A nanoantibody for detecting aflatoxin B1 and its application

Abstract

The present invention discloses a nano-antibody for detecting aflatoxin B1 and its application, belonging to the field of food detection. The amino acid sequence of the nano-antibody is shown in SEQ ID NO: 1. The present invention constructs a Bactrian camel immune antibody library, uses phage display technology to solid-phase-coat the detection antigen on an ELISA plate, and then puts the Bactrian camel immune antibody library into the plate for affinity panning to obtain a nano-antibody that specifically binds to aflatoxin B1. The nano-antibody is then applied to the immunological detection and analysis of aflatoxin B1, and a rapid, sensitive, and stable method for detecting aflatoxin B1 is established through this immunoassay. The present invention provides a new method for the specific and rapid detection of aflatoxin B1 in the food field.

Description

Technical Field

[0001] The present invention relates to the field of food detection, and in particular to a nano antibody for detecting aflatoxin B1 and applications thereof. Background Art

[0002] Aflatoxin is a highly toxic liver-carcinogen. Aflatoxin B1 can cause cells to incorrectly repair DNA, leading to severe DNA mutagenesis. It can also inhibit DNA and RNA synthesis, thereby inhibiting protein synthesis. Epidemiological studies of liver cancer in my country have found that dietary aflatoxin contamination levels in certain regions are positively correlated with the incidence of primary liver cancer. The maximum limit for aflatoxin B1 in cereals and their products in my country is 20 μg / kg.

[0003] Extensive work has been done on the detection of AFB1 using high-performance liquid chromatography, chromatography, and mass spectrometry, with some success. However, these methods, along with time-consuming pretreatment steps and specialized operators, make AFB1 detection challenging. Immunoassays that utilize the specific binding of antibodies to antigens have gained widespread recognition and adoption due to their universal applicability, high sensitivity, simplicity, and low cost. Antibodies are central to molecular recognition and detection. The most widely used polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs) face several key drawbacks that need to be overcome, including large molecular weight, expensive preparation, poor reproducibility, and susceptibility to inactivation. The variable domain of a heavy-chain antibody (VHH, also known as a Nanobody) is the smallest antibody (only 15 kDa) and is a fragment of an antibody derived from camels. This novel antibody exhibits low immunogenicity, high stability, high affinity, and high specificity. Nanobodies (Nb) combine the advantages of monoclonal antibodies with superior stability and ease of expression. Due to their ease of genetic manipulation and superior soluble expression in prokaryotic systems, different bifunctional Nbs have been explored for food environmental analysis and human health diagnosis, but their applications are limited by problems such as poor specificity, sensitivity, and stability. Summary of the Invention

[0004] The purpose of the present invention is to provide a nanobody for detecting aflatoxin B1 and its application to solve the problems existing in the above-mentioned prior art. The nanobody can specifically bind to aflatoxin B1, providing a new immunological detection method for rapid, sensitive and specific detection of aflatoxin B1.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a nanobody for detecting aflatoxin B1, wherein the amino acid sequence of the nanobody is shown in SEQ ID NO: 1:

[0007] VLAALLQGVQAQVQLVDSGGGSVQAGGSLRLSCVASGYTLSNYCMGWFRQVSGKEREGVAGIWTGGGSIWYADSVKGRFTISQDKDKKTLYLQMNSLKPEDTAVYYCAARWSGSWSGCSGRDYNYWGQGTQVTVSS.

[0008] More preferably, Figure 7 As shown, the amino acid of the framework region FR1 of the nanobody is: VLAALLQGVQAQVQLVDSGGGSVQAGGSLRLS; the amino acid of the framework region FR2 of the nanobody is: WFRQVSGKEREGVAG; the amino acid of the framework region FR3 of the nanobody is: IWYADSVKGRFTISQDKDKKTLYLQMNSLKPEDTAVYY; the amino acid of the framework region FR4 of the nanobody is: QGTQVTVSS; the amino acid of the complementary determining region CDR1 of the nanobody is: CVASGYTLSNYCMG; the amino acid of the complementary determining region CDR2 of the nanobody is: IWTGGGS; the amino acid of the complementary determining region CDR3 of the nanobody is: CAARWSGSWSGCSGRDYNYWG.

[0009] The present invention also provides a gene encoding the nanobody, the nucleotide sequence of which is shown in SEQ ID NO: 2:

[0010] GTCCTGGCTGCTCTTCTACAAGGTGTCCAGGCTCAGGTGCAACTGGTGGACTCTGGGGGGGGCTCAGTGCAGCTGGAGGGTCTCTGAGACTCTCCTGTGTAGCCTCTGGATACACCTTGAGTAAC TACTGCATGGGCTGGTTCCGCCAGGTTTCAGGGAAGGAGCGCGAGGGGGTCGCAGGTATTTGGACTGGCGGTGGTAGCATATGGTATGCCGACTCCGTGAAGGGCCGATTCACCATCTCTCAAGAC AAGGACAAGAAGACGCTGTATCTGCAAATGAACAGCCTGAAACC TGAGGACACTGCCGTGTATTACTGTGCGGCAAGGTGGTCAGGCTCTTGGTCTGGGTGTTCAGGAAGGGACTATAAC TACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA CTCGAG. (The underlined constant region gene sequence corresponding to the nanobody).

[0011] The present invention also provides a recombinant vector comprising the gene.

[0012] The present invention also provides a host cell comprising the recombinant vector.

[0013] The present invention also provides a kit for detecting the nanobody, wherein the kit detects aflatoxin B1 based on an indirect ELISA method, and the kit uses the nanobody according to claim 1 as a detection antibody.

[0014] The present invention also provides a method for detecting aflatoxin B1, comprising the following steps:

[0015] (1) Aflatoxin B1 hapten is coupled to a carrier protein to obtain aflatoxin B1 complete antigen, and the aflatoxin B1 complete antigen is used as a detection antigen;

[0016] (2) adding the sample to be tested and the nanobody to the solid phase carrier coated with the detection antigen, discarding the liquid after sufficient reaction and washing;

[0017] (3) After adding enzyme-labeled secondary antibody and fully reacting, the liquid is discarded and washed, and then a color reaction is performed. The reaction is terminated and the OD value is measured at 450 nm. The content of aflatoxin B1 can be obtained based on the OD value.

[0018] Preferably, the carrier protein is bovine serum albumin, and the enzyme-labeled secondary antibody is an HRP-labeled secondary antibody.

[0019] The present invention also provides the use of the nanobody in preparing an immunological detection kit for aflatoxin B1.

[0020] Preferably, the immunological detection kit uses aflatoxin B1 complete antigen obtained by coupling aflatoxin B1 hapten with a carrier protein as the detection antigen, and the nanobody is the detection antibody.

[0021] The present invention discloses the following technical effects:

[0022] The present invention immunizes Bactrian camels and uses the immunized Bactrian camel lymphocytes to establish an antibody gene library. This antibody gene library has good nano-antibody gene diversity. By screening this antibody gene library, the present invention obtains a nano-antibody against aflatoxin B1. This nano-antibody has high sensitivity and specificity, can be stored and used for a long time at room temperature, is simple to operate, and takes a short time. It has good application prospects and broad development space in the rapid and effective detection of aflatoxin B1. This nano-antibody can be prepared in large quantities by genetic engineering recombinant expression. After the nano-antibody of the present invention is expressed in prokaryotes, it is subjected to immunological detection and analysis in the form of protein. Through this immunological test and analysis, a rapid, sensitive and stable method for detecting aflatoxin B1 is established. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 The electrophoresis test results of the first round of PCR amplification products; 1. Amplification product; M. marker;

[0025] Figure 2 The electrophoresis test results of the second round of PCR amplification products; 1. Amplification product; M. marker;

[0026] Figure 3 To count the number of colonies on the culture dish;

[0027] Figure 4 For enzyme-linked immunosorbent assay to identify positive clones;

[0028] Figure 5 Draw a standard curve of aflatoxin B1 standard for indirect competitive ELISA;

[0029] Figure 6 Standard curves for different aflatoxin B1 analogs and metabolites;

[0030] Figure 7 Schematic diagram of the amino acid sequence of nanobodies. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Example 1 Construction of Bactrian camel immune antibody library

[0037] 1. Experimental methods

[0038] 1.1 Construction of aflatoxin B1 nanoantibody gene library

[0039] (1) Double hump immunization method

[0040] Healthy Bactrian camels were immunized with a complete antigen, aflatoxin B1-BSA (purchased from Shandong Ludu Biotechnology Co., Ltd.), administered subcutaneously in the neck of the camels. Each immunization dose was 0.5 mg. The first immunization was emulsified with 0.5 mL of complete Freund's adjuvant and the antigen. Subsequent booster immunizations were emulsified with 0.5 mL of incomplete Freund's adjuvant and the antigen. Each booster immunization was repeated two weeks apart for a total of four immunizations.

[0041] Starting from the second immunization, 1 mL of Bactrian camel blood was collected one week after each immunization to separate serum for immune response testing. One week after the third and fourth immunizations, 20 mL of Bactrian camel peripheral blood was collected to separate lymphocytes for later use.

[0042] (2) Monitoring of immune response

[0043] The immune response was monitored using the indirect ELISA method. The specific procedures are as follows:

[0044] Immobilization of the detection antigen: Using the complete antigen aflatoxin B1-BSA as the detection antigen and BSA as the control antigen, dilute the coating solution (0.375g Na2CO3 and 0.7325g NaHCO3, dilute to 250mL) to 1μg / mL. Add 100μL per well to a 96-well ELISA plate and incubate at 4°C overnight. The next day, wash the plate three times with a 20-fold dilution of PBST (0.01M PBS, 0.05% Tween-20) and pat dry on absorbent paper. Block the plate with 160μL of 3% skim milk powder in PBS (w / v) per well and incubate at 37°C for 1 hour. Pour the liquid from the wells and wash three times with a 20-fold dilution of PBST (0.01M PBS, 0.05% Tween-20). Pat dry on absorbent paper and use for ELISA testing. Alternatively, place the plate in a sealed bag and store at 4°C.

[0045] Immunoreaction: Dilute Bactrian camel serum 100-fold, 1,000-fold, 10,000-fold, and 100,000-fold. Add the diluted serum to the titer column and control wells in descending order of concentration, adding 100 μL of each concentration to one titer well and one control well. Bring the final two wells to a total volume of 100 μL with PBS to serve as a blank control. After mixing, incubate at 37°C for 60 minutes. Wash the plate three times with 20-fold diluted PBST (0.01 M PBS, 0.05% Tween-20) and pat dry on absorbent paper.

[0046] Add enzyme-labeled secondary antibody: Anti-VHH-HRP secondary antibody diluted 3000 times with PBST, add 100 μL to each well, incubate at 37°C for 60 min, wash the plate three times with 20-fold diluted washing solution PBST (0.01 M PBS, 0.05% Tween-20), and pat dry on absorbent paper.

[0047] TMB substrate color development reaction and termination: Mix equal volumes of TMB substrate solution A and solution B in advance, add 100 μL to each well, incubate at 37°C for 3-5 minutes, and add 50 μL H2SO4 to each well to terminate the reaction.

[0048] Reading and data analysis: The absorbance value (OD) at 450 nm was read using a microplate reader. 450nm ), based on the immune response results, lymphocytes with the highest titer and the lowest control binding were selected for the preparation of nanobodies.

[0049] (3) Isolation of Bactrian camel lymphocytes

[0050] Mix whole blood from a Bactrian camel with an equal volume of normal saline in a 1:1 ratio to create diluted blood, and store at room temperature. Add 20 mL of lymph separation fluid to a sterile 50 mL centrifuge tube and slowly add 20 mL of diluted blood along the tube wall using a sterile Pasteur pipette. Centrifuge at 3000 g for 20 minutes. Transfer the lymphocyte layer to a new 50 mL centrifuge tube and centrifuge at 3000 g for 15 minutes at 4°C. Discard the supernatant. Add lysis buffer (TRNsol) to each lymphocyte aliquot, and aliquot 1 mL into 2 mL centrifuge tubes. Store at -80°C until needed.

[0051] (4) Extraction of total RNA

[0052] Add 0.2 mL of chloroform per 1 mL of the lysate. Cap the centrifuge tube, shake vigorously for 15 seconds, and incubate on ice for 5 minutes. Centrifuge at 12,000 rpm for 10 minutes at room temperature. Transfer no more than 80% of the upper aqueous phase to a new centrifuge tube, slowly add 0.7 volumes of anhydrous ethanol, and mix thoroughly. Transfer the resulting solution and precipitate to a GBC adsorption column, centrifuge at 12,000 rpm for 30 seconds, and discard the waste solution. Add 500 μL of Wash Buffer I to the GBC adsorption column, centrifuge at 12,000 rpm for 1 minute, and discard the waste solution. Add 600 μL of Wash Buffer II to the GBC adsorption column, centrifuge at 12,000 rpm for 30 seconds, and discard the waste solution. Centrifuge at 12,000 rpm for 1 minute, discard the waste solution, and open the cap to air dry any remaining rinse solution in the adsorption column at room temperature. Transfer the GBC adsorption column to a new centrifuge tube, add 30-100 μL of ddH2O, incubate at room temperature for 2 minutes, and then centrifuge at 12,000 rpm for 1 minute at 4°C. Collect the liquid in the tube and store it at -80℃.

[0053] (5) cDNA synthesis

[0054] Using RNA as a template, cDNA was synthesized according to the instructions of Takara's first-strand reverse transcription kit. The specific method is as follows:

[0055] PrimeScript cDNA kit TM IV 1st strand cDNA Synthesis Mix (purchased from TaKaRa), according to the cDNA synthesis reaction system shown below, the reagents were mixed in a nuclease-free centrifuge tube and operated on ice;

[0056] Table 1 Reaction system for cDNA synthesis

[0057] Reagents volume RNA 15 μl Radom 6mers 2 μl 5×PrimeScript Iv cDNA synthesis Mix 4 μl

[0058] The above reaction system was incubated at 30°C for 10 min, incubated at 42°C for 15 min, incubated at 95°C for 5 min, cooled in an ice water bath, and frozen at -20°C.

[0059] (6) Amplification of the target gene VHH of the nanobody

[0060] Primer (5'-3'):

[0061] Call-F:GTCCTGGCTGCTCTTCTACAAGG;

[0062] Call-R:GGTACGTGCTGTTGAACTGTTCC;

[0063] VHH-F:cggggtaccccGTCCTGGCTGCTCTTCTACA;

[0064] VHH-R:tccccccgggTGAGGAGAYGGTGACCWGGGT.

[0065] Degenerate primers: Y = C / T; W = A / T

[0066] The first round of PCR uses cDNA as its PCR template. The specific reaction parameters are as follows:

[0067] Table 2 First round PCR reaction system

[0068]

[0069]

[0070] The reaction conditions were as follows: 98°C for 30s; 98°C for 10s, 55°C for 30s, 72°C for 30s, 36 cycles; 72°C for 5min; 4°C forever.

[0071] The first PCR amplification product has a product band at 750bp (such as Figure 1 As shown), the glue is recycled.

[0072] The second round of PCR uses the product recovered from the first round of PCR as a template for the second round of PCR amplification. The specific reaction parameters are as follows:

[0073] Table 3 Second round PCR reaction system

[0074] Reagents volume 750bp template 2 μl VHH-F 1 μl VHH-R 1 μl 2×Q5 High-Fidelity 2×Master Mix 25 μl <![CDATA[Sterile H2O]]> 21 μl

[0075] The reaction conditions were: 98°C for 30 seconds; 98°C for 10 seconds, 55°C for 30 seconds, 72°C for 30 seconds, 36 cycles; 72°C for 5 minutes; 4°C forever.

[0076] The second PCR amplification product has a product band at 450bp (such as Figure 2 As shown), the glue is recycled.

[0077] (7) Construction of anti-AFB1 nanoantibody gene library

[0078] ① Enzyme digestion of VHH target gene and vector

[0079] The VHH target gene and pCantab 5E vector were digested using X-MAI and KpnI-HF enzymes at 37°C for 12 hours.

[0080] The VHH gene and pCantab 5E vector digestion products were directly cleaned and recovered using a DNA recovery kit.

[0081] ② Ligation of enzyme digestion products

[0082] The vector pCantab 5E and the VHH fragment were mixed (molar ratio 1:3), reacted at 16°C for 12 h, and then cleaned and recovered using a DNA recovery kit.

[0083] ③Electroshock conversion

[0084] Add 5 μL of the ligation product to 50 μL of electrocompetent E. coli TG1, mix gently, and transfer to a 0.2 cm cuvette for electroporation (1.8 kV). Immediately after electroporation, add 3 mL of SOC medium preheated to 37°C to the cuvette and shake at 250 rpm at 37°C for 1 hour to revive the cells. Prepare a serial dilution series of 10 μL of the recovered culture. Spread 50 μL of each serial dilution series onto a 90 mm diameter 2YT-Amp dish, which will serve as a counting plate. Incubate at 37°C overnight. Incubate the remaining undiluted culture at 250 rpm at 37°C overnight.

[0085] Count the number of colonies on the culture dish (such as Figure 3 Calculate the total number of bacteria in the resuscitated bacterial solution and perform multiple electric shock transformations until the total number of transformed colonies reaches 10 7 This number is the library capacity of the nanobody gene library.

[0086] The genetically modified Escherichia coli cultured overnight was added to glycerol (v / v) with a final concentration of 18%, and frozen at -20°C to obtain the aflatoxin B1 nanoantibody gene library.

[0087] 1.2 Phage rescue

[0088] Inoculate cells 10 times larger than the reservoir volume in 200 mL 2YT (Amp) and culture at 37°C, 250 rpm until OD 600Approximately 0.4 to 0.6; add helper phage M13K07 (20:1 multiplicity of infection), incubate at 37°C for 30 minutes, then incubate at 250 rpm for 30 minutes. Add kanamycin (1:1000) and incubate overnight at 30°C and 250 rpm. Centrifuge at 12,000 rpm for 15 minutes at 4°C, remove the supernatant, add 1 / 5 volume of PEG / NaCl (100 g PEG8000 and 73.05 g sodium chloride, dilute to 500 mL with water), and incubate on ice for 1 hour. Centrifuge at 10,000 rpm for 15 minutes at 4°C, discard the supernatant, resuspend the pellet in 1 mL of PBS, transfer to a 2 mL centrifuge tube, and centrifuge at 12,000 rpm for 15 minutes at 4°C. Take 10 μL of the aliquot for volume determination, and add the remainder to a final concentration of 20% glycerol and store at -80°C.

[0089] The immunogen was used to immunize Bactrian camels, and the detection antigen was used to monitor the immune response. A total of four immunizations were conducted on Bactrian camels. The immune responses are shown in Table 4. Starting from the second immunization, the titer of Bactrian camel serum increased significantly compared to the negative serum before immunization, reaching a titer of 1:1000. Therefore, blood from the fourth immunization was collected for library construction.

[0090] Table 4 Immune response

[0091] Serum dilution multiple AFB1-BSA BSA 10 times 2.791 0.133 100 times 2.622 0.059 1000 times 0.67 0.046 10000 times 0.13 0.046 100000 times 0.054 0.046 Negative control 0.046 0.049

[0092] 1.3 Affinity panning of nanobodies

[0093] Aflatoxin B1-BSA was diluted to 10 μg / mL using the coating solution and added to the microwells of the ELISA plate, 100 μL per well, and allowed to stand at 4°C overnight. The next day, the plate was washed three times with PBST (0.01M PBS, 0.05% Tween-20), and 160 μL of 3% skim milk powder-PBS (w / v) solution was added to each well and allowed to stand at 37°C for 1 hour. The liquid in the wells was poured out, and the plate was washed three times with PBST (0.01M PBS, 0.05% Tween-20), patted dry on absorbent paper, and stored at 4°C until use. BSA was added to the above-mentioned aflatoxin B1 nanobody phage library to a final BSA concentration of 1% (w / v). The phage library (w / v) containing 1% BSA was added to three microwells with immobilized antigen, 100 μL was added to each well, and incubated at 37°C for 1 hour. Unbound phage in the wells were discarded and the microwells were washed 10 times with PBST. The wells were incubated with 100 μL of glycine (0.2 M) and shaken for 30 minutes at 37°C. The elution reaction was then performed with 100 μL of Tris-HCl solution and shaken for 5 minutes. The liquid in the microwells was collected into a sterile centrifuge tube. The phage at this point is called "bound output," and the first round of screening is complete. 10 μL of eluted phage was titered, and the remainder was used to infect 20 mL of E. coli TG1 strain grown to the logarithmic phase for amplification. The second round of panning was performed in the same manner as the first round. In the third and fourth rounds of panning, 100 μL of 50 ng / mL AFB1 standard was used for competitive elution. The wells were incubated with shaken for 1 hour at 37°C. The liquid in the microwells was collected into a 1.5 mL sterile centrifuge tube. The phage at this point is called "competitive output." The above screening steps were carried out for a total of 4 rounds, and the coating antigen concentration in rounds 1-4 was reduced from 10ug / mL to 5ug / mL, 2.5ug / mL, and 1.25ug / mL.

[0094] 1.4 Identification of positive clones

[0095] Positive phage clones were identified using an indirect enzyme-linked immunosorbent assay (ELISA). The specific method was as follows: 96 single colonies were randomly selected from the output titer measurement plate of the fourth round of competitive elution, inoculated into 96-well plates containing 200 μL of 2YT-Amp per well, and incubated overnight at 37°C to serve as the bacterial culture "master plate."

[0096] Take out 10 μL of bacterial solution from each well of the mother plate and inoculate it into another 96-well deep-well plate with 100 μL of 2YT-Amp in each well. The inoculated well numbers should correspond to those of the mother plate. Incubate at 37°C, 180 rpm for 1.5 h. Dilute M13K07 phage and add it to the wells. Incubate at 30°C, 180 rpm overnight. Store the mother plate at 4°C for later use.

[0097] For antigen immobilization, dilute AFB1-BSA to 10 μg / mL using coating solution. Simultaneously, dilute BSA standard to 10 μg / mL, add 100 μL per well, and incubate at 4°C overnight. The next day, wash the microwells three times with PBST, pat dry, and add 160 μL of 3% skim milk powder (w / v) diluted in PBS to each well. Block at 37°C for 1 hour, discard the blocking solution, wash the microwells three times with PBST, pat dry, and store at 4°C until ready to use.

[0098] ELISA positive clones:

[0099] Centrifuge the deep-well plate at 4000 rpm for 10 minutes. Remove the ELISA plate containing the immobilized antigen and aspirate the supernatant from the centrifuged 96-well plate. Add 100 μL of the supernatant to each numbered ELISA well. Incubate at 37°C for 60 minutes, wash three times with PBST, and pat dry. Dilute the Anti-M13-HRP secondary antibody 4000-fold with PBS, add 100 μL of the solution to each well, and incubate at 37°C for 60 minutes. Wash three times with PBST (0.01 M PBS, 0.05% Tween-20), pat dry, and add 100 μL of TMB colorimetric solution (previously mixed with equal volumes of Colorimetric Solution A and Colorimetric Solution B) to each well. Develop at 37°C for 10 minutes. Terminate the reaction by adding 50 μL of 10% H2SO4 (v / v) stop solution. Measure the absorbance at 450 nm using a microplate reader.

[0100] The deep-well plate was centrifuged at 4000 rpm for 10 minutes. The ELISA plate containing the immobilized antigen was removed. The supernatant from the centrifuged 96-well plate was aspirated and 50 μL of the solution was added to each well of the corresponding numbered ELISA well. A 50 μL AFB1 standard (ng / mL) was also added. Incubate at 37°C for 60 minutes, wash three times with PBST, and pat dry. Anti-M13-HRP secondary antibody was diluted 4000-fold with PBS and 100 μL was added to each well. Incubate at 37°C for 60 minutes. Wash three times with PBST, pat dry, and add 100 μL of TMB colorimetric solution (previously mixed with equal volumes of colorimetric solution A and colorimetric solution B) to each well. Develop at 37°C for 10 minutes. The reaction was terminated by adding 50 μL of 10% H2SO4 (v / v) stop solution. The absorbance at 450 nm was measured using a microplate reader.

[0101] The absorbance value measured by enzyme-labeled Figure 4 The inhibition rate of each positive clone can be calculated using the following formula. Select clones in the deep-well plate whose OD value is 3 times greater than that of the negative control well and that exhibit significant inhibition (inhibition rate > 20%). Record the corresponding well number and transfer the bacterial solution from the corresponding well in the master plate to a sterile centrifuge tube. Add glycerol and freeze for later use.

[0102] The phage clone of the nanobody obtained through indirect competitive ELISA was sent to a sequencing company for gene sequencing. Based on the DNA sequencing results and the codon table, the amino acid sequence of the nanobody was obtained. The results showed that a nanobody against aflatoxin B1 was obtained and named Nb10E.

[0103] Example 2 Preparation of Nanobody Nb10E

[0104] Nanobody Nb10E was prepared by protein expression. The specific method was as follows: the obtained phage clone of nanobody Nb10E was used to extract its plasmid using a kit, and the plasmid was chemically transformed into E. coli Trans B (DE3). A single colony was selected from the transformation plate and inoculated into 5 mL of 2YT (Amp) medium and incubated at 37°C, 220 rpm for 6 hours. The culture was inoculated into 200 mL of 2YT (Amp) medium and incubated at 37°C, 250 rpm until the OD reached 0. 600 When the pH value was approximately 0.4-0.6, IPTG was added to a working concentration of 0.1 mM and cultured overnight at 37°C, 250 rpm. The next day, the cells were centrifuged at 8000 g for 10 minutes at 4°C. The pellet was collected and disrupted by ultrasonication. The cell wall was then centrifuged at 8000 g for 10 minutes. The supernatant was collected and purified on a nickel gravity column to obtain the nanobody Nb10E.

[0105] Example 3 Determination of activity and sensitivity of nanobody Nb10E

[0106] (1) Dilute the complete aflatoxin B1-BSA antigen to 10 μg / mL using the coating solution. Simultaneously, set up a control group with a 10 μg / ml BSA standard solution and add 100 μL per well to the microwells of the ELISA plate. Incubate at 4°C overnight. The next day, wash the plate twice with PBST, then add 160 μL of 3% skim milk powder (w / v) solution to each well and incubate at 37°C for 1 hour. Pour out the liquid in the wells, wash the plate twice with PBST, pat dry on absorbent paper, and store at 4°C for later use.

[0107] After purification, the nanoantibody Nb10E was serially diluted with PBS (10, 100, 1000, 5000, 10000 times) to obtain a series of nanoantibodies at different concentrations. 100 μL of the serially diluted antibody was first added to each well of the ELISA plate. Similarly, the nanoantibody at the same dilution factor was added to the wells immobilized with BSA. A blank control (100 μL PBS) was also used in one well. The plate was incubated at 37°C for 60 minutes, washed three times with PBST, and patted dry on absorbent paper before adding 100 μL of anti-VHH-HRP secondary antibody diluted 3000 times with PBST to each well. Incubate at 37°C for 60 min, wash the plate three times with PBST, pat dry on absorbent paper, add 100 μL of TMB colorimetric solution to each well, incubate at 37°C in the dark for 10 min, add 50 μL of 10% H2SO4 (v / v) stop solution to each well, and read the OD value at 450 nm on a microplate reader.

[0108] (2) Determine the working concentration of antibodies and antigens:

[0109] Aflatoxin B1-BSA complete antigen was diluted with coating solution to different concentrations (10, 5, 2.5, 1.25, 0.625, 0.3125 μg / mL) and added to the microwells of the ELISA plate, 100 μL per well, and incubated at 4°C overnight. The next day, the plate was washed twice with PBST, and 160 μL of 3% skim milk powder (w / v) solution was added to each well and incubated at 37°C for 1 hour. The liquid in the wells was poured out, and the plate was washed twice with PBST, patted dry on absorbent paper, and stored at 4°C until use. The purified nanobody Nb10E was serially diluted with PBS (1000, 2500, 4000, 6000, 8000, 10000 times) to obtain a series of nanobody concentrations. 100 μL of serially diluted antibody was added to each well of the ELISA plate. Similarly, the nanoantibody with the same dilution was added to the wells immobilized with BSA. A blank control (100 μL PBS) was also used in one well. The plate was incubated at 37°C for 60 min, washed three times with PBST, and patted dry on absorbent paper. 100 μL of anti-VHH-HRP secondary antibody diluted 3000-fold with PBST was added to each well. The plate was incubated at 37°C for 60 min, washed three times with PBST, and patted dry on absorbent paper. 100 μL of TMB colorimetric solution was added to each well. The plate was incubated at 37°C in the dark for 10 min. 50 μL of 10% H2SO4 (v / v) stop solution was added to each well, and the OD value was read at 450 nm on a microplate reader.

[0110] OD 450nm The antibody concentration between 1 and 1.5 is the working concentration of the nanobody. Under the above experimental conditions, the working concentration of Nb10E is 1000 times diluted and the working concentration of the antigen is 2.5 μg / ml.

[0111] (3) Drawing the standard curve of indirect competitive ELISA

[0112] Aflatoxin B1 standard was diluted with 10% methanol in PBS to create a series of different aflatoxin B1 concentrations. 50 μL of the solution was added to a microplate plate, with three replicates of each concentration tested. Three blank wells (50 μL of PBS) were also prepared. Nanobody Nb10E was diluted 500-fold with PBS to a working concentration, and 50 μL of the diluted antibody was added to each well. The plate was incubated at 37°C for 30 minutes, washed three times with PBST, and patted dry on absorbent paper. 100 μL of anti-VHH-HRP secondary antibody diluted 5000-fold in PBST was added to each well. The plate was incubated at 37°C for 60 minutes, washed three times with PBST, and patted dry on absorbent paper. 100 μL of TMB colorimetric solution was added to each well. The plate was incubated at 37°C in the dark for 10 minutes. 50 μL of 10% H₂SO₄ (v / v) stop solution was added to each well, and the OD value was read at 450 nm on a microplate reader. The OD of the drug blank group 450 The average value of the values ​​was recorded as B0, and the OD values ​​at different drug concentrations were recorded as 450 The mean value was recorded as Bx, and the Bx / B0 ratio at different drug concentrations and the standard deviation of each replicate data set were calculated using Excel. An indirect competitive standard curve was constructed using a scatter plot in Origin software, with drug concentration as the horizontal axis and the Bx / B0 ratio as the vertical axis, and a logistic function fit was performed.

[0113] The standard curve of the indirect competitive ELISA established based on the antibody Nb10E is shown in the figure below. Figure 5 As shown in the figure, it can be seen that the standard curve is S-shaped, with good linear correlation, and the detection range is 0.592ng / mL to 13.711ng / mL, IC 50 It is 3.657ng / mL, which can meet the maximum limit detection requirements in my country.

[0114] Prepare standard solutions of various aflatoxin B1 analogs, use Nb10E to detect gradient concentrations of various aflatoxin B1 analogs, and draw standard curves for each aflatoxin B1 analog and metabolite. The results are as follows: Figure 6 As shown, the nanobody Nb10E can specifically recognize aflatoxin B1.

[0115] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A nanobody for detecting aflatoxin B1, characterized in that: The amino acid sequence of the nanobody is shown in SEQ ID NO:

1.

2. A gene encoding a nanobody for detecting aflatoxin B1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

3. A recombinant vector comprising the gene according to claim 2. A host cell comprising the recombinant vector according to claim 3 .

5. A method for detecting aflatoxin B1 for non-diagnostic purposes, characterized in that: The following steps are involved: (1) Aflatoxin B1 hapten is coupled to a carrier protein to obtain aflatoxin B1 complete antigen, and the aflatoxin B1 complete antigen is used as a detection antigen; (2) adding the sample to be tested and the nanobody according to claim 1 to the solid phase carrier coated with the detection antigen, discarding the liquid after sufficient reaction, and washing; (3) After adding enzyme-labeled secondary antibody and fully reacting, the liquid is discarded and washed, and then a color reaction is performed. The reaction is terminated and the OD value is measured at 450 nm. The content of aflatoxin B1 can be obtained based on the OD value.

6. The method according to claim 5, wherein The carrier protein is bovine serum albumin, and the enzyme-labeled secondary antibody is an HRP-labeled secondary antibody.

7. Use of the nanobody according to claim 1 in the preparation of an immunological detection kit for aflatoxin B1.

8. The use according to claim 7, characterized in that The immunological detection kit uses aflatoxin B1 complete antigen obtained by coupling aflatoxin B1 hapten with a carrier protein as the detection antigen, and the nanobody is the detection antibody.

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