An antibody for non-competitive detection of aflatoxin b1 and application thereof

Abstract

The application belongs to the technical field of genetic engineering antibodies, and particularly relates to an antibody for non-competitive detection of aflatoxin B1 and application thereof. The antibody comprises a nanobody of an aflatoxin B1 antigen-antibody immune complex and a monoclonal antibody against aflatoxin B1. The amino acid sequence of the nanobody of the aflatoxin B1 antigen-antibody immune complex is shown in SEQ ID NO. 1. The monoclonal antibody against aflatoxin B1 comprises a heavy chain and a light chain. The amino acid of the heavy chain is shown in SEQ ID NO. 10, and the amino acid of the light chain is shown in SEQ ID NO. 11. The antibody for non-competitive detection of aflatoxin B1 has low cost, can be prepared in a large amount through a prokaryotic expression system, is simple to prepare, has strong operability, and can be applied to non-competitive immunological analysis of aflatoxin B1.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering antibody technology, specifically relating to an antibody for non-competitive detection of aflatoxin B1 and its application. Background Technology

[0002] Aflatoxin B1 (AFB1) is a highly toxic and carcinogenic small-molecule mycotoxin produced by fungi such as Aspergillus flavus and Aspergillus parasiticus. It widely contaminates agricultural products such as corn, peanuts, rice, and their products. AFB1 exhibits extremely high hepatotoxicity and carcinogenicity, and is classified as a Group 1 carcinogen by the World Health Organization, posing a serious threat to human and animal health. Due to the prevalence and high toxicity of AFB1 contamination, establishing rapid and sensitive AFB1 detection methods is crucial for ensuring food safety and public health. Among numerous AFB1 detection methods, immunological detection methods have been widely used for rapid screening of actual samples due to their advantages of simple operation, high specificity, and high sensitivity. However, as a small-molecule hapten, AFB1 has a small molecular weight and a single antigenic epitope, making it impossible for it to be simultaneously bound by two conventional antibodies like large-molecule protein antigens. This leads to the fact that most current immunological analysis methods for AFB1 are based on competitive reaction modes. Although competitive immunoassays are widely used, they typically suffer from inherent drawbacks such as relatively cumbersome operation procedures, limited sensitivity improvement, and narrow detection range. In contrast, non-competitive immunoassay modalities offer significant advantages such as ease of operation, higher sensitivity, and a wider detection kinetic range. Therefore, developing non-competitive immunoassay methods for AFB1 has important practical significance and application value. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide an antibody for non-competitive detection of aflatoxin B1 and its application, specifically adopting the following technical solution:

[0004] In a first aspect, the present invention provides an antibody for non-competitive detection of aflatoxin B1, the antibody comprising a nanobody of an anti-aflatoxin B1 antigen-antibody immune complex and a monoclonal antibody against aflatoxin B1.

[0005] The amino acid sequence of the nanobody of the anti-aflatoxin B1 antigen-antibody immune complex is shown in SEQ ID NO. 1;

[0006] The monoclonal antibody against aflatoxin B1 comprises a heavy chain and a light chain; the amino acids of the heavy chain are shown in SEQ ID NO. 10, and the amino acids of the light chain are shown in SEQ ID NO. 11.

[0007] SEQ ID NO.1:

[0008] QVQLVESGGGLVQPGGSLRLACTASGFNLDIYDVGWYRQAPGNQREVVARITTRGSTYYADSVKGRFTISSRDNAKNTVYLQMNNLKPEDTAVYYCNTWPNWRLSWGQGTQVTVSS.

[0009] SEQ ID NO.10:

[0010] EVQLLQSGTELVKPGASVQLSCTASGLNIKDTYIHWVKQRPEQGLEWIGRIDPANGHTKYDPKFQGKATMTADTSSNTAYLQLSSLTSEDSAVYYCAREGEWLLRGDYWGQGTTLTVSS.

[0011] SEQ ID NO.11:

[0012] NIVLTQSPASLAVSLGQRATISCRASESVDGYGNSFMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCHQNNEDPWTFGGGTKLEIK.

[0013] The nanobody (A-2G) of the anti-aflatoxin B1 antigen-antibody immune complex provided by this invention includes four framework regions (FR) and three complementarity-determining regions (CDR), wherein the amino acid sequences of the framework regions (FR1, FR2, FR3, FR4) are as follows:

[0014] FR1: QVQLVESGGGLVQPGGSLRLACTAS (SEQ ID NO.2);

[0015] FR2: VGWYRQAPGNQREVVAR (SEQ ID NO.4);

[0016] FR3: YYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYC (SEQ ID NO.6);

[0017] FR4: WGQGTQVTVSS (SEQ ID NO. 8); The amino acid sequences of the complementarity-determining regions (CDR1, CDR2, CDR3) of A-2G are as follows:

[0018] CDR1:GFNLDIYD (SEQ ID NO.3);

[0019] CDR2: ITTRGST (SEQ ID NO.5);

[0020] CDR3: NTWPNWRLS (SEQ ID NO.7).

[0021] The framework region has a relatively conserved structure and mainly plays a role in maintaining the protein structure; the complementarity-determining region has a relatively diverse structure and is mainly responsible for the recognition of antigen-antibody immune complexes.

[0022] The monoclonal antibody (11Y12) against aflatoxin B1 provided by this invention comprises four framework regions (FRs) and three complementarity-determining regions (CDRs) in its heavy chain variable region. The amino acid sequences of the framework regions (FR1, FR2, FR3, and FR4) are as follows:

[0023] FR1: EVQLLQSGTELVKPGASVQLSCTAS (SEQ ID NO.12);

[0024] FR2:IHWVKQRPEQGLEWIGR (SEQ ID NO.13);

[0025] FR3:KYDPKFQGKATMTADTSSNTAYLQLSSLTSEDSAVYYC (SEQ ID NO.14);

[0026] FR4: WGQGTTLTVSS (SEQ ID NO.15);

[0027] The amino acid sequences of the heavy chain complementarity-determining regions (CDR1, CDR2, CDR3) of 11Y12 are as follows:

[0028] CDR1:GLNIKDTY (SEQ ID NO.16);

[0029] CDR2: IDPANGHT (SEQ ID NO.17);

[0030] CDR3: AREGEWLLRGDY (SEQ ID NO. 18).

[0031] The monoclonal antibody (11Y12) against aflatoxin B1 provided by this invention comprises four framework regions (FRs) and three complementarity-determining regions (CDRs) in its light chain variable region. The amino acid sequences of the framework regions (FR1, FR2, FR3, and FR4) are as follows:

[0032] FR1: NIVLTQSPASLAVSLGQRATISCRAS (SEQ ID NO.19);

[0033] FR2:MHWYQQKPGQPPKLLIY (SEQ ID NO.20);

[0034] FR3: NLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYC (SEQ ID NO .21);

[0035] FR4: FGGGTKLEIK (SEQ ID NO.22);

[0036] The amino acid sequences of the light chain complementarity-determining regions (CDR1, CDR2, CDR3) of 11Y12 are as follows:

[0037] CDR1: ESVDGYGNSF (SEQ ID NO.23);

[0038] CDR2: LAS (SEQ ID NO.24);

[0039] CDR3:HQNNEDPWT (SEQ ID NO.25).

[0040] The present invention may also provide proteins or polypeptides comprising one or more amino acid sequences of SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.8, and having at least 90% homology with one of the amino acid sequences thereof. Alternatively, it may comprise one or more amino acid sequences of SEQ ID NO.3, SEQ ID NO.5, and SEQ ID NO.7, and having at least 80% homology with one of the amino acid sequences thereof.

[0041] This invention employs phage display of a nanobody library technology, using the AFB1 antigen-antibody immune complex as the target molecule. Nanobodies containing the AFB1 antigen-antibody immune complex are screened from a library of naturally derived single-domain heavy chain antibodies, and then applied to a non-competitive immunoassay system for AFB1. By using phage display of a nanobody library technology to screen for nanobodies that specifically bind to the target molecule, this method avoids the animal immunization process required for traditional antibody preparation. The steps are simple, convenient, and rapid, and the screened nanobodies can be used for non-competitive immunoassay of AFB1.

[0042] The nanobodies provided by this invention can be prepared in large quantities through prokaryotic expression.

[0043] The nucleotide sequences or partial sequences provided by this invention can be expressed using suitable expression systems to obtain the corresponding proteins or peptides. These expression systems include bacterial, yeast, filamentous fungi, bacteriophages, animal cells, insect cells, plant cells, or cell-free expression systems.

[0044] The nanobodies provided by this invention can be applied to immunological detection and analysis. Types of immunological detection include enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography, and immunodot hybridization, all of which are immunological analysis detection types based on antigen-antibody specific reactions.

[0045] Secondly, the present invention provides a gene encoding a nanobody for detecting the above-mentioned anti-aflatoxin B1 antigen-antibody immune complex, the nucleotide sequence of which is shown in SEQ ID NO.9.

[0046] SEQ ID NO.9:

[0047] GGCCCAGGCGGCCCAGGTGCAGCTCGTGGAGTCAGGGGGAGGCTTGGTGCAGCCTGGGGGCTCTCTGAGACTCGCCTGTACAGCCTCTGGATTCAATTTGGACATTTATGACGTGGGCTGGTACCGCCAGGTCCAGGGAACCAGCGCGAAGTGGTCGCACGGATTACTACTCGTGGTAGCACCTACTATGCAGACTCC GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTACAAATGAACAACCTGAAACCTGAGGACACGGCCGTCTATTATTGTAACACATGGCCAAATTGGAGGCTAAGCTGGGGCCAGGGGACCCAGGTCACCGTGTCCTCAGAACCCAAGACACCAAAACCACAACCA GGCCAGGCCGGCC (The underlined part indicates the restriction endonuclease recognition site.)

[0048] Thirdly, the present invention provides the application of the above-mentioned aflatoxin B1 antigen-antibody immune complex nanobody in the preparation of a kit for immunological detection.

[0049] Fourthly, the present invention provides a kit for immunological detection, the kit comprising the above-described antibody for non-competitive detection of aflatoxin B1.

[0050] As a further preferred embodiment, the kit is an enzyme-linked immunosorbent assay kit or an immunochromatographic assay kit.

[0051] As a further preferred embodiment, the kit is used to detect aflatoxin B1.

[0052] In a seventh aspect, the present invention provides a method for detecting aflatoxin B1 for non-diagnostic purposes, the method comprising using the antibody described above for non-competitive detection of aflatoxin B1 to determine the presence of aflatoxin B1 in a sample by immunochromatography or ELISA.

[0053] As a further preferred embodiment, the ELISA method specifically includes the following steps:

[0054] S1. The monoclonal antibody 11Y12 was coated in a well plate at 4°C for 12 h and washed 3 times with PBST.

[0055] S2, after adding 5% skim milk and blocking for 1.5-2 hours, wash 3 times with 0.05% PBST;

[0056] S3. Simultaneously add the sample to be tested and the nanobody of the anti-aflatoxin B1 antigen-antibody immune complex, then incubate with 0.05% PBST and wash three times.

[0057] S4. After incubation with HRP-labeled detection antibody, wash three times with 0.05% PBST.

[0058] S5. Add TMB colorimetric solution, and after color development, add H2SO4 to stop. Take the reading at a wavelength of 450 nm.

[0059] As a further preferred embodiment, the immunochromatography method specifically includes the following steps:

[0060] Add the sample solution to be tested to the sample pad well of the immunochromatographic test strip prepared from the antibody for non-competitive detection of aflatoxin B1. Let it stand at room temperature for 10-15 minutes to allow the liquid to completely flow through the observation area. Then place the test strip under a UV lamp to observe the fluorescence bands and determine the result based on the fluorescence color development of the detection line and the control line.

[0061] As a further preferred embodiment, the immunochromatographic test strip uses an anti-aflatoxin B1 monoclonal antibody as the detection line, an anti-VHH monoclonal antibody as the quality control line, and an anti-aflatoxin B1 antigen-antibody immune complex nanobody as the detection probe.

[0062] The beneficial effects of this invention are as follows:

[0063] (1) The present invention provides an antibody for non-competitive detection of aflatoxin B1, which is low in cost, easy to prepare, highly operable, and can be applied to non-competitive immunological analysis of aflatoxin B1.

[0064] (2) The amino acid sequence of the aflatoxin B1 antigen-antibody immune complex nanobody or the anti-aflatoxin B1 monoclonal antibody provided by the present invention can be used as a precursor and modified by random or site-directed mutagenesis techniques to obtain mutants with better properties, which can be used to develop proteins or peptides for further use in the industrial and food safety fields. Attached Figure Description

[0065] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0066] Figure 1This is a schematic diagram of the amino acid numbering and structural domains of nanobody A-2G provided in some embodiments of this application.

[0067] Figure 2 These are SDS-PAGE identification images of nanobody A-2G prokaryotic expression provided in some embodiments of this application.

[0068] Figure 3 These are specific identification diagrams of nanobody A-2G provided in some embodiments of this application.

[0069] Figure 4 This is the AFB1 curve for non-competitive ELISA analysis established in this application.

[0070] Figure 5 This is a physical image of AFB1 obtained by non-competitive immunochromatographic analysis based on time-resolved fluorescence immunochromatography established in this application. Detailed Implementation

[0071] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0072] Example 1

[0073] Obtaining anti-AFB1 antigen-antibody immune complex nanobodies

[0074] (1) Obtaining monoclonal antibodies against aflatoxin B1

[0075] First, aflatoxin B1 (AFB1) was derivatized using chemical synthesis methods and conjugated with a carrier protein (BSA) to construct a synthetically produced AFB1 complete antigen. Subsequently, BALB / c mice were immunized with this complete antigen according to an immunization schedule. After the mouse serum titer reached a predetermined standard, spleen cells were fused with mouse myeloma cells (SP2 / 0) using a fusion agent. Following screening with HAT selective medium, specificity detection by ELISA, and subcloning using limiting dilution, a hybridoma cell line capable of stably secreting a highly specific and high-affinity anti-aflatoxin B1 monoclonal antibody was finally selected and named 11Y12. The amino acid composition of the heavy chain of the corresponding monoclonal antibody 11Y12 is shown in SEQ ID NO. 10, and the amino acid composition of the light chain is shown in SEQ ID NO. 11.

[0076] SEQ ID NO.10:

[0077] EVQLLQSGTELVKPGASVQLSCTASGLNIKDTYIHWVKQRPEQGLEWIGRIDPANGHTKYDPKFQGKATMTADTSSNTAYLQLSSLTSEDSAVYYCAREGEWLLRGDYWGQGTTLTVSS.

[0078] SEQ ID NO.11:

[0079] NIVLTQSPASLAVSLGQRATISCRASESVDGYGNSFMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCHQNNEDPWTFGGGTKLEIK.

[0080] The heavy chain variable region of the anti-aflatoxin B1 monoclonal antibody (11Y12) provided by this invention includes four framework regions (FRs) and three complementarity-determining regions (CDRs), wherein the amino acid sequences of the framework regions (FR1, FR2, FR3, FR4) are as follows:

[0081] FR1: EVQLLQSGTELVKPGASVQLSCTAS (SEQ ID NO.12);

[0082] FR2:IHWVKQRPEQGLEWIGR (SEQ ID NO.13);

[0083] FR3:KYDPKFQGKATMTADTSSNTAYLQLSSLTSEDSAVYYC (SEQ ID NO.14);

[0084] FR4: WGQGTTLTVSS (SEQ ID NO.15);

[0085] The amino acid sequences of the heavy chain complementarity-determining regions (CDR1, CDR2, CDR3) of 11Y12 are as follows:

[0086] CDR1:GLNIKDTY (SEQ ID NO.16);

[0087] CDR2: IDPANGHT (SEQ ID NO.17);

[0088] CDR3: AREGEWLLRGDY (SEQ ID NO. 18).

[0089] The light chain variable region of the anti-aflatoxin B1 monoclonal antibody (11Y12) provided by this invention includes four framework regions (FRs) and three complementarity-determining regions (CDRs), wherein the amino acid sequences of the framework regions (FR1, FR2, FR3, FR4) are as follows:

[0090] FR1: NIVLTQSPASLAVSLGQRATISCRAS (SEQ ID NO.19);

[0091] FR2:MHWYQQKPGQPPKLLIY (SEQ ID NO.20);

[0092] FR3: NLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYC (SEQ ID NO .21);

[0093] FR4: FGGGTKLEIK (SEQ ID NO.22);

[0094] The amino acid sequences of the light chain complementarity-determining regions (CDR1, CDR2, CDR3) of 11Y12 are as follows:

[0095] CDR1: ESVDGYGNSF (SEQ ID NO.23);

[0096] CDR2: LAS (SEQ ID NO.24);

[0097] CDR3:HQNNEDPWT (SEQ ID NO.25).

[0098] (2) Affinity panning and identification of anti-AFB1 antigen-antibody immune complex nanobodies

[0099] Nanobodies targeting AFB1 antigen-antibody immune complexes were screened from a camel-derived natural heavy chain phage antibody library using Protein A solid-phase affinity panning. The specific process is as follows:

[0100] 1. Ascites fluid containing anti-AFB1 monoclonal antibody was purified using an affinity column to obtain anti-AFB1 monoclonal antibody 11Y12; the anti-AFB1 monoclonal antibody was diluted to a final concentration of 20 μg / mL with PBS (pH 7.4), added to two wells of an ELISA plate, and coated overnight at 4°C.

[0101] 2. Aspirate the coating solution, wash 5 times with PBST (10mM PBS, 1.5% Tween-20 (v / v)), then add 1% BSA-PBS (or 2% OVA-PBS) and block at 37℃ for 2h.

[0102] 3. Remove the blocking solution, wash 5 times with PBST, and add 100 μL of camel-derived natural single-domain heavy chain phage antibody library (titer approximately 1.0 × 10⁻⁶) to one well (well A). 12 Add 100 μL of 100 ng / mL AFB1 standard to another well (well B) to form an AFB1 antigen-antibody complex, and incubate at 37°C for 1 h.

[0103] 4. Transfer the unbound phage in well A to well B, where an AFB1 antigen-antibody complex has been formed, and add AFB1 standard. Incubate at 37°C for 1 hour.

[0104] 5. Discard the unbound phages in well B, wash 10 times with PBST, add 100 μL of Glycine-HCl (0.2M, pH 2.2) to elute for 8 min, and immediately neutralize with 15 μL of Tris-HCl (1M, pH 9.1).

[0105] 6. Take 10 μL of the eluted phage to determine the titer, and use the remaining eluent for amplification in the next round of screening.

[0106] 7. In the second and third screening processes, blocking was performed alternately with 1% BSA and 2% OVA. In each round of screening, the coating amount of AFB1 monoclonal antibody 11Y12 and the amount of AFB1 standard added were gradually reduced.

[0107] 8. After three rounds of screening, the randomly selected monoclonal antibodies were rescued using helper phage M13KO7 to obtain phage particles displaying the antibody variable region. The binding activity of the phage particles was then determined by non-competitive phage-ELISA. Background control was set up for the experiment. The specific sample addition steps are shown in Table 1.

[0108] Table 1. Non-competitive phage-ELISA sample loading table

[0109]

[0110] ELISA-positive clones were sent to a sequencing company for sequencing, yielding a single, independent DNA insert sequence, named A-2G. The coding sequence for the nanobody is as follows:

[0111] GGCCCAGGCGGCCCAGGTGCAGCTCGTGGAGTCAGGGGGAGGCTTGGTGCAGCCTGGGGGCTCTCTGAGACTCGCCTGTACAGCCTCTGGATTCAATTTGGACATTTATGACGTGGGCTGGTACCGCCAGGTCCAGGGAACCAGCGCGAAGTGGTCGCACGGATTACTACTCGTGGTAGCACCTACTATGCAGACTCC GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTACAAATGAACAACCTGAAACCTGAGGACACGGCCGTCTATTATTGTAACACATGGCCAAATTGGAGGCTAAGCTGGGGCCAGGGGACCCAGGTCACCGTGTCCTCAGAACCCAAGACACCAAAACCACAACCA GGCCAGGCCGGCC

[0112] (Underlined areas indicate restriction endonuclease recognition sites)

[0113] According to the codon, the corresponding amino acid series is as shown in SEQ ID NO.1.

[0114] The amino acid sequence of nanobody A-2G, which is a complex of anti-AFB1 antigen and monoclonal antibody, was obtained.

[0115] A-2G ​​amino acid sequence: QVQLVESGGGLVQPGGSLRLACTASGFNLDIYDVGWYRQAPGNQREVVARITTRGSTYYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCNTWPNWRLSWGQGTQVTVSS (SEQ ID NO.1);

[0116] The nanobody A-2G is a complex of anti-AFB1 antigen and monoclonal antibody, wherein the amino acid sequences FR1, FR2, FR3, and FR4 in the framework region of A-2G are as follows:

[0117] FR1: QVQLVESGGGLVQPGGSLRLACTAS (SEQ ID NO.2);

[0118] FR2: VGWYRQAPGNQREVVAR (SEQ ID NO.4);

[0119] FR3: YYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYC (SEQ ID NO.6);

[0120] FR4: WGQGTQVTVSS (SEQ ID NO.8);

[0121] The nanobody A-2G is a complex of anti-AFB1 antigen and monoclonal antibody, wherein the amino acid sequences CDR1, CDR2, and CDR3 of the complementarity-determining region of A-2G are as follows:

[0122] CDR1:GFNLDIYD (SEQ ID NO.3);

[0123] CDR2: ITTRGST (SEQ ID NO.5);

[0124] CDR3: NTWPNWRLS (SEQ ID NO.7).

[0125] (3) Expression of anti-AFB1 antigen-antibody complex nanobody in Escherichia coli TOP10F'

[0126] 1. Extract the phage plasmid pComb3Xss-A-2G from positive E. coli, transform the plasmid into E. coli TOP10F', and induce expression.

[0127] 2. Inoculate a single colony into 5 mL of liquid LB / Amp medium and incubate overnight at 37°C and 200 rpm with shaking.

[0128] 3. Inoculate the above culture medium at a rate of 1% into 50 mL of liquid LB medium, and culture at 37°C and 200 rpm with shaking until the OD reaches 0.5. Then add IPTG to a final concentration of 1 mM and incubate overnight at 37°C and 200 rpm for induction culture.

[0129] 4. After centrifuging the induced culture at 8000 rpm for 10 min, the bacterial cells were resuspended in 15 mL of PBS and sonicated. The sonication conditions were 220 W, with a 2-second sonication cycle followed by a 3-second interval, for a total of 50 cycles. The lysed bacterial cells were then centrifuged at 8000 rpm for 15 min at 4 °C. The supernatant was collected and purified by affinity column chromatography to obtain soluble nanobodies. After elution with gradient concentrations of imidazole and concentration, the nanobodies were identified by SDS-PAGE. Figure 2 ).

[0130] Example 2

[0131] Specificity identification of nanobodies (denoted as A-2G) based on anti-aflatoxin B1 antigen-antibody immune complexes.

[0132] (1) Identification of the specificity of monoclonal antibodies against different fungal toxins

[0133] 1. Dilute the anti-AFB1 monoclonal antibody 11Y12, anti-ochratoxin A (OTA) monoclonal antibody, anti-fumonisin B1 (FB1) monoclonal antibody, anti-zearalenone toxin (ZEN) monoclonal antibody, and anti-vomiting toxin (DON) monoclonal antibody to 2 μg / mL with PBS (pH 7.4) and add them to the wells of the ELISA plate, 100 μL / well, and coat overnight at 4°C.

[0134] 2. Discard the coating solution, wash three times with 0.05% PBST, add 300 μL of 5% skim milk, and block at 37°C for 1.5 hours.

[0135] 3. Discard the blocking solution, wash the plate three times with 0.05% PBST, add 50 μL of 100 ng / mL AFB1 standard and 50 μL of 10 μg / mL anti-aflatoxin B1 antigen-antibody immune complex nanobody A-2G to each well, and incubate at 37°C for 45 minutes.

[0136] 4. Discard the liquid in the wells, wash the plate three times with 0.05% PBST, add 100 μL of HRP-labeled anti-HA secondary antibody / anti-influenza virus hemagglutinin-tagged secondary antibody diluted 1:7500 to each well, and incubate at 37°C for 45 minutes.

[0137] 5. Discard the liquid in the wells, wash the plate three times with 0.05% PBST, develop the color with TMB substrate, terminate with 2M H2SO4, and then read the OD. 450 Signal.

[0138] 6. Plotting different monoclonal antibodies as the x-axis, OD 450nm Create a bar chart with the vertical axis as the ordinate. Figure 3 A). The results showed that the nanobody A-2G of the aflatoxin B1 antigen-antibody immune complex obtained by panning only had binding activity and responsiveness against the AFB1 monoclonal antibody 11Y12 and the AFB1 complex, and had no response to other fungal toxin monoclonal antibodies, showing good specificity.

[0139] (2) Identification of the specificity of different fungal toxin small molecules

[0140] 1. Dilute the anti-AFB1 monoclonal antibody 11Y12 to 2 μg / mL with PBS (pH 7.4) and add it to the wells of the microplate, 100 μL / well, and coat overnight at 4°C.

[0141] 2. Discard the coating solution, wash three times with 0.05% PBST, add 300 μL of 5% skim milk, and block at 37°C for 1.5 hours.

[0142] 3. Discard the blocking solution, wash the plate three times with 0.05% PBST, add 50 μL of 100 ng / mL AFB1 standard, OTA standard, FB1 standard, ZEN standard, DON standard and 50 μL of 10 μg / mL anti-aflatoxin B1 antigen-antibody immune complex nanobody A-2G to each well, and incubate at 37°C for 45 minutes.

[0143] 4. Discard the liquid in the wells, wash the plate three times with 0.05% PBST, add 100 μL of HRP-labeled anti-HA secondary antibody / anti-influenza virus hemagglutinin-tagged secondary antibody diluted 1:7500 to each well, and incubate at 37°C for 45 minutes.

[0144] 5. Discard the liquid in the wells, wash the plate three times with 0.05% PBST, develop the color with TMB substrate, terminate with 2M H2SO4, and then read the OD. 450 Signal.

[0145] 6. Construct a bar chart with different monoclonal antibodies as the x-axis and OD450nm as the y-axis. Figure 3 B). The results showed that the nanobody A-2G of the aflatoxin B1 antigen-antibody immune complex obtained by panning only had binding activity and responsiveness against the AFB1 monoclonal antibody 11Y12 and the AFB1 complex, and had no response to other mycotoxin standards, demonstrating good specificity.

[0146] Example 3

[0147] A non-competitive ELISA analysis for the detection of AFB1 based on a nanobody (denoted as A-2G) of an anti-aflatoxin B1 antigen-antibody immune complex.

[0148] 1. Dilute the anti-AFB1 monoclonal antibody 11Y12 to 2 μg / mL with PBS (pH 7.4) and add it to the wells of the microplate, 100 μL / well, and coat overnight at 4°C.

[0149] 2. Discard the coating solution, wash three times with 0.05% PBST, add 300 μL of 5% skim milk, and block at 37°C for 1.5 hours.

[0150] 3. Discard the blocking solution, wash the plate three times with 0.05% PBST, add 50 μL of AFB1 standard at different concentrations and 50 μL of nanobody A-2G with a concentration of 10 μg / mL anti-aflatoxin B1 antigen-antibody immune complex to each well, and incubate at 37°C for 45 minutes.

[0151] 4. Discard the liquid in the wells, wash the plate three times with 0.05% PBST, add 100 μL of HRP-labeled anti-HA secondary antibody / anti-influenza virus hemagglutinin-tagged secondary antibody diluted 1:7500 to each well, and incubate at 37°C for 45 minutes.

[0152] 5. Discard the liquid in the wells, wash the plate three times with 0.05% PBST, develop the color with TMB substrate, terminate with 2M H2SO4, and then read the OD. 450 Signal.

[0153] 6. Plot the logarithm of AFB1 concentration on the x-axis and P / N on the y-axis to establish a non-competitive ELISA curve for AFB1 analysis based on nanobodies. Figure 4 The results showed that the nanobodies obtained by panning exhibited binding activity and responsiveness to the AFB1 antigen-antibody complex; the limit of detection (EC50) was [missing information]. 10 The concentration was 2.65 ng / mL, and the linear range was (EC50) 20 -EC 80 The concentrations ranged from 5.24 to 53.78 ng / mL.

[0154] Example 4

[0155] A non-competitive immunochromatographic strip for the analysis and detection of AFB1 based on a nanobody (denoted as A-2G) of an anti-aflatoxin B1 antigen-antibody immune complex.

[0156] 1. Take an appropriate amount of carboxyl-modified europium (Eu) elemental time-resolved fluorescent microspheres (particle size 100-200 nm), add EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and NHS (N-hydroxysuccinimide) to activate the carboxyl groups on the surface of the microspheres, and react with shaking at room temperature for 15-20 minutes. Centrifuge to remove excess activator, resuspend in labeling buffer, add purified anti-aflatoxin B1 antigen-antibody complex nanobody A-2G, and couple at room temperature in the dark for 2 hours. Then add blocking buffer containing BSA to block unbound active sites, centrifuge, resuspend the precipitate in storage buffer, and obtain the fluorescent microsphere-labeled nanobody A-2G probe. Spray the probe evenly onto a glass fiber binding pad and freeze-dry under vacuum for later use.

[0157] 2. Assembly of Immunochromatographic Test Strips: Nitrocellulose membrane (NC membrane) is used as the solid-phase carrier. Detection line (T line): Dilute anti-AFB1 monoclonal antibody 11Y12 to an appropriate concentration (e.g., 1-2 mg / mL) and apply it to the test area of ​​the NC membrane using a membrane scribing device. Control line (C line): Dilute anti-VHH nanobody monoclonal antibody (or anti-HA / His-tagged antibody, depending on the A-2G tag) to an appropriate concentration and apply it to the control area distal to the T line. Stack the prepared NC membrane, gold-labeled conjugate pad, sample pad, and absorbent pad sequentially onto a PVC substrate according to the standard chromatography structure, and cut into 3-4 mm wide test strips.

[0158] 3. Detection Method: Add 100 μL of the sample solution to be tested (or a standard solution containing different concentrations of AFB1) to the sample pad of the test strip. The liquid flows towards the absorbent pad under the action of the capillary. Observe the results after reacting at room temperature for 10-15 minutes.

[0159] 4. Result Judgment and Principle: This embodiment adopts the non-competitive method (forward detection) mode, and the principle is as follows:

[0160] Positive result (+): When the sample contains AFB1, AFB1 flows with the chromatography solvent to the T line and is captured by the monoclonal antibody 11Y12 immobilized at the T line, forming a solid-phase "antibody-antigen complex". At this point, the colloidal gold-labeled nanobody A-2G, which flows in with the solvent, specifically recognizes and binds to this "antibody-antigen complex", thus accumulating at the T line and appearing as a red band. The gold-labeled probe continues to flow to the C line and is captured and colored. The result is: red at the T line and red at the C line.

[0161] Negative result (-): When the sample does not contain AFB1, the monoclonal antibody 11Y12 at the T line is in a free, unloaded state and cannot be recognized and bound by the nanobody A-2G. The colloidal gold probe is not retained at this point and flows directly through the T line. The result is: the T line is uncolored, and the C line is red.

[0162] Validity determination: Regardless of whether AFB1 is present in the sample, line C should be red; otherwise, the test strip is invalid.

[0163] 5. Detection performance

[0164] Using this test strip to test a series of AFB1 standards, the results showed that as the AFB1 concentration increased, the red band on the T line gradually deepened, exhibiting a good positive correlation. This method overcomes the difficulty of visually interpreting the "line disappearance method" of traditional small molecule competitive test strips, achieving intuitive "color development equals positive" detection. Figure 5 As shown, when the concentration of AFB1 exceeds 0.68 ng / mL, AFB1 contamination can be visually detected.

[0165] The embodiments of this application have been described above with reference to the accompanying drawings. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the core ideas of this application. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. An antibody for non-competitive detection of aflatoxin B1, characterized in that, The antibodies include nanobodies of anti-aflatoxin B1 antigen-antibody immune complexes and monoclonal antibodies against aflatoxin B1. The amino acid sequence of the nanobody of the anti-aflatoxin B1 antigen-antibody immune complex is shown in SEQ ID NO.1; The monoclonal antibody against aflatoxin B1 comprises a heavy chain and a light chain; the amino acids of the heavy chain are shown in SEQ ID NO. 10, and the amino acids of the light chain are shown in SEQ ID NO.

11.

2. A gene encoding a nanobody that detects the anti-aflatoxin B1 antigen-antibody immune complex of claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

9.

3. The use of the antibody for non-competitive detection of aflatoxin B1 as described in claim 1 in the preparation of a kit for the immunological detection of aflatoxin B1.

4. A kit for the immunological detection of aflatoxin B1, characterized in that, The kit includes the antibody for non-competitive detection of aflatoxin B1 as described in claim 1.

5. The reagent kit according to claim 4, characterized in that, The kit is an enzyme-linked immunosorbent assay kit or an immunochromatographic assay kit.

6. The reagent kit according to claim 5, characterized in that, The kit is used to detect aflatoxin B1.

7. A method for detecting aflatoxin B1 for non-diagnostic purposes, characterized in that, The method includes determining aflatoxin B1 in a sample by immunochromatography or ELISA using the antibody for non-competitive detection of aflatoxin B1 as described in claim 1.

8. The method according to claim 7, characterized in that, The ELISA method specifically includes the following steps: S1. The monoclonal antibody against aflatoxin B1 was coated in a well plate at 4°C for 12 h and washed 3 times with PBST. S2, after adding 5% skim milk and blocking for 1.5-2 hours, wash 3 times with 0.05% PBST; S3. Simultaneously add the sample to be tested and the nanobody of the anti-aflatoxin B1 antigen-antibody immune complex, then incubate with 0.05% PBST and wash three times. S4. After incubation with HRP-labeled detection antibody, wash three times with 0.05% PBST. S5. Add TMB colorimetric solution, and after color development, add H2SO4 to stop. Take the reading at a wavelength of 450 nm.

9. The method according to claim 7, characterized in that, The immunochromatographic method specifically includes the following steps: Add the sample solution to be tested to the sample pad well of the immunochromatographic test strip prepared from the antibody for non-competitive detection of aflatoxin B1. Let it stand at room temperature for 10-15 minutes to allow the liquid to completely flow through the observation area. Then place the test strip under a UV lamp to observe the fluorescence bands and determine the result based on the fluorescence color development of the detection line and the control line.

10. The method according to claim 9, characterized in that, The immunochromatographic test strip uses an anti-aflatoxin B1 monoclonal antibody as the detection line, an anti-VHH monoclonal antibody as the quality control line, and a nanobody coupled with a signal-labeled anti-aflatoxin B1 antigen-antibody immune complex as the detection probe.

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