Anti-ochratoxin A antibody and application thereof

Abstract

The invention discloses an antibody for resisting ochratoxin A and application of the antibody. According to the invention, an artificial antigen obtained by coupling the ochratoxin A and a carrier protein is used as an immunogen to prepare the monoclonal antibody against the ochratoxin A. The ochratoxin A can be sensitively and specifically detected by using the monoclonal antibody, and the lowest limit of detection (LOD) of the monoclonal antibody is 0.18 ng / mL. In addition, the half inhibition (IC50) of icELISA (Enzyme-Linked Immunosorbent Assay), which is established by utilizing the monoclonal antibody for resisting the ochratoxin A, on the ochratoxin A is 1.42 ng / mL, and the linear range is 0.34 ng / mL to 5.40 ng / mL. The invention provides the antibody capable of being used for detecting the ochratoxin A and the method for detecting the ochratoxin A, and the antibody and the method have the advantages of being simple, convenient, rapid, high in specificity, wide in linear range and high in sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme-linked immunosorbent assay. More specifically, it relates to an antibody against ochratoxin A and its application. Background Art

[0002] Ochratoxin A (OTA) is a secondary metabolite of certain strains of Aspergillus and Penicillium, belonging to the isocoumarin compounds. Compared with other mycotoxins, OTA stays in the human circulation for the longest time. When the accumulation amount of OTA reaches a certain level, combined with its long-term chronic toxic effects, it may lead to the occurrence of tumors. Since ochratoxin A is often present in foods such as grains and milk powder, detecting foods that may be contaminated with ochratoxin A is of great significance for ensuring food safety and maintaining the health of the body.

[0003] The existing common methods for detecting ochratoxin A mainly include instrumental analysis methods such as high-performance liquid chromatography, gas chromatography, ultraviolet spectroscopy, liquid chromatography-tandem mass spectrometry, etc. Although instrumental analysis methods have good repeatability, high detection accuracy and sensitivity, such methods require expensive instruments, and their sample pretreatment process is complex, the operation is cumbersome, and it takes a long time, which cannot meet the needs of rapid on-site detection of a large number of samples. Therefore, there is an urgent need for a simple, rapid method that can specifically and sensitively detect OTA. Summary of the Invention

[0004] In view of the above deficiencies in the prior art, the present invention provides an antibody against ochratoxin A and its application.

[0005] The first object of the present invention is to provide an antibody against ochratoxin A.

[0006] The second object of the present invention is to provide the application of the antibody in enriching ochratoxin A.

[0007] The third object of the present invention is to provide the application of the antibody in preparing a product for enriching ochratoxin A.

[0008] The fourth object of the present invention is to provide the application of the antibody in detecting ochratoxin A.

[0009] The fifth object of the present invention is to provide the application of the antibody in preparing a product for detecting ochratoxin A.

[0010] The sixth object of the present invention is to provide an enzyme-linked immunosorbent assay kit for detecting ochratoxin A.

[0011] The above objects of the present invention are achieved by the following technical solutions:

[0012] The present invention uses an artificial antigen obtained by conjugating ochratoxin A with a carrier protein as an immunogen to prepare a monoclonal antibody against ochratoxin A. Using the monoclonal antibody, ochratoxin A can be sensitively and specifically detected, and its lowest detection limit is 0.18 ng / mL. In addition, the half-inhibition (IC 50 ) of the icELISA for ochratoxin A established using the monoclonal antibody against ochratoxin A is 1.42 ng / mL, and the linear range is 0.34 - 5.40 ng / mL. That is, the detection sensitivity of ochratoxin A using the antibody of the present invention is high and the linear range is wide, which can meet the requirements of rapid and sensitive detection of ochratoxin A. Therefore, the present invention claims the protection of the antibody and its applications.

[0013] The present invention provides an antibody against ochratoxin A. Specifically, the antibody includes the following complementarity-determining regions:

[0014] VL-CDR1: KSVSTSGYSY;

[0015] VL-CDR2: LVS;

[0016] VL-CDR3: QHIRELT;

[0017] VH-CDR1: GFDFSSYD;

[0018] VH-CDR2: ISSGGRYS;

[0019] VH-CDR3: ARQNDYEAWFAY.

[0020] Specifically, the amino acid sequence of the variable region of the light chain of the antibody is as shown in SEQ ID NO.1; the amino acid sequence of the variable region of the heavy chain of the antibody is as shown in SEQ ID NO.2.

[0021] The present invention also provides the gene sequence encoding the antibody.

[0022] Specifically, the nucleotide sequence of the gene encoding the variable region of the light chain of the antibody is as shown in SEQ ID NO.3; the nucleotide sequence of the gene encoding the variable region of the heavy chain of the antibody is as shown in SEQ ID NO.4.

[0023] By conjugating the antibody with magnetic beads to prepare immunomagnetic beads and testing their adsorption rate for ochratoxin A, the present invention finds that the antibody of the present invention can effectively adsorb ochratoxin A. Therefore, the present invention claims the protection of the application of the antibody in enriching ochratoxin A.

[0024] The present invention also claims the protection of the application of the antibody in preparing a product for enriching ochratoxin A.

[0025] The present invention also claims the use of the antibody in detecting ochratoxin A.

[0026] The present invention also claims the use of the antibody in preparing a product for detecting ochratoxin A.

[0027] The present invention also provides an enzyme-linked immunosorbent assay (ELISA) kit for detecting ochratoxin A, which contains the antibody of the present invention.

[0028] Specifically, it further contains an enzyme-labeled plate coated with a coating antigen.

[0029] Specifically, the coating antigen is an artificial antigen obtained by conjugating ochratoxin A with ovalbumin.

[0030] Specifically, the ELISA kit further contains reagents required for the color reaction.

[0031] Specifically, the reagents required for the color reaction include an enzyme-labeled secondary antibody and a chromogenic solution.

[0032] More specifically, the enzyme-labeled secondary antibody is goat anti-mouse IgG-HRP; the chromogenic solution is a TMB chromogenic solution.

[0033] The present invention has the following beneficial effects:

[0034] The present invention uses an artificial antigen obtained by conjugating ochratoxin A with a carrier protein as an immunogen to prepare a monoclonal antibody against ochratoxin A. Using the monoclonal antibody, ochratoxin A can be detected sensitively and specifically, and its lowest detection limit (LOD) is 0.18 ng / mL. In addition, the half-inhibition (IC 50 ) of the icELISA for ochratoxin A established using the monoclonal antibody against ochratoxin A is 1.42 ng / mL, and the linear range is 0.34 - 5.40 ng / mL.

[0035] The present invention provides an antibody capable of detecting ochratoxin A and a method for detecting ochratoxin A, which has the advantages of simplicity, rapidity, strong specificity, wide linear range, and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 For the ultraviolet scanning identification results of OTA, carrier proteins BSA, LF, OVA, and artificial antigens OTA-BSA, OTA-LF, and OTA-OVA

[0037] Figure 2 For the analysis results of the framework region and complementary determining region of the light chain of the monoclonal antibody OTA-E4 against ochratoxin A.

[0038] Figure 3 Analysis results of the framework region and complementarity-determining region of the heavy chain of monoclonal antibody OTA-E4 against ochratoxin A.

[0039] Figure 4 An indirect competitive ELISA standard curve established using monoclonal antibody OTA-E4 against ochratoxin A with OTA-OVA as the coating antigen. Detailed implementation methods

[0040] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0041] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0042] Example 1 Preparation of monoclonal antibody against ochratoxin A

[0043] 1. Preparation of artificial antigen

[0044] The artificial antigen used in the present invention to prepare monoclonal antibody against ochratoxin A is prepared by coupling ochratoxin A with carrier proteins (ovalbumin (OVA), bovine serum albumin (BSA), or lactoferrin (LF)) by the active ester method; among them, the structural formula of ochratoxin A is shown in formula (I):

[0045]

[0046] The structural formula of the prepared ochratoxin A artificial antigen is shown in formula (II):

[0047]

[0048] The preparation method of the artificial antigen is as follows:

[0049] Dissolve ochratoxin A in DMF, add EDC and NHS (the molar ratio of OTA:NHS:EDC is 1:1.5:1.5), stir and react at 4°C for 8 h, denoted as solution A; dissolve the carrier proteins in phosphate buffer respectively, denoted as solution B; dropwise add solution A into solution B, mix and stir and react at 4°C for 8 h, the molar ratio of carrier protein to ochratoxin A is 1:80, and the reaction solution is dialyzed at 4°C for 3 days using an 8000-13000 dialysis membrane, changing the dialysis solution (0.01M PBS) 2 times a day. After dialysis, artificial antigens OTA-BSA, OTA-LF, and OTA-OVA are obtained, and they, the used carrier proteins (BSA, LF, OVA), and OTA are identified by ultraviolet scanning (190-450 nm).

[0050] The UV scanning identification results of OTA, carrier proteins BSA, LF, OVA and artificial antigens OTA-BSA, OTA-LF and OTA-OVA are as follows Figure 1 shown. It can be seen from Figure 1 that compared with the carrier proteins (BSA, LF, OVA) and OTA, the characteristic absorption peaks of the artificial antigens prepared in the present invention have an obvious blue shift, and the artificial antigens have the characteristic absorption peaks of OTA and carrier proteins (BSA, LF, OVA), indicating that the artificial antigens are successfully prepared.

[0051] 2. Detection of the immune effect of artificial antigens

[0052] (1) Animal immunization

[0053] Using healthy 6-week-old female Balb / c mice as experimental animals, the identified artificial antigens OTA-BSA and OTA-LF were used as immunogens and emulsified completely with an equal amount of immune adjuvant (Freund's complete adjuvant for the first immunization, and Freund's incomplete adjuvant for subsequent booster immunizations), and immunized by multiple injection methods including subcutaneous injection on the back, subcutaneous injection at various sites, intraperitoneal injection and foot injection; immunization was carried out every 2 weeks after the first immunization, and a small amount of tail vein blood was taken for antibody quality identification after the fourth immunization; after the antibodies were stable, the mice with the best performance were selected for cell fusion, and 3 days before cell fusion, 0.5 mg of immunogen was directly injected into the abdominal cavity of the mice for an additional immunization.

[0054] (2) Antibody quality identification

[0055] Using the identified artificial antigen OTA-OVA as the coating antigen, taking the above-mentioned mouse tail vein blood as the detected antibody, and using the indirect competitive ELISA method to determine the antiserum titer and inhibition rate of the mouse serum, and evaluating them by comprehensively considering the titer and inhibition rate of each antiserum.

[0056] The specific operation steps for antibody quality identification are as follows:

[0057] ① Coating the plate: Dilute the artificial antigen OTA-OVA with 0.05M carbonate buffer (pH 9.6) to 1000 ng / mL, and coat it overnight at 4℃ at 100 μL / well; discard the coating solution, wash it 2 times with PBST, add 120 μL of blocking solution (5% skim milk) to each well, block it at 37℃ for 3 h, discard the blocking solution, dry it at 37℃ and store it in a sealed bag at 4℃ for use, to obtain the coated enzyme-linked immunosorbent assay (ELISA) plate.

[0058] ② Serum titer and inhibition rate detection: The enzyme-linked immunosorbent assay (ELISA) plate wrapped in step ① is set as the titer column and the inhibition column horizontally and vertically, respectively. Titer column: 50 μL of PBS and 50 μL of mouse serum diluted by gradient multiples (1K, 2K, 4K, 8K, 16K, 32K, 64K) are added to each well. Inhibition column: 50 μL of diluted 1000 ng / mL OTA and 50 μL of serum diluted by gradient multiples (1K, 2K, 4K, 8K, 16K, 32K, 64K) are added to each well, and two groups of parallel samples are made. Incubate at 37 °C for 40 min, wash 5 times with PBST, pat dry the liquid in the wells, add enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP) diluted 1:5000, incubate at 37 °C for 30 min, then wash 5 times with PBST, pat dry the liquid in the wells, add 100 μL of TMB substrate solution, develop color at 37 °C in the dark for 10 min, and then add 50 μL of termination solution (2M H2SO4) to terminate the reaction. Read the absorbance value at 450 nm with an enzyme-labeled immunosorbent assay reader.

[0059] The detection results of the titer and inhibition rate of the antiserum obtained by immunizing Balb / c female mice with the prepared artificial antigen are shown in Table 1. From the results shown in Table 1, it can be seen that both artificial antigens can induce the production of murine polyclonal antibodies in immunized mice, and the obtained antiserum has an inhibitory effect on the target analyte ochratoxin A. The inhibitory effect of the OTA-LF mice is the most obvious, indicating that the artificial antigen prepared in the present invention can be used for the subsequent preparation of monoclonal antibodies against ochratoxin A and the establishment of immunoassay methods.

[0060] Table 1 Titer and inhibition rate of OTA mouse antiserum

[0061]

[0062] 3. Preparation of monoclonal antibodies against OTA

[0063] (1) Cell fusion

[0064] Mix the spleen cells of immunized OTA-LF mice with mouse myeloma cells (SP2 / 0) in the logarithmic growth phase, slowly add preheated fusogen (PEG1500) within 45 s for fusion, suspend evenly with HAT medium, and then add an appropriate amount of feeder cells (peritoneal macrophages of non-immunized mice), culture in a 96-well culture plate, and culture in a 37 °C, 5% CO2 incubator. Replace half of the medium with HT medium after 5 days and replace all the medium after 9 days.

[0065] (2) Screening of positive hybridomas

[0066] After cell fusion, when the cells grow to 1 / 4 of the culture well area, the hybridoma cells are screened by a stepwise screening method; for the primary screening, the indirect ELISA method is used, with OTA-OVA as the coating antigen (its optimal coating concentration and positive serum dilution are routinely titrated by the checkerboard method in advance) to coat the enzyme-linked immunosorbent assay (ELISA) plate, add the culture supernatant of the well to be tested for incubation, after washing, add goat anti-mouse IgG-HRP, and add TMB substrate solution for color reaction; the positive wells screened are further screened by the indirect competitive ELISA method. First, mix the cell supernatant with 1000 ng / mL of ochratoxin A in equal volume, incubate at 37 °C in a water bath for 30 min, then add it to the pre-coated ELISA plate. At the same time, use PBS to replace ochratoxin A as a control, and the remaining steps are the same as above; if the OD450nm value after being blocked by ochratoxin A drops to less than 50% of the control well, it is judged as positive. The wells that are positive after 2 to 3 detections are immediately subcloned by the limiting dilution method.

[0067] (3) Expansion culture of hybridoma cells

[0068] Expand the culture of the hybridoma cells after 2 to 3 subclonings and establishment of cell lines, collect the supernatant and determine the titer by indirect ELISA, and store it frozen; inject 0.5 mL of liquid paraffin intraperitoneally into 8- to 10-week-old Balb / c mice at a dose of 0.5 mL / mouse. After 7 to 10 days, inject 1 to 2 × 10 6 / mouse of hybridoma cells. After 7 to 10 days, draw the ascites of the mice, centrifuge and take the supernatant, determine the titer, and store it frozen for later use.

[0069] The titer and inhibition rate of the OTA monoclonal cell line screened by the present invention are shown in Table 2, and OTA-E4 is selected for subsequent experiments.

[0070] Table 2 Titer and inhibition rate of OTA monoclonal cell line

[0071]

[0072] 4. Sequence analysis of the monoclonal antibody OTA-E4 against OTA

[0073] (1) Total RNA extraction

[0074] Take the monoclonal antibody cell line OTA-E4 to extract its total RNA, and the extraction method of total RNA is carried out according to the method of Trizol reagent of Guangzhou Jiebeisi Biotechnology Co., Ltd.

[0075] (2) cDNA synthesis

[0076] Using the extracted total cell RNA as a template, refer to the instruction manual of the reverse transcription kit of Takara Company to synthesize the first strand of cDNA.

[0077] (3) Amplification and sequencing of the variable region gene sequence of the monoclonal antibody

[0078] ①PCR cloning of heavy chain (VH) and light chain (VL) variable region genes

[0079] Using the cDNA synthesized by reverse transcription as a template, the heavy chain and light chain variable region genes of the monoclonal antibody were cloned respectively using universal primers. The universal primers used are shown in Table 3.

[0080] Table 3 Universal primers for amplifying the heavy chain and light chain variable regions of single-chain antibodies

[0081]

[0082] The specific process is as follows:

[0083] First, 6 primers of LF were mixed in equal proportion, and then the mixed LF primers were combined with LB1 - LB19 primers respectively to form 19 primer groups. Using the first-strand cDNA as a template, the light chain variable region was amplified with these 19 primer groups respectively. The reaction system and reaction conditions are shown in Table 4.

[0084] Table 4 PCR amplification system and reaction conditions for light chain variable region

[0085]

[0086] The primer groups capable of amplifying the sequence were screened out, and the numbers of their LB primers were determined as positive LB primers. Then each positive LB primer was combined with 6 LF primers respectively, and PCR reactions were carried out using the first-strand cDNA as a template. The reaction system and reaction conditions were the same as those in Table 4. After PCR, the bands were identified by gel electrophoresis, and the bands in the range of 300bp - 500bp were recovered by cutting the gel with a kit. The PCR gel running products were recovered by the OMEGA gel extraction kit and transformed into Escherichia coli DH5α. Multiple single colonies were selected for colony PCR and DNA sequencing identification and analysis.

[0087] The cloning of the heavy chain variable region gene sequence was the same as above.

[0088] After adjusting the sequencing results by DNAman software, the complete forward sequence was obtained and input in FASTA format into IMGT (https: / / www.imgt.org / IMGT_vquest / analysis) for murine antibody variable region gene sequence analysis.

[0089] It can be seen from the sequencing results that the amino acid sequence of the light chain variable region of the anti-ochratoxin A monoclonal antibody OTA-E4 described in the present invention is as follows (shown in SEQ ID NO.1):

[0090] DIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWKSN

[0091] The amino acid sequence of the heavy chain variable region of the anti-OTA monoclonal antibody OTA-E4 is as follows (shown in SEQ ID NO.2):

[0092] EVQGVESGGGLVKPGGSLKFSCVASGFDFSSYDMSWVRQTPEKRLEWVASISSGGRYSYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCARQNDYEAWFAYWGLGTLVTVSA

[0093] The nucleotide sequence encoding the light chain variable region of the anti-OTA monoclonal antibody OTA-E4 is as follows (shown in SEQ ID NO.3):

[0094] GACATTGTGATGACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAC

[0095] The nucleotide sequence encoding the heavy chain variable region of the anti-OTA monoclonal antibody OTA-E4 is as follows (shown in SEQ ID NO.4):

[0096] GAGGTGCAGGGGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAATTCTCCTGTGTAGCCTCTGGATTCGATTTCAGTTCCTATGACATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAAGTATTAGTAGTGGTGGCCGTTATTCTTATTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAGGAACACCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAAGACACGGCCTTGTATTACTGTGCAAGACAGAATGATTATGAGGCCTGGTTTGCTTACTGGGGCCTAGGGACTCTGGTCACTGTCTCTGCG

[0097] In the present invention, the structures of the variable regions of the light and heavy chains of the anti-ochratoxin A monoclonal antibody OTA-E4 were analyzed simultaneously, and the results are shown in Figure 2 and Figure 3 respectively. From Figure 2 and Figure 3 , it can be seen that the light and heavy chains of the OTA-E4 respectively include 4 framework regions (FR1, FR2, FR3, FR4) and 3 complementarity-determining regions (CDR1, CDR2, CDR3). The amino acid sequences of the 3 complementarity-determining regions of the light and heavy chains of the OTA-E4 are shown as follows:

[0098] VL-CDR1: KSVSTSGYSY (SEQ ID NO.5);

[0099] VL-CDR2: LVS;

[0100] VL-CDR3: QHIRELTR (SEQ ID NO.6);

[0101] VH-CDR1: GFDFSSYD (SEQ ID NO.7);

[0102] VH-CDR2: ISSGGRYS (SEQ ID NO.8);

[0103] VH-CDR3: ARQNDYEAWFAY (SEQ ID NO.9).

[0104] Example 2 Preparation of immunomagnetic beads and detection of the adsorption rate of ochratoxin A

[0105] In the present invention, an immunomagnetic bead was prepared by conjugating a monoclonal antibody OTA-E4 against ochratoxin A with magnetic beads, and its adsorption rate for ochratoxin A was tested.

[0106] 1. Preparation of Immunomagnetic Beads

[0107] (1) Preparation of Protein Solution

[0108] An appropriate amount of monoclonal antibody OTA-E4 was diluted with sodium methyl ester sulfonate (MES, 0.1 mol / L, pH 4.8) to prepare a protein solution with a concentration of 1.0 mg / mL. The prepared protein solution was stored at 4 °C for later use.

[0109] (2) Washing of Magnetic Beads

[0110] Take 500 μL of magnetic beads (BEAVER, product number 70703-5, NHS magnetic bead kit, average particle size 200 nm) into a 1.5 mL EP centrifuge tube (the magnetic beads should be repeatedly inverted, mixed evenly using a vortex shaker or a vertical mixer before sampling to ensure the uniformity of the experiment). Place the EP tube in a magnetic separation rack to enrich the magnetic beads and remove the supernatant; add 1 mL of pre-cooled 1 mmol / L hydrochloric acid solution at 2-8 °C, vortex for 15 s to mix the magnetic beads evenly, place the EP tube in the magnetic separation rack to enrich the magnetic beads, and remove the supernatant.

[0111] (3) Immobilization of Bioligand

[0112] Add 500 μL of protein solution to the EP tube (the protein solution should be added immediately after washing the magnetic beads), vortex for 30 s to mix evenly, vortex the EP tube for 15 s, place it on a vertical mixer, and mix at room temperature for 1-2 h. Use a magnetic separation rack to enrich the magnetic beads and save the flow-through solution.

[0113] (4) Blocking of Magnetic Beads

[0114] Add 500 μL of blocking solution (3 mol / L ethanolamine) to the EP tube, vortex for 30 s, place the EP tube in a magnetic separation rack to enrich the magnetic beads, discard the supernatant, repeat the above steps 4 times, add 500 μL of blocking solution again, vortex for 30 s, place the EP tube in a vertical mixer and react at room temperature for 2 h, place the EP tube in a magnetic separation rack to enrich the magnetic beads, remove the supernatant, add 1 mL of ultrapure water to the EP tube, mix well, use a magnetic separation rack to enrich the magnetic beads, and discard the supernatant.

[0115] (5) Storage

[0116] Add 1 mL of PBS buffer solution containing 0.05% sodium azide to the EP tube, mix well, use a magnetic rack to enrich the magnetic beads, and discard the supernatant. Add 500 μL of PBS buffer solution containing 0.05% sodium azide to the EP tube, mix well, and store at 4 °C for later use.

[0117] 2. Detection of the adsorption rate of immunomagnetic beads to OTA

[0118] The immunomagnetic beads prepared by the present invention are used to adsorb OTA in the sample, and then the eluent is used to elute the OTA adsorbed on the magnetic beads. The eluent can be used for the analysis and detection of OTA. That is, the immunomagnetic beads prepared by the present invention using the antibody can be used for the enrichment of OTA and the pretreatment method in the process of OTA analysis and detection.

[0119] (1) Enrichment of OTA by immunomagnetic beads

[0120] Take 4 1 mL centrifuge tubes, add 0.2 mg of immunomagnetic beads, aspirate 1 mL of OTA standard drug with a concentration of 25 ng / mL prepared with 40% methanol, mix well and place on a shaker at 25 °C, shake and enrich at 250 r / min for 15 min, and retain the supernatant after magnetic separation; add 500 μL of 80% methanol to the magnetic beads that have completed enrichment, place on a shaker at 37 °C, shake and elute at 250 r / min for 5 min, and retain the supernatant after magnetic separation;

[0121] (2) Detection of adsorption rate

[0122] Use an ELISA detection kit to measure the residual amount of unadsorbed OTA in the supernatant and the content of OTA in the eluent in the above steps respectively, indirectly calculate the actual adsorption amount, and thus calculate the adsorption rate and elution rate.

[0123] The calculation formulas for the adsorption rate and elution rate are as follows:

[0124] Adsorption rate = (drug addition amount - supernatant residue amount) / drug addition amount × 100%;

[0125] Elution rate = drug content in eluent / (drug addition amount - supernatant residue amount) × 100%.

[0126] On the basis of the above enrichment method, the present invention optimizes factors such as extraction reagents, the amount of immunomagnetic beads used, adsorption time, and elution volume.

[0127] The optimized method for enriching OTA in the sample using immunomagnetic beads and establishing the immunomagnetic bead pretreatment method is as follows:

[0128] Weigh 5.0 g (±0.1 g) of the experimental sample to be tested into a 50 mL centrifuge tube, add 25 mL of 80% methanol aqueous solution, mix well at 2500 rpm for 5 min, centrifuge at 4000 r / min for 5 - 10 min. After solid-liquid separation, take 2 mL of the supernatant, and dilute the sample extract with V(supernatant):V(ultrapure water) = 1:3 or V(supernatant):V(ultrapure water) = 1:7; add 4 mg of magnetic beads to the diluted extract, mix well and place on a shaker at 25°C, shake and enrich at 250 r / min for 5 - 10 min, add 400 μL of 80% methanol to the magnetic beads after enrichment, place on a shaker at 37°C, shake and elute at 250 r / min for 5 min, and retain the supernatant after magnetic separation for subsequent detection.

[0129] Based on the optimized immunomagnetic bead pretreatment method, the present invention tested the adsorption rate of the prepared immunomagnetic beads for OTA, and the adsorption rate was 90.4%.

[0130] Meanwhile, the present invention used the immunomagnetic bead pretreatment method to adsorb other mycotoxins to test the specificity of the immunomagnetic beads. The results are shown in Table 5. As can be seen from Table 5, the adsorption efficiency of the immunomagnetic beads of the present invention for other mycotoxins is less than 0.1%, indicating that this pretreatment method has good method specificity for OTA.

[0131] Table 5 Specificity evaluation of OTA immunomagnetic beads (n = 3)

[0132]

[0133] Note: The drug addition concentration is 25 ng / mL for all.

[0134] Example 3 Method for detecting ochratoxin A

[0135] Based on the monoclonal antibody OTA-E4 against ochratoxin A described in Example 1, the present invention constructed a method for detecting ochratoxin A and established an indirect competitive ELISA standard curve for the monoclonal antibody OTA-E4 against ochratoxin A.

[0136] 1. Coating and blocking

[0137] Dilute the OTA-OVA coating antigen to 1000 ng / mL with the coating solution (pH 9.6, 0.1 mol / L carbonate buffer, 1.65 g of Na2CO3, 2.65 g of NaHCO3, 1000 mL of distilled water), coat overnight at 37°C. The next day, after washing twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), add 2% skim milk powder, 120 μL per well, block at 37°C for 3 h, discard the blocking solution, dry at 37°C for 60 min, and store in a sealed bag at 4°C for use.

[0138] 2. Establishment of standard curve

[0139] (1) Experimental method

[0140] Add 50 μL of anti - ochratoxin A monoclonal antibody with a concentration of 3.5 μg / mL and a series of 50 μL of ochratoxin A standards with different concentrations into each well of the coated ELISA plate. Incubate at 37 °C for 40 min, wash five times with PBST, pat dry the liquid in the wells, add the enzyme - labeled secondary antibody (goat anti - mouse IgG - HRP) diluted 1:5000, incubate at 37 °C for 40 min, wash five times with PBST, pat dry the liquid in the wells, add 100 μL of TMB substrate solution, and develop color in the dark at 37 °C for 10 min; add 50 μL of termination solution (2 M H2SO4) to terminate the reaction; read the absorbance at 450 nm with an ELISA reader. Establish an indirect competitive standard curve with the concentration of ochratoxin A standard on the abscissa and B / B0 (absorbance of the well with added ochratoxin A / absorbance of the well without added ochratoxin A) on the ordinate.

[0141] (2) Experimental results

[0142] The indirect competitive ELISA standard curve established by using the prepared anti - ochratoxin A monoclonal antibody OTA - E4 of the present invention is as Figure 4 shown. As can be Figure 4 seen, the standard curve is S - shaped. The IC 50 of the prepared monoclonal antibody OTA - E4 for ochratoxin A is 1.42 ng / mL, the detection limit (LOD) is 0.18 ng / mL, and the linear range is 0.34 - 5.40 ng / mL.

[0143] Example 4 Specificity detection

[0144] Based on the method described in Example 3, the present invention also carried out specificity detection. The coating and blocking processes of the ELISA plate were the same as those in Example 3.

[0145] For the well - wrapped enzyme - linked immunosorbent assay (ELISA) plates, add 50 μL of anti - ochratoxin A monoclonal antibody with a concentration of 3.5 μg / mL and 50 μL of a series of ochratoxin A structural analogs and functional analogs standards with different concentrations (as shown in Table 6) to each well. Incubate at 37 °C for 40 min, wash five times with PBST, pat dry the liquid in the wells, add the enzyme - labeled secondary antibody (goat anti - mouse IgG - HRP) diluted 1:5000, incubate at 37 °C for 40 min, wash five times with PBST, pat dry the liquid in the wells, add 100 μL of TMB substrate solution, and develop color in the dark at 37 °C for 10 min; add 50 μL of the stop solution (2 M H2SO4) to terminate the reaction; read the absorbance value at 450 nm using an enzyme - linked immunosorbent assay reader. Use the concentration of the drug standard on the abscissa and B / B0 (absorbance of the well with the drug / absorbance of the well without the drug) on the ordinate to calculate the IC 50 of each drug, and then calculate the cross - reactivity (CR) of each substance with ochratoxin A to evaluate the specificity of the antibody. The results are shown in Table 6, indicating good specificity.

[0146]

[0147] Table 6 Cross - reactivity of OTA and its structural analogs by icELISA method

[0148]

[0149] The above - mentioned embodiments are preferred embodiments of the present invention. However, the embodiments of the present invention are not limited by the above - mentioned embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. An antibody against ochratoxin A, characterized in that, The antibody comprises the following complementary determining regions: VL-CDR1: KSVSTSGYSY; VL-CDR2: LVS; VL-CDR3: QHIRELTR; VH-CDR1: GFDFSSYD; VH-CDR2: ISSGGRYS; VH-CDR3: ARQNDYEAWFAY.

2. The antibody according to claim 1, wherein The amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO.1; the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO.

2.

3. Use of the antibody according to claim 1 or 2 for enriching ochratoxin A.

4. Use of the antibody according to claim 1 or 2 for preparing a product for enriching ochratoxin A.

5. Use of the antibody according to claim 1 or 2 for detecting ochratoxin A.

6. Use of the antibody according to claim 1 or 2 for preparing a product for detecting ochratoxin A.

7. An enzyme-linked immunosorbent assay kit for detecting ochratoxin A, characterized in that, Contains the antibody according to claim 1 or 2.

8. The enzyme-linked immunosorbent assay kit according to claim 7, wherein Also contains an enzyme-labeled plate coated with a coating antigen.

9. The enzyme-linked immunosorbent assay kit according to claim 8, wherein The coating antigen is an artificial antigen obtained by conjugating ochratoxin A with ovalbumin.

10. The enzyme-linked immunosorbent assay kit according to claim 7, characterized in that, Also contains reagents required for the color reaction.

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