Antibodies against ochratoxin a and uses thereof

Abstract

The application discloses an antibody against ochratoxin A and application thereof. The application uses an artificial antigen obtained by coupling ochratoxin A and a carrier protein as an immunogen to prepare a monoclonal antibody against ochratoxin A. The monoclonal antibody can sensitively and specifically detect ochratoxin A, and the minimum detection limit (LOD) is 0.18 ng / mL. In addition, the half inhibition (IC 50 ) of the icELISA for ochratoxin A established by 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 application provides an antibody capable of detecting ochratoxin A and a method for detecting ochratoxin A, and has the advantages of simplicity, rapidness, strong specificity, wide linear range and high sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of enzyme-linked immunosorbent assay (ELISA) technology. More specifically, it relates to an antibody against ochratoxin A and its application. Background Technology

[0002] Ochratoxin A (OTA) is a secondary metabolite produced by certain strains of Aspergillus and Penicillium, belonging to the isocoumarin class of compounds. Compared to other fungal toxins, OTA has the longest residence time in the human circulation. When OTA accumulates to a certain level, its long-term chronic toxic effects may lead to tumors. Since ochratoxin A is commonly found in foods such as grains and milk powder, detecting potentially contaminated foods is crucial for ensuring food safety and maintaining public health.

[0003] Existing common methods for detecting ochratoxin A (OTA) mainly include instrumental analytical methods such as high-performance liquid chromatography (HPLC), gas chromatography (GC), ultraviolet spectroscopy (UV spectroscopy), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). While instrumental analytical methods offer good repeatability and high accuracy and sensitivity, they require expensive equipment, and their sample pretreatment processes are complex, cumbersome, and time-consuming, failing to meet the needs of rapid on-site testing of large batches of samples. Therefore, there is an urgent need for a simple, rapid, specific, and sensitive method for detecting OTA. Summary of the Invention

[0004] To address the shortcomings of the prior art, this invention provides an antibody against ochratoxin A and its application.

[0005] The first objective of this invention is to provide an antibody against ochratoxin A.

[0006] A second object of the present invention is to provide the use of the antibody in the enrichment of ochratoxin A.

[0007] A third object of the present invention is to provide the use of the antibody in the preparation of products for enriching ochratoxin A.

[0008] A fourth object of the present invention is to provide the use of the antibody in the detection of ochratoxin A.

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

[0010] The sixth objective of this invention is to provide an enzyme-linked immunosorbent assay (ELISA) kit for detecting ochratoxin A.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution:

[0012] This invention utilizes an artificial antigen conjugated with ochratoxin A and a carrier protein as an immunogen to prepare a monoclonal antibody against ochratoxin A. This monoclonal antibody can sensitively and specifically detect ochratoxin A, with a limit of detection of 0.18 ng / mL. Furthermore, the half-maximal inhibition (IC50) of an icELISA against ochratoxin A established using this anti-ochratoxin A monoclonal antibody is shown to be [percentage missing]. 50 The effective concentration was 1.42 ng / mL, and the linear range was 0.34–5.40 ng / mL. Therefore, the antibody described in this invention exhibits high detection sensitivity and a wide linear range for the detection of ochratoxin A, meeting the requirements for rapid and sensitive detection of ochratoxin A. Consequently, this invention seeks protection for the antibody and its applications.

[0013] This 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 light chain variable region of the antibody is shown in SEQ ID NO.1; the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.2.

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

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

[0023] This invention, through the coupling of the antibody with magnetic beads to form immunomagnetic beads and testing their adsorption rate for ochratoxin A, found that the antibody of this invention can effectively adsorb ochratoxin A. Therefore, this invention seeks protection for the use of the antibody in the enrichment of ochratoxin A.

[0024] The present invention also claims protection for the use of the antibody in the preparation of products for enriching ochratoxin A.

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

[0026] The present invention also claims protection for the use of the antibody in the preparation of products for the detection of ochratoxin A.

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

[0028] Specifically, it also contains ELISA plates coated with coated antigens.

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

[0030] Specifically, the enzyme-linked immunosorbent assay kit also contains reagents required for the colorimetric reaction.

[0031] Specifically, the reagents required for the colorimetric reaction include enzyme-labeled secondary antibody and colorimetric solution.

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

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

[0034] This invention utilizes an artificial antigen obtained by conjugating ochratoxin A with a carrier protein as an immunogen to prepare a monoclonal antibody against ochratoxin A. The monoclonal antibody can sensitively and specifically detect ochratoxin A, with a limit of detection (LOD) of 0.18 ng / mL. Furthermore, the half-maximal inhibition (ICP-C) of an icELISA against ochratoxin A established using the monoclonal antibody against ochratoxin A is shown to be [percentage missing]. 50 The effective concentration was 1.42 ng / mL, and the linear range was 0.34–5.40 ng / mL.

[0035] This invention provides an antibody and a method for detecting ochratoxin A, which are simple, rapid, highly specific, have a wide linear range, and are highly sensitive. Attached Figure Description

[0036] Figure 1 UV scanning identification results for OTA, carrier proteins BSA, LF, OVA, and artificial antigens OTA-BSA, OTA-LF, and OTA-OVA.

[0037] Figure 2 The results of analysis of the framework region and complementarity-determining region of the light chain of the anti-ochratoxin A monoclonal antibody OTA-E4 are presented.

[0038] Figure 3 The results of analysis of the framework region and complementarity-determining region of the heavy chain of the anti-ochratoxin A monoclonal antibody OTA-E4 are presented.

[0039] Figure 4 The indirect competitive ELISA standard curve was established using OTA-OVA as the coating agent and the anti-ochratoxin A monoclonal antibody OTA-E4. Detailed Implementation

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

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

[0042] Example 1: Preparation of monoclonal antibodies against ochratoxin A

[0043] 1. Preparation of artificial antigens

[0044] The artificial antigen used in this invention to prepare the monoclonal antibody against ochratoxin A is prepared by coupling ochratoxin A with a carrier protein (ovalbumin (OVA), bovine serum albumin (BSA), or lactoferrin (LF)) via an active ester method; wherein, 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 method for preparing the artificial antigen is as follows:

[0049] Ochratoxin A was dissolved in DMF, and EDC and NHS were added (the molar ratio of OTA:NHS:EDC was 1:1.5:1.5). The mixture was stirred at 4°C for 8 hours and denoted as solution A. The carrier proteins were dissolved separately in phosphate buffer and denoted as solution B. Solution A was added dropwise to solution B and the mixture was stirred at 4°C for 8 hours. The molar ratio of carrier protein to ochratoxin A was 1:80. The reaction solution was dialyzed at 4°C for 3 days using an 8000–13000 dialysis membrane, with the dialysate (0.01M PBS) changed twice a day. After dialysis, artificial antigens OTA-BSA, OTA-LF, and OTA-OVA were obtained and identified by ultraviolet scanning (190–450 nm) along with the carrier proteins (BSA, LF, OVA) and OTA.

[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 As shown. By Figure 1 It can be seen that the artificial antigen prepared by this invention has a significant blue shift in characteristic absorption peaks compared with carrier proteins (BSA, LF, OVA) and OTA, and the artificial antigen possesses the characteristic absorption peaks of OTA and carrier proteins (BSA, LF, OVA), indicating that the artificial antigen was successfully prepared.

[0051] 2. Detection of the immunogenicity of artificial antigens

[0052] (1) Animal Immunization

[0053] Healthy 6-week-old Balb / c female mice were used as experimental animals. The identified artificial antigens OTA-BSA and OTA-LF were used as immunogens and emulsified with equal amounts of adjuvants (French complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent booster immunizations). Immunization was carried out by various injection methods, including subcutaneous injection on the back, subcutaneous injection at various sites, intraperitoneal injection, and foot injection. After the first immunization, mice were immunized every 2 weeks. After the fourth immunization, a small amount of tail vein blood was collected for antibody quality identification. After the antibodies stabilized, the mice with the best performance were selected for cell fusion. Three days before cell fusion, 0.5 mg of immunogen was directly injected into the intraperitoneal cavity of the mice for a booster immunization.

[0054] (2) Antibody quality identification

[0055] Using the identified artificial antigen OTA-OVA as the coating antigen, tail vein blood from the above-mentioned mice was used as the detection antibody. The antiserum titer and inhibition rate of mouse serum were determined by indirect competitive ELISA. The titer and inhibition rate of each antiserum were comprehensively considered to evaluate them.

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

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

[0058] ② Serum titer and inhibition rate detection: The enzyme-labeled plate prepared in step ① was set up as a titer column and an inhibition column in the horizontal and vertical directions, respectively. For the titer column: 50 μL of PBS and 50 μL of serially diluted mouse serum (1K, 2K, 4K, 8K, 16K, 32K, 64K) were added to each well. For the inhibition column: 50 μL of diluted 1000 ng / mL OTA and 50 μL of serially diluted serum (1K, 2K, 4K, 8K, 16K, 32K, 64K) were added to each well. Two parallel groups were prepared. The plates were incubated at 37℃ for 40 min, washed five times with PBST, the liquid in the wells was blotted dry, and 1:5000 diluted enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP) was added. After incubating at 37℃ for 30 min, the plates were washed five times with PBST, the liquid in the wells was blotted dry, and 100 μL of... After developing the TMB substrate solution at 37°C in the dark for 10 min, add 50 μL of stop solution (2M H2SO4) to terminate the reaction; read the absorbance at 450 nm using an ELISA reader.

[0059] 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. As shown in Table 1, mice immunized with both artificial antigens produced mouse polyclonal antibodies, and the resulting antiserum had an inhibitory effect on the target analyte ochratoxin A. The inhibitory effect was most significant in OTA-LF mice, indicating that the artificial antigen prepared in this invention can be used for the subsequent preparation of ochratoxin A monoclonal antibodies and the establishment of immunoassay methods.

[0060] Table 1. Antiserum titer and inhibition rate of OTA mice

[0061]

[0062] 3. Preparation of anti-OTA monoclonal antibodies

[0063] (1) Cell fusion

[0064] Spleen cells from immunized OTA-LF mice were mixed with myeloma cells from mice in logarithmic growth phase (SP2 / 0). Preheated fusion agent (PEG1500) was slowly added over 45 seconds to induce fusion. The cells were then suspended in HAT medium and homogenized before adding an appropriate amount of feeder cells (peritoneal macrophages from unimmunized mice). The mixture was cultured in 96-well plates at 37°C in a 5% CO2 incubator. After 5 days, the medium was partially replaced with HT medium, and after 9 days, the medium was completely replaced.

[0065] (2) Screening for positive hybridomas

[0066] After cell fusion, when the cells grew to 1 / 4 of the culture well area, hybridoma cells were screened using a stepwise screening method. Initial selection was performed using an indirect ELISA method. OTA-OVA was used as the coating antigen (its optimal coating concentration and positive serum dilution were pre-determined using a checkerboard method). The ELISA plate was coated with the culture supernatant from the test wells, incubated, washed, and then goat anti-mouse IgG-HRP was added, followed by TMB substrate solution for colorimetric reaction. Positive wells were then screened again using an indirect competitive ELISA method. The cell supernatant was mixed with an equal volume of 1000 ng / mL ochratoxin A, incubated at 37°C for 30 min, and then added to the coated ELISA plate. PBS was used as a control instead of ochratoxin A. The remaining steps were the same as above. If the OD450nm value after ochratoxin A blockade decreased to below 50% of the control well, it was considered positive. Wells that showed positive results after 2 or 3 tests were immediately subcloned using a limiting dilution method.

[0067] (3) Large-scale culture of hybridoma cells

[0068] Hybridoma cells obtained after 2-3 subclonings were cultured and expanded. The supernatant was collected and its titer was determined by indirect ELISA. The cells were then frozen. 8-10 week old Balb / c mice were intraperitoneally injected with 0.5 mL of liquid paraffin per mouse. 7-10 days later, 1-2 × 10⁶ hybridoma cells were injected intraperitoneally. 6 / mouse, 7-10 days later, ascites fluid was extracted from mice, centrifuged to obtain the supernatant, the potency was determined, and the fluid was frozen for later use.

[0069] The titers and inhibition rates of the OTA monoclonal cell lines screened in this invention are shown in Table 2. OTA-E4 was selected for subsequent experiments.

[0070] Table 2. Titer and Inhibition Rate of OTA Monoclonal Cell Lines

[0071]

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

[0073] (1) Total RNA extraction

[0074] Total RNA was extracted from the monoclonal antibody cell line OTA-E4. The extraction method for total RNA was based on the Trizol reagent method of Guangzhou Jiebes Biotechnology Co., Ltd.

[0075] (2) cDNA synthesis

[0076] Using extracted total cellular RNA as a template, cDNA first strand was synthesized according to the instructions of the Takara reverse transcription kit.

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

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

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

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

[0081]

[0082] The specific process is as follows:

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

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

[0085]

[0086] Primer sets capable of amplifying sequences were selected, and their LB primer numbers were determined as positive LB primers. Each positive LB primer was then combined with six LF primers, and PCR was performed using the first strand of cDNA as a template. The reaction system and conditions were the same as in Table 4. After PCR, the bands were identified by gel electrophoresis. Bands in the range of 300bp to 500bp were excised using a kit. The PCR gel products were recovered using the OMEGA gel extraction kit and transformed into E. coli DH5α. Multiple single colonies were selected for colony PCR and DNA sequencing identification and analysis.

[0087] Cloning of the heavy chain variable region gene sequence is the same as above.

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

[0089] The sequencing results show that the amino acid sequence of the light chain variable region of the anti-ochratoxin A monoclonal antibody OTA-E4 described in this invention is as follows (SEQ ID NO.1):

[0090] DIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWKSN

[0091] The amino acid sequence of the variable region of the OTA-E4 heavy chain of the anti-OTA monoclonal antibody is shown below (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 shown below (SEQ ID NO.3):

[0094] GACATTTGTGATGACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTA TCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAC

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

[0096] GAGGTGCAGGGGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAATTCTCCTGTGTAGCCTCTGGATTCGATTTCAGTTCCTATGACATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAAGTATTAGTAGTGGTGGCCGTTATTCTTATT ATCCAGACAGTGTGAAGGGCCGATTCACCATCCCAGAGACAATGCCAGGAACACCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAAGACACGGCCTTGTATTACTGTGCAAGACAGAATGATTATGAGGCCTGGTTTGCTTACTGGGGCCTAGGGACTCTGGTCACTGTCTCTGCG

[0097] The present invention analyzed the structures of the light and heavy chain variable regions of the monoclonal antibody OTA-E4, which is simultaneously antagonistic to ochratoxin A. The results are as follows: Figure 2 and Figure 3 As shown. By Figure 2 and Figure 3 It is known that the light and heavy chains of OTA-E4 each include four framework regions (FR1, FR2, FR3, FR4) and three complementarity-determining regions (CDR1, CDR2, CDR3). The amino acid sequences of the three complementarity-determining regions of the light and heavy chains of OTA-E4 are shown below:

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

[0099] VL-CDR2: LVS;

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

[0101] VH-CDR1: GDFFSSYD (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 their adsorption rate for ochratoxin A

[0105] This invention prepares immunomagnetic beads by coupling the monoclonal antibody OTA-E4 against ochratoxin A with magnetic beads, and tests their adsorption rate for ochratoxin A.

[0106] 1. Preparation of immunomagnetic beads

[0107] (1) Preparation of protein solution

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

[0109] (2) Cleaning of magnetic beads

[0110] Take 500 μL of magnetic beads (BEAVER, catalog number 70703-5, NHS magnetic bead kit, average particle size 200 nm) into a 1.5 mL EP centrifuge tube (before sampling, repeatedly invert the tube and use a vortex mixer or vertical mixer to ensure uniform mixing to guarantee experimental consistency). Place the EP tube in a magnetic separator 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 a magnetic separator to enrich the magnetic beads, and remove the supernatant.

[0111] (3) Biological ligand fixation

[0112] Add 500 μL of protein solution to an EP tube (add the protein solution immediately after washing the magnetic beads), vortex for 30 seconds to mix thoroughly, vortex the EP tube for 15 seconds, place it on a vertical mixer, mix at room temperature for 1–2 hours, enrich the magnetic beads using a magnetic separation rack, and preserve the flow-through solution.

[0113] (4) Magnetic bead sealing

[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 separator 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 to react at room temperature for 2 h, place the EP tube in a magnetic separator to enrich the magnetic beads, remove the supernatant, add 1 mL of ultrapure water to the EP tube, mix thoroughly, enrich the magnetic beads using a magnetic separator, discard the supernatant.

[0115] (5)Save

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

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

[0118] The immunomagnetic beads prepared according to this invention adsorb OTA in a sample, and then the OTA adsorbed on the magnetic beads is eluted using an eluent. The eluent can be used for the analysis and detection of OTA. In other words, the immunomagnetic beads prepared using the antibody described in this invention can be used for OTA enrichment and as a pretreatment method in the OTA analysis and detection process.

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

[0120] Take four 1mL centrifuge tubes, add 0.2mg of immunomagnetic beads, and inhale 1mL of OTA standard drug with a concentration of 25ng / mL prepared in 40% methanol. After mixing, place the tubes on a shaker at 25℃ and shake at 250r / min for 15min. After magnetic separation, retain the supernatant. Add 500μL of 80% methanol to the enriched magnetic beads, place the tubes on a shaker at 37℃ and shake at 250r / min for 5min, and retain the supernatant after magnetic separation.

[0121] (2) Adsorption rate detection

[0122] The amount of unadsorbed OTA residue in the supernatant and the content of OTA in the eluent were determined by using an ELISA assay kit to indirectly calculate the actual adsorption amount, thereby calculating the adsorption rate and elution rate.

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

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

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

[0126] Based on the above enrichment method, this invention optimizes factors such as extraction reagent, amount of immunomagnetic beads, adsorption time, and elution volume.

[0127] The optimized immunomagnetic bead enrichment method for OTA in samples is as follows:

[0128] Weigh 5.0 g (±0.1 g) of the sample to be tested into a 50 mL centrifuge tube, add 25 mL of 80% methanol aqueous solution, mix at 2500 rpm for 5 min, centrifuge at 4000 rpm for 5-10 min, after solid-liquid separation, take 2 mL of 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 in a shaker at 25℃, shake at 250 rpm for 5-10 min to enrich, add 400 μL of 80% methanol to the enriched magnetic beads, place in a shaker at 37℃, shake at 250 rpm for 5 min to elute, and retain the supernatant for subsequent detection after magnetic separation.

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

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

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

[0132]

[0133] Note: The concentration of each drug added is 25 ng / mL.

[0134] Example 3: Method for detecting ochratoxin A

[0135] Based on the anti-ochratoxin A monoclonal antibody OTA-E4 described in Example 1, this invention constructs a method for detecting ochratoxin A and establishes an indirect competitive ELISA standard curve for the anti-ochratoxin A monoclonal antibody OTA-E4.

[0136] 1. Wrapping and sealing

[0137] The OTA-OVA coating stock was diluted to 1000 ng / mL with coating buffer (pH 9.6, 0.1 mol / L carbonate buffer, 1.65 g Na2CO3, 2.65 g NaHCO3, 1000 mL distilled water), and coated overnight at 37°C. The next day, the sample was washed twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)), and 120 μL of 2% skim milk powder was added to each well. The sample was blocked at 37°C for 3 h, the blocking solution was discarded, and the sample was dried at 37°C for 60 min. The sample was then stored in a sealed bag at 4°C for later use.

[0138] 2. Establishment of the standard curve

[0139] (1) Experimental methods

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

[0141] (2) Experimental Results

[0142] The indirect competitive ELISA standard curve established by the prepared anti-ochratoxin A monoclonal antibody OTA-E4 in this invention is shown in the figure below. Figure 4 As shown. By Figure 4 It can be seen that the standard curve is S-shaped, and the IC50 of the prepared monoclonal antibody OTA-E4 against ochratoxin A is... 50 The limit of detection (LOD) was 1.42 ng / mL, the limit of detection (LOD) was 0.18 ng / mL, and the linear range was 0.34–5.40 ng / mL.

[0143] Example 4 Specificity Detection

[0144] Based on the method described in Example 3, the present invention also performed specific detection, and the coating and blocking process of the ELISA plate was the same as in Example 3.

[0145] Add 50 μL of 3.5 μg / mL anti-ochratoxin A monoclonal antibody and a series of 50 μL of ochratoxin A structural and functional analog standards at different concentrations to each well of the packaged ELISA plate (as shown in Table 6). Incubate at 37°C for 40 min, wash five times with PBST, blot dry the liquid in the wells, add 1:5000 diluted enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP), incubate at 37°C for 40 min, wash five times with PBST, blot dry the liquid in the wells, add 100 μL of TMB substrate solution, and develop color at 37°C in the dark for 10 min. Add 50 μL of stop solution (2M H2SO4) to terminate the reaction. Read the absorbance at 450 nm using an ELISA reader. Calculate the IC50 of each drug with the drug standard concentration on the x-axis and B / B0 (absorbance of wells with drug added / absorbance of wells without drug added) on the y-axis. 50 Then, the cross reactivity (CR) of each substance with ochratoxin A was calculated to assess the specificity of the antibody. The results are shown in Table 6, which shows that the specificity is good.

[0146]

[0147] Table 6 shows the cross-reactivity of OTA and structurally similar cross-reactivity using the icELISA method.

[0148]

[0149] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An antibody against ochratoxin A, characterized in that, The antibody includes the following complementarity-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, characterized in that, The amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO.1; the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.

2.

3. The use of the antibody according to claim 1 or 2 in the enrichment of ochratoxin A.

4. The use of the antibody according to claim 1 or 2 in the preparation of a product for enriching ochratoxin A.

5. The use of the antibody according to claim 1 or 2 in the preparation of a product for detecting ochratoxin A.

6. An enzyme-linked immunosorbent assay (ELISA) kit for detecting ochratoxin A, characterized in that, Contains the antibody as described in claim 1 or 2.

7. The enzyme-linked immunosorbent assay kit according to claim 6, characterized in that, It also contains ELISA plates coated with coating antigens.

8. The enzyme-linked immunosorbent assay kit according to claim 7, characterized in that, The coating was originally an artificial antigen obtained by conjugating ochratoxin A with ovalbumin.

9. The enzyme-linked immunosorbent assay kit according to claim 6, characterized in that, It also contains reagents needed for the colorimetric reaction.

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