solution, incubate at 37°C in the dark for 15 min, and measure the OD450 value using a microplate reader. The sandwich ELISA standard curve can be fitted using the following formula:

[0048]

[0049] The OD value and Aspergillus ochra concentration curve was used to determine the Aspergillus ochra content in the sample. The established Sandwich-ELISA method achieved detection limits of 0.118 μg / mL and 0.1285 μg / mL for Aspergillus ochra in peanut and corn samples, respectively.

[0050] The markers in the above-mentioned 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 a series of immunoassay methods can be established 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 1 g of CNBr-activated Sepharose lyophilized matrix powder (one gram of lyophilized matrix powder will yield a final volume of 3.5 mL of swollen matrix) and dissolve it in 1 mmol / L HCl. The matrix will swell immediately, then wash the sample in a sintered glass filter with 1 mmol / L HCl for 15 minutes.

[0054] 2. Ligand (antibody) coupling

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

[0056] b. Calculation of coupling efficiency: Centrifuge the agarose gel at 2000 rpm until it reaches the bottom of the tube. Transfer the supernatant to a fresh tube and measure the protein content. The coupling efficiency is calculated to be 98.5%. Wash the agarose gel at the bottom of the tube with coupling buffer to remove excess ligand.

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

[0058] d) To remove excess uncoupled ligand, wash the matrix for 3-5 cycles with pH 4 and 8 buffers: 0.1 mol / L acetic acid / sodium acetate buffer and 0.1 mol / L Tris-HCl buffer, using 3-5 times the volume of the matrix for each buffer. Each wash cycle begins with a 0.1 mol / L acetic acid / sodium acetate buffer, followed by a 0.1 mol / L Tris-HCl buffer wash.

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

[0060] 3. Prepare the slurry using binding buffer and mix it 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 operate and 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 matrix surface, the opening below the affinity column is opened, 3-5 times the column bed volume of sterile-filtered 0.01% NaN3-PBS is passed through the column, and the column is preserved with 0.01% NaN3-PBS. At this point, the affinity column has been filled and equilibrated and can be used directly; for example, the sample pretreatment in HPLC detection methods and HPLC-MS / MS detection methods effectively reduces or even eliminates the matrix effect, achieving a high recovery rate of Aspergillus ochraceus and low matrix effect.

Claims

1. Hybridoma cell line zqm4B4 was deposited with the China Center for Type Culture Collection (CCTCC) on March 22, 2023, at Wuhan University, Wuhan, China, with the deposit number CCTCC NO: C202365.

2. The anti-Aspergillus ochraceus monoclonal antibody encoded by the hybridoma cell line zqm4B4 according to claim 1, 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 according to claim 1 is injected into BALB / C mice that have been 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 Aspergillus ochraceus content for non-disease diagnosis purposes.

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

7. The product for detecting Aspergillus ochraceus according to claim 6, characterized in that: ELISA kit or immunoaffinity column for Aspergillus ochraceus immunodetection.

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