CN113637080A - AFM1 anti-idiotype nano-antibody substituted antigen-based VHH-ELISA method - Google Patents

Abstract

The invention belongs to the field of molecular biology, and particularly relates to a VHH-ELISA method based on AFM1 anti-idiotype nano antibody substituted antigen. The invention adopts VHH C4 as a substitute for the VHH-ELISA established by the coating antigen to carry out the sample addition recovery experiment, which shows that the aflatoxin M₁The anti-idiotype nano antibody replaces the antigen VHH-ELISA method, has accurate and reliable result, and is an effective and feasible immunoassay method. Can be applied to the pollution detection of aflatoxin M1.

Description

AFM1 anti-idiotype nano-antibody substituted antigen-based VHH-ELISA method

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to aflatoxin M₁Anti-idiotype nano antibody as aflatoxin M₁Use of an antigen substitute.

Background

Aflatoxins are metabolites produced by aspergillus flavus or aspergillus parasiticus that contaminate a variety of agricultural products and are found during the growth, harvesting, storage and processing of agricultural crops. These metabolites are highly toxic and, therefore,mutagenic, teratogenic and carcinogenic compounds have been identified as causative agents of carcinogenesis in human liver and extrahepatic. Aflatoxin B₁(AFB₁) The highest toxicity is classified as class 1 human carcinogen by the international agency for research on cancer. AFM₁Is AFB₁The hydroxylated metabolite of (a). Ingestion quilt AFB₁AFM will be produced by lactating animals of contaminated feed₁Is excreted into milk and can therefore be found in a wide variety of milk products. AFM₁Also has toxic and carcinogenic effects and has been classified as a class 1 carcinogen. Due to AFM₁The risk of contamination to human health, particularly to infants and children, has become a global concern. AFM in milk by European Union₁The limit of (2) is 0.05 ng/mL. However, the standards for the definitions in China, the United states and Brazil are all 0.5 ng/mL.

Many applications for AFM have been previously established₁And (3) a detection method. High Performance Liquid Chromatography (HPLC) and Fluorescence Detection (FD) have successfully replaced Thin Layer Chromatography (TLC), and remain one of the most widely used methods. Currently, liquid chromatography tandem mass spectrometry (LC-MS/MS) and electrospray ionization quadrupole time-of-flight mass spectrometry have been developed. All these steps rely on expensive, well-equipped laboratories and several hours, thereby weakening them in AFM₁Application in detection. On the other hand, enzyme-linked immunosorbent assays (ELISAs) are becoming increasingly popular due to their low cost and ease of application. The immunological method is suitable for quickly detecting aflatoxin. The ELISA detection method needs less samples, and is a high-throughput AFM₁Conventional analytical methods of (3). Due to AFM₁Since small molecules cannot be used alone as antigens, they are often used as complete antigens after being coupled with Bovine Serum Albumin (BSA), Ovalbumin (OVA), or the like. However, the synthesis of complete antigens is complicated and expensive, requiring the use of large quantities of AFM₁The standard substance not only poses great threat to operators, but also possibly causes secondary pollution to the environment. Thus, the proposal of Anti-Idiotype antibodies (Anti-Idiopype) may provide a solution for this purpose, the Anti-Idiotype antibodies recognizing the variable regions of the Idiotype antibodies mimicking the three-dimensional structure of antigens expressing Jerne and mimics the antigen conformation, so that anti-idiotype antibodies can be used as AFMs₁Antigen substitutes were developed to develop a safe format for ELISA.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide the AFM 1-based anti-idiotypic nanobody for replacing aflatoxin M₁ELISA immunoassay method of the standard substance.

In order to achieve the purpose, the invention adopts the technical scheme that:

providing aflatoxins M₁Anti-idiotype nano antibody as aflatoxin M₁Application of antigen substitute, aflatoxin M₁The anti-idiotype nano antibody is aflatoxin M₁The amino acid sequence of the anti-idiotype nano antibody VHH C4 is shown in SEQ ID NO. 1.

According to the scheme, the aflatoxin M is coded₁The nucleotide sequence of the anti-idiotype nano antibody amino acid is shown in SEQ ID NO. 2.

AFM 1-based anti-idiotype nano antibody for replacing aflatoxin M₁The ELISA immunoassay method of the standard substance comprises the following steps: subjecting aflatoxin M₁Anti-idiotype nano antibody VHH C4 replacing aflatoxin M₁Antigen coating enzyme label plate, and detecting the sample (containing aflatoxin M)₁Sample of (1) as a competitor to perform a competitive inhibition ELISA reaction, and detecting to obtain a binding rate B/B₀Value based on aflatoxin M₁Concentration-binding ratio B/B₀Obtaining the aflatoxin M by a value standard curve₁The concentration of (c).

According to the scheme, the competitive inhibition ELISA reaction is specifically as follows: subjecting aflatoxin M₁Diluting the anti-idiotype nano antibody VHH C4, coating the diluted anti-idiotype nano antibody on an enzyme label plate, and coating the enzyme label plate overnight at 4 ℃; the next day, sealing with sealing solution, and incubating at 37 deg.C for 1 h; adding methanol/PBS buffer solution into the enzyme labeling hole, and adding aflatoxin M into the enzyme labeling hole₁Diluting the monoclonal antibody and the competitor by PBS7.4 buffer solution, adding the diluted monoclonal antibody and the competitor into an enzyme-labeled hole, and incubating for 1h at 37 ℃; adding horse radish peroxidase labeled goat anti-mouse antibody,incubating at 37 ℃ for 1 h; adding color developing solution, reacting at 37 deg.C for 15min, and measuring OD₄₅₀Obtaining a binding rate B/B₀The value is obtained.

According to the scheme, the aflatoxin M₁The coating concentration of the anti-idiotype nano antibody is 20 mu g/mL-1.25 mu g/mL.

According to the scheme, the blocking solution is 3% BSA, 1.5% OVA or 3% skimmed milk powder.

According to the scheme, the pH value of the methanol/PBS buffer solution is 7.0-7.4.

According to the scheme, the concentration of methanol in the methanol/PBS buffer solution is 5-10%.

According to the scheme, the sample to be detected (containing aflatoxin M)₁Sample of (d) is a dairy product, including yogurt, milk or milk powder, and the like.

The invention provides aflatoxins M₁Anti-idiotype nano antibody VHH C4 as aflatoxin M₁Use of an antigen substitute. Provides a VHH-ELISA method based on the nano antibody to replace the antigen, and simultaneously optimizes the coating concentration and the aflatoxin M used in the VHH-ELISA method based on the nano antibody to replace the antigen₁Working concentration of monoclonal antibody, blocking reagent, pH, methanol concentration.

The invention has the following beneficial effects:

the invention provides aflatoxins M₁Anti-idiotype nano antibody VHH C4 as aflatoxin M₁The application of antigen substitute and the establishment of aflatoxin M₁Anti-idiotype VHH-ELISA method based on nanobody replacement antigen. The invention selects the sample to determine the aflatoxin M₁The addition recovery rate of the anti-idiotype nano antibody VELISA is 84.1-119.1%, the accuracy is good, and the aflatoxin M is proved₁The anti-idiotype nano antibody replaces the antigen VHH-ELISA method, has accurate and reliable result, and is an effective and feasible immunoassay method.

Drawings

FIG. 1 is the test results for determining diversity of phage display nanobody immune libraries.

FIG. 2 shows positive clones identified by phase-ELISA.

FIG. 3 shows VHH C4 and anti-aflatoxin B₁(abbreviated as AFB)₁) And ochratoxin A (OTA for short) and T-2 toxin (T-2 for short) monoclonal antibodies.

FIG. 4 is a chessboard optimized VHH C4 coating concentration and aflatoxin M₁Working concentration of monoclonal antibody.

FIG. 5 is a graph of the effect of blocking reagents on VELISA.

FIG. 6 is a graph of the effect of phosphate buffer pH on VELISA.

Figure 7 is a graph of the effect of salt ion concentration on VELISA.

FIG. 8 is a graph of the effect of methanol concentration on VELISA.

FIG. 9 is a VHH C4-based VELISA on aflatoxin B₁Aflatoxins B₂Aflatoxin G₁Aflatoxin G₁And aflatoxin M₂Cross-reactive conditions of (1).

FIG. 10 VHH C4 vs AFM in different sample matrices₁The competition curve of (c).

Detailed Description

In order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.

In the following examples, the aflatoxins M₁The monoclonal antibody is secreted and produced by a hybridoma cell strain 2C9 with the preservation number of CCTCC NO. C201018, the hybridoma cell strain 2C9 is preserved in China Center for Type Culture Collection (CCTCC) at 7.13.2010, and the anti-aflatoxin M produced by the hybridoma cell strain 2C9₁Monoclonal antibodies and uses thereof, application No.: 201110108230.6, respectively.

Example 1 construction of phage display Nanobody immune libraries

1. Alpaca immunization

Taking anti-aflatoxin M₁Monoclonal antibody (dissolved in PBS7.4) 200. mu.g, emulsified with an equal volume of Freund's incomplete adjuvant, to a three-year-old male alpaca (Alpa)ca) were injected subcutaneously in multiple spots, and then immunized every two weeks for 8 times, and blood sampling was started after quadruplicate immunization. And 7-10 days after the fourth immunization, collecting 10mL of blood by using an EDTA vacuum blood collection tube jugular vein, simultaneously, slightly reversing the blood collection tube to avoid blood coagulation, treating the blood by using a filter in a LeukoLOCK kit, sequentially washing the filter by using 3mL of LPBS buffer solution and 3mL of RNAlater buffer solution, retaining the needed leucocytes in the filter, and finally sealing and storing the filter at-80 ℃ for RNA extraction.

2. Total RNA extraction

Total RNA in alpaca blood was isolated according to the manual of the LeukoLOCK total RNA extraction kit of Life Technology, as follows:

(1) adding 70 mu LpH of adjusting buffer solution into 2.5mL of lysis/binding solution for use; (2) the sealed filter was allowed to stand to return to room temperature, the filter cap was opened, and the residual RNAlater buffer in the filter was flushed out with a 2mL syringe; (3) 2.5mL of the prepared lysis/binding buffer solution is sucked by a 2.5mL syringe to wash the filter once, and the effluent is collected by a 15mLRNAse-free centrifuge tube; (4) addition of nuclease-free ddH₂O2.5 mL, vortex and mix evenly, add 25 uL proteinase K, shake the centrifuge tube for 5min at room temperature 250 g; (5) taking out the RNA-bonded magnetic beads stored at 4 ℃, uniformly mixing by vortex, absorbing 50 mu L of the mixture into the centrifuge tube, vortexing the centrifuge tube, adding 2.5mL of isopropanol into the centrifuge tube, and shaking for 5min at room temperature; (6) centrifuging the centrifuge tube at 3200g for 3min, carefully sucking out the supernatant, and discarding, taking care not to suck the precipitated magnetic beads; (7) adding 600 mu L of lotion I, repeatedly blowing and beating the precipitated magnetic beads to uniformly disperse the magnetic beads, transferring the suspension to a 1.5mL centrifuge tube, rinsing the 15mL centrifuge tube once with 600 mu L of lotion I, and transferring to the same 1.5mL centrifuge tube; (8) centrifuging a 1.5mL centrifuge tube at 16000g for 30s, and carefully discarding the supernatant; (9) adding 750 mu L of washing solution 2/3, violently vortexing and shaking for 30s to disperse the aggregated and precipitated magnetic beads, centrifuging 16000g for 30s to collect the magnetic beads, and carefully discarding the supernatant; (10) standing the centrifuge tube in an ultra-clean bench with the cover open for 2min, volatilizing residual alcohol in the washing solution 2/3, mixing with TURBO Nase master, adding 4 μ L of LTURBO DNase (20U/. mu.L) into 296 μ L of LeukoLOCK DNase buffer, mixing wellTransferring to the centrifuge tube, repeatedly blowing and beating the precipitated magnetic beads by using a pipette gun to uniformly disperse the magnetic beads, shaking for 10min at 1000g at room temperature, and slightly reversing and mixing for several times in the midway; (11) sequentially adding 300 mu L of lysis/binding solution (without pH regulation buffer solution) and isopropanol into the centrifuge tube, mixing uniformly, inching and centrifuging for 2s, and then incubating for 3min at room temperature; (12) centrifuging at 16000g for 30s, discarding the supernatant, adding 750 μ L of lotion 2/3, violently vortexing for 30s, 16000g for 30s, discarding the supernatant, adding 750 μ L of lotion 2/3 again, violently vortexing for 30s, 16000g for 1min, discarding the supernatant as much as possible, standing at room temperature for 3min with an opening, volatilizing the residual lotion, and taking care that the solution cannot be left for too long to avoid the magnetic beads from being excessively dried; (13) adding 60 mu L of eluent, carrying out vortex oscillation for 30s, centrifuging 16000g for 2min, transferring the eluted RNA solution to another 1.5mL centrifugal tube without nuclease, reserving 1 mu LRNA solution, measuring the concentration by nanodrop, taking about 3 mu L RNA solution for agarose gel electrophoresis analysis, and immediately and completely inverting the remaining RNA solution into cDNA to prevent degradation.

Synthesis of cDNA

First strand cDNA was synthesized according to the reverse transcription kit instructions, as follows:

(1) two 200. mu.L PCR tubes without nuclease were taken, 3. mu.L of 10mM dNTPmix, 3. mu.L of 50. mu.M oligo (dT)20, and 24. mu.L of LRNA were added, and gently mixed;

(2) heating at 65 deg.C for 5min to denature the template and open the secondary structure;

(3) immediately cooling on ice for at least 1 min;

(4) pre-mix for cDNA synthesis in situ:

(5) adding 30 mu L of premix into each of the two PCR tubes, and uniformly mixing by using a micropipette;

(6) heating the reaction mixture: annealing at 50 deg.C for 50min, and heating at 85 deg.C for 5min to terminate the reaction;

(7) adding 3 mu LRNAseH into the reaction mixed solution respectively, mixing uniformly, heating at 37 ℃ for 20min, and decomposing the incompletely reacted RNA;

(8) the first strand cDNA was synthesized and stored at-20 ℃ after packaging.

4. Amplification of heavy chain antibody variable region VHH genes

Taking respective degenerate primer pairs of IgG2 and IgG3 variable region VHH genes, respectively carrying out Polymerase Chain Reaction (PCR) amplification by taking cDNA as a template, wherein the primer pair F, R2 is used for cloning an IgG2 subtype, and the primer pair F, R1 is used for cloning an IgG3 subtype, and the reaction system is as follows:

the PCR procedure was:

wherein, the upstream primers corresponding to the amplified IgG2 and IgG3 are respectively primers R2 and R1, and the primer sequences are shown in Table 1.

TABLE 1 VHH antibody Gene amplification and sequencing primer sequence Listing

5. Construction of phage display Nanobody library

Carrying out enzyme digestion on the vector pComb3X and the vector VHH by adopting SfiI, connecting the enzyme digested VHH fragment with the vector pComb3X by using T4 ligase, taking 3 mu L of a connecting product to 25 mu L of E.coli ER2738 competent cells, sucking out all the connecting product after light mixing, transferring the connecting product to a precooled electric rotating cup (the inner diameter is 1mm), and quickly placing the electric rotating cup in an electric rotating instrument for electric conversion; immediately adding 1mL of SOC culture medium preheated at 37 ℃ into an electric transfer cup after electric shock, gently sucking and uniformly mixing by using a pipette gun, transferring into a bacteria shaking tube, and performing shake recovery culture for 1h at 250g in a shaking table at 37 ℃; repeat the electricityAfter 10 transfers of 3. mu.L of the ligation product, 10 transfers of the resulting mixture were pooled and 1. mu.L of the resulting mixture was diluted 10-fold with sterile water and spread on LB-Amp plates, which were incubated overnight in an incubator at 37 ℃ for estimation of the stock volume. Transferring all the transformed bacteria to 200mLSB culture medium, adding carbenicillin to 50 μ g/mL, and tetracycline to 20 μ g/mL; 250g, cultured to OD at 37 ℃₆₀₀Is 0.6; add 1mL of helper phage (1X 10)¹³pfu/mL), standing at 37 ℃ for 30min for infection; 250g, culturing for 2h at 37 ℃, adding kanamycin to the concentration of 70 mu g/mL, and continuing to culture overnight; centrifuging the bacterial solution at 4 deg.C for 15min at 10000g the next day; taking the supernatant into a sterile centrifuge tube, adding 1/4 volumes of PEG/NaCl solution, carrying out ice bath for 2h, centrifuging again, discarding the supernatant, and adding 10mL of resuspension (containing 1 Xprotease inhibitor and 0.02% NaN)₃0.5% BSA in PBS buffer) resuspend the pellet; filtering the phage solution with a 0.22 μm filter to remove residual bacteria and the like; the obtained phage solution is a phage display nano antibody library, which is subpackaged, marked and stored at-70 ℃.

6. Identification of phage display Nanobody immune libraries

Randomly picking 30 single clones from the plate to 1mLSB culture medium, and culturing at 37 ℃ until bacterial liquid OD₆₀₀About 0.8, the bacterial solution was taken out, with gback as primer (see table 1), sent to the company for sequencing, the clone sequence was analyzed, and the diversity degree of the library was determined. As shown in FIG. 1, it can be seen from the amino acid sequences of the inserts of the selected 30 monoclonal antibodies that the conserved regions FR1, FR2, FR3 and FR4 of the nanobody have high similarity, while the variable regions CDR1, CDR2 and CDR3 are different. Particularly, the high diversity of the CDR3 region (the region from 100 th to 120 th of amino acid) ensures that the phage display library has rich diversity, which indicates that the constructed phage display nano antibody library has good diversity and can be used for subsequent screening work.

Example 2 Aflatoxin M₁Panning and identification of anti-idiotype nanobodies

(I) Aflatoxin M₁Panning of anti-idiotype Nanobodies

Aflatoxin M₁Monoclonal antibody as targetThe aflatoxin M₁The monoclonal antibody is secreted and produced by a hybridoma cell strain 2C9 with the preservation number of CCTCC NO. C201018, the hybridoma cell strain 2C9 is preserved in China Center for Type Culture Collection (CCTCC) at 7.13.2010, and the anti-aflatoxin M produced by the hybridoma cell strain 2C9₁Monoclonal antibodies and uses thereof, application No.: 201110108230.6) by reducing the concentration of the coating source, the aflatoxins M that are eluted competitively, in rounds₁Alternatively using a blocking reagent to perform affinity enrichment panning on the concentration of the standard substance to obtain the aflatoxin M₁Antibodies to monoclonal antibodies, i.e. anti-antibodies, also called anti-idiotype antibodies, were panned as follows:

(1) coating: coating aflatoxin M₁The monoclonal antibody is 50 mu g/mL, 100 mu L/hole, 6-hole coating and diluted by coating buffer; removing adsorption holes, coating 3% BSA/PBS, 300 mu L/hole, and coating 6 holes; the effect of coating at 4 ℃ over night is better than that of incubation at 37 ℃ for 2 h;

(2) and (3) sealing: 3% PBSTM, 300. mu.L/well, incubating at 37 ℃ for 2h, and washing the plate by hand for 3 times;

(3) adding a library to the coated antigen wells: reacting at 37 ℃ for 1h at a concentration of 100 mu L/hole, then reacting at room temperature for 1h by a shaking table, and then manually washing the 6 holes by using a gun head with a filter element for 10 times;

(4) and (3) elution: 100ng/mL aflatoxin M is prepared₁Reacting the standard product for 30min in a shaking table at room temperature;

(5) desorption: transferring the eluent to a desorption hole with the concentration of 100 mu L/hole, and reacting for 1h at room temperature;

(6) the 600 μ L eluate, called "1 st output", was pooled and the first round of panning was completed; 1st output needs to be amplified before it can be used for the second round of panning;

(7) the "1 st output" titer was determined by taking 10. mu.L of the "1 st output" and diluting the solution in a gradient to 10³、10⁴、10⁵10. mu.L of each of the 3 dilutions was infected with 90. mu.L of ER2738(OD 0.8), allowed to stand at 37 ℃ for 30min, plated on LB-Amp (ampicillin) plates, cultured overnight at 37 ℃ and the number of single clones on the next day of plate was counted to estimate titer.

In subsequent panning, coating antibody concentrationGradually reducing aflatoxin M in eluent₁The concentration was gradually reduced, three rounds of elutriation were performed, the elutriation process is shown in table 2, and the elutriation enrichment results are shown in table 3.

TABLE 2 panning of phage Nanobody libraries

TABLE 3 enrichment results of phage from each round of panning

(II) identification of Positive phage clones

(1) Randomly picking 30 monoclonals from the output titer plate of the last round of panning, preserving bacteria on an LB-Amp plate, and inoculating 3mL of SB culture medium;

(2) shake culturing at 37 deg.C for 4-5h until OD value is 0.8, adding 30 μ L M13KO7 helper phage, and standing at 37 deg.C for 30 min;

(3) shake culturing at 37 deg.C for 1h, adding Kana with final concentration of 70 μ g/mL, and culturing at 37 deg.C;

(4) the next day, respectively taking 500 mu L of bacterial liquid for centrifugation, 5000g and 10min, and directly using the supernatant in Phage-ELISA;

(5) blocking an ELISA plate, incubating at 37 ℃ for 1h by using 3% PBSTM (phosphoenolpyruvate succinate) and 300 mu L/hole, and washing the plate for 3 times by using a machine;

(6) adding the phage supernatant of each clone into 3 corresponding holes, adding 50 mu L of standard substance into 50 mu L of each hole, adding 50 mu L of 10% methanol/PBS buffer solution into 1,4,7 and 10 columns and adding 50 mu L of 10% methanol/PBS buffer solution into 2,3,5,6,8,9,11 and 12 columns, and shaking and mixing uniformly by using a microplate reader after adding;

(7) standing for 1h at 37 ℃, and washing the plate for 10 times by hands;

(8) a second antibody, Anti-M13 horseradish peroxidase, is diluted at 1:5000 and incubated for 1h at 37 ℃;

(9) color development, incubation for 15min at 37 ℃ and detection of absorbance at 450 nm.

30 clones were subjected to phase-ELISA and found to react with aflatoxin M₁Differential binding response of monoclonal antibodiesIs relatively large. OD reaction with antibody in Phage-ELISA assay₄₅₀Higher value with addition of AFM₁Rear OD₄₅₀Phage clones with significantly decreased values were judged to be positive, and as a result, 21 positive clones (clones nos. 1, 2,3, 4, 6, 7, 8, 11,12, 14, 16, 17, 20, 21, 23, 24, 25, 27, 28, 29, and 30 in order) were obtained by four rounds of panning as shown in fig. 2. According to the ELISA result, the positive clones are picked out from the bacteria-protecting plate, after activation culture, the positive clones are sent to Shanghai's institute for sequence analysis, the sequencing primer is gback, gene sequencing is carried out on 21 positive clones, the result shows that 21 monoclonals have the same amino acid sequence, 1 nano antibody which can be specifically combined with the aflatoxin M1 monoclonal antibody is successfully enriched in the screening process, and the nano antibody is named as aflatoxin M₁The amino acid sequence of the anti-idiotype nano antibody VHH C4 is shown as SEQ ID NO. 1, and the nucleotide sequence is shown as SEQ ID NO. 2.

Example 3 Aflatoxin M₁Expression and purification of anti-idiotype nano-antibody

Firstly, preparing Top 10F' competent cells:

(1) selecting a proper amount of Top 10F', streaking on an LB-tetracycline plate, and culturing at 37 ℃ overnight;

(2) the Top 10F' was picked up and cultured in 5mL LB medium at 37 ℃ and 250rpm to OD₆₀₀0.6 to 0.8;

(3) standing the bacterial liquid on ice for 30 min;

(4) centrifuging at 4 deg.C for 5min at 5000g, rapidly freezing, discarding supernatant, adding 1mL precooled 0.1M CaCl₂Resuspending the thallus in the solution, sucking, beating, mixing uniformly, and carrying out ice bath for 7 min;

(5) repeating the above operation once;

(6) centrifuging at 4 deg.C for 5min at 5000g, quickly freezing, completely sucking out liquid, and pre-cooling precipitate with 100 μ L of 0.1M CaCl₂The solution is resuspended, and then Top 10F' competent cells are obtained.

(II) extracting and transforming positive phage cloning plasmid:

(1) selecting a proper amount of glycerol bacteria of VHH C4/ER2738, streaking the glycerol bacteria on an LB-aminobenzyl plate, and culturing the glycerol bacteria at 37 ℃ overnight;

(2) selecting VHH C4/ER2738 single colony to 2mL SB culture medium, culturing at 37 deg.C to OD₆₀₀0.8, extracting a plasmid of VHH C4;

(3) adding 1.5. mu.L of LVHH C4 plasmid to 100. mu.L of Top 10F' competent cells prepared above on ice, mixing gently, and standing on ice for 30 min;

(4) thermally shocking at 42 deg.C for 90s, rapidly putting back on ice, and standing for 5 min;

(5) adding 700 μ L LB culture medium into each centrifuge tube in a super clean bench, resuscitating and culturing at 37 deg.C and 200rpm for 45 min;

(6) centrifuging at 12000g for 4min at 4 deg.C, removing supernatant, adding 100 μ L LB medium to the centrifuge tube, resuspending, mixing, spreading LB-ampicillin plate, and culturing at 37 deg.C overnight.

Induced expression and purification of (III) nano antibody

Selecting single colony on VHH C4/Top 10F' plate, shaking culturing at 37 deg.C and 250rpm overnight, transferring 200 μ L overnight bacteria into 200mLSB culture medium, and continuously culturing to OD₆₀₀At 0.6, 200. mu.L of 1M IPTG was added and induction was carried out overnight at 37 ℃. Centrifuging at 4 deg.C for 8000g and 15min to collect bacteria, adding lysis solution B-PER at a ratio of 20mL/g according to thallus precipitation quality, dispersing precipitate completely, slowly shaking at room temperature for 10min, centrifuging at 12000g for 20min, and collecting supernatant as crude extract of nanometer antibody. The crude extract was dialyzed against 0.01M PBS7.4 and then passed through a 0.22 μ M aqueous filter for nickel column purification.

The crude extract of the nano antibody is purified by using a Ni-NTA His bond Resin kit, and the steps are as follows:

(1) sequentially using sterile ddH of 10 times the column volume₂O, 0.01mol/L PBS7.4 equilibrium column;

(2) adding the filtered and sterilized crude extract into a chromatographic column, mixing the crude extract with a filler, and shaking the mixture at room temperature for 1 hour to fully combine the nano antibody with the filler;

(3) the mixture was loaded into a column, which was equilibrated with 10 column volumes of 0.01mol/L PBS 7.4;

(4) preparing 10mL of 20mM, 40mM and 300mM imidazole-PBS respectively from 1M imidazole, filtering the solution through a 0.22 mu M aqueous phase filter membrane to be used as eluent for gradient elution, and collecting the effluent by using a 2mL centrifuge tube;

(5) the column was filtered through 10mL of 20mM imidazole-PBS 7.4 without collecting the effluent;

(6) the first 3mL of effluent was collected by passing 10mL of 40mM imidazole-PBS 7.4 through the column

(7) The column was filtered with 10mL of 300mM imidazole-PBS 7.4 and the whole flow-through was collected;

(8) washing the column with 10 times column volume of 1M imidazole-PBS 7.4, without collecting effluent, eluting all non-specifically bound heteroproteins;

(9) sequentially using 10 column volumes of PBS, 10 column volumes of ddH₂And O, washing the column with 20% ethanol with 10 times of the column volume, and finally sealing the column with 20% ethanol water with the same volume for storage.

Performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on the effluent of each tube, mixing the effluent with obvious nano antibody bands, dialyzing in PBS (phosphate buffer solution) at 4 ℃ overnight, concentrating by using an ultrafiltration tube to obtain a VHH C4 nano antibody solution, and subpackaging at-20 ℃ for storage.

Example 4 specificity analysis of the anti-idiotype Nanobody VHH C4

(1) Specific analysis of the anti-idiotype nanobody VHH C4: aflatoxin B with a concentration of 0.2 mug/mL is prepared by using coating buffer solution₁Antigen namely AFB₁-BSA, ochratoxin A antigen i.e. OTA-BSA, T-2 toxin antigen i.e. T-2-BSA and aflatoxin M₁Antigen namely AFM₁BSA, overnight coating of ELISA plates at 4 ℃; the next day, blocking with 3% skimmed milk powder/conventional phosphate buffer solution/conventional phosphate Tween buffer solution, incubating at 37 deg.C for 1 hr, and adding anti-aflatoxin B₁Aflatoxin M₁Starting from 10 mu g/mL of four monoclonal antibodies of ochratoxin OTA and T-2 toxin, carrying out multiple dilution by using a conventional phosphate buffer solution, and incubating for 1h at 37 ℃; adding a goat anti-mouse monoclonal antibody marked by horseradish peroxidase, and incubating for 1h at 37 ℃; adding color developing solution, developing at 37 deg.C for 15min, adding stop solution, and measuring OD₄₅₀。

After coating and blocking according to the above method, VHH C4 was sequentially treated with conventional phosphate buffer solutionDiluting, and preparing the anti-aflatoxin B according to the optimized concentration₁Aflatoxin M₁Adding 50 mu LVHH C4 diluent and 50 mu L of corresponding monoclonal antibody into each hole of the four monoclonal antibodies of ochratoxin OTA and T-2 toxin, vibrating and uniformly mixing by using an enzyme-labeling instrument, and incubating for 1h at 37 ℃; diluting according to the ratio of 1:5000, adding horse radish peroxidase labeled goat anti-mouse antibody, and reacting for 1h at 37 ℃; developing the color by the same method as above, and determining OD₄₅₀The value is obtained.

The test results are shown in FIG. 3, and FIG. 3 shows that VHH C4 can inhibit aflatoxin M₁Monoclonal antibody and antigen AFM₁BSA binding, with increasing VHH C4 concentration, shows more and more significant inhibition against aflatoxin B₁The combination of monoclonal antibodies of ochratoxin OTA and T-2 toxin and corresponding antigens has no inhibition effect, which indicates that the VHH C4 has good selectivity and only has the effect of combining with aflatoxin M₁The monoclonal antibody variable regions are specifically bound.

Example 5 Nanobody VHH C4 as Aflatoxin M₁Establishment of ELISA (VELISA) method for substituting antigen

Nano antibody VHH C4 as aflatoxin M₁The procedure for competitive inhibition of ELISA reaction with surrogate antigens was: (1) subjecting aflatoxin M₁Substitution of aflatoxin M after dilution of anti-idiotype nano antibody₁Coating the antigen on an enzyme label plate, and coating overnight at 4 ℃; the next day, sealing with sealing solution, and incubating at 37 deg.C for 1 h; (2) adding methanol/PBS 7.4 buffer solution into the enzyme labeling hole, and adding aflatoxin M₁Diluting the monoclonal antibody and the competitor by PBS7.4 buffer solution, adding the diluted monoclonal antibody and the competitor into an enzyme-labeled hole, and incubating for 1h at 37 ℃; (3) adding a horse radish peroxidase-labeled goat anti-mouse antibody, and incubating for 1h at 37 ℃; (4) adding color developing solution, reacting at 37 deg.C for 15min, and measuring OD₄₅₀。

(I) VELISA Condition optimization

(1) Optimal VHH coating concentration and aflatoxin M₁Working concentration of monoclonal antibody: sequentially diluting VHH C4 to the concentration of 20, 10, 5, 0.06, 2.5 and 1.25 mu g/mL respectively, coating an enzyme label plate, and respectively coating the nano antibody VHH C4 with a chessboard methodDegree and aflatoxin M₁Optimizing the working concentration of the monoclonal antibody, and taking OD₄₅₀The nanometer antibody concentration and the aflatoxin M with the value of about 1.0₁The concentration of the monoclonal antibody is the optimal working concentration, and the aflatoxin M₁The monoclonal antibody was diluted 9-fold to concentrations of 100, 11.11, 1.23, 0.14, 0.02. mu.g/mL, and the indirect ELISA assay was performed at these concentrations to establish a standard curve (FIG. 4). According to different color rendering values of all the combinations, the working concentration of the selected VHH C4 is 5 mug/mL and corresponds to the aflatoxin M₁The concentration of the monoclonal antibody was 2. mu.g/mL.

(2) The best blocking reagent: aflatoxin M at optimal VHH C4 coating concentration₁The effect of different blocking reagents on the VELISA response was investigated at the working concentration of the monoclonal antibody. A standard curve was established with 3% BSA, 1.5% OVA and 3% skimmed milk powder, respectively (FIG. 5), and 3% skimmed milk powder was determined as the optimal blocking solution.

(3) Optimum pH: diluting aflatoxin M with phosphate buffer solutions with pH of 5,6, 7 and 8 respectively₁Monoclonal antibody, aflatoxin M was diluted in 10% methanol in each buffer at double ratio₁The standard, the established VELISA standard curve is shown in fig. 6, and the reaction sensitivity is highest when the pH is 7.

(4) Optimum salt ion concentration: separately adapted ddH₂Diluting aflatoxin M by four solutions of O, 10mmol/LPBS, 20mmol/L PBS and 40mmol/L PBS₁Monoclonal antibody, aflatoxin M was diluted in 10% methanol in each buffer at double ratio₁Standard product for detecting aflatoxin M by using VELISA₁The standard curve of (2). As shown in FIG. 7,10 mmol/L PBS was the best reaction buffer.

(5) Optimum methanol concentration: the optimized optimal nano antibody coating concentration and aflatoxin M₁Under the conditions of the working concentration of the monoclonal antibody, the blocking reagent and the optimal phosphate buffer solution, the influence of different methanol concentrations on the VELISA reaction is continuously discussed. Diluting aflatoxin M with conventional phosphate buffer solution and conventional phosphate Tween buffer solution with methanol concentration of 5%, 10%, 20% and 40%, respectively₁And aflatoxin M₁Monoclonal antibody, preparationVertical inhibition curves (FIG. 8), where the IC is derived from the curves at methanol concentrations of 5% and 10%₅₀IC very close to, but at 10% methanol concentration₅₀Smaller and the trend of competing fit curves is relatively better.

(6) Cross-reactivity of VELISA: selecting AFB (immune globulin) by taking VHH C4 as a substitute antigen according to optimized conditions₁、AFB₂、 AFG₁、AFG₁、AFM₁And AFM₂Six mycotoxins were used as competitors to determine the cross-reactivity of VELISA. As can be seen in FIG. 9, the ELISA was aligned with AFM₁IC of VHH C4 with higher sensitivity₅₀The value reaches 0.25ng/mL, and is compared with AFB₁、 AFB₂、AFG₁、AFG₁And AFM₂No cross reaction exists, so the specificity of the VELISA is good, and the aflatoxin M in the dairy product can be detected in a targeted manner₁。

Example 6 establishment of the analysis method for VHH-ELISA samples

(1) Competition curves of nanobody VHH C4 against AFM1 in different sample matrices

Directly taking 10mL of blank samples of milk and yoghourt, centrifuging for 10min at the temperature of 4 ℃ and 5000g, and directly diluting the aflatoxin M by using a lower layer clear liquid without a butter layer₁A standard substance, wherein a VELISA competitive inhibition curve of the matrix extracting solution is established; for the blank sample of milk powder, accurately weighing 10g on an analytical balance, dissolving in preheated pure water at 50 ℃, fixing the volume to 100mL, fully mixing uniformly, taking 10mL, centrifuging 5000g at 4 ℃ for 10min, and directly diluting aflatoxin M with the supernatant without the milk fat layer₁The results of the standard substance and the standard curve established under the reaction system without the matrix show that the three samples have small matrix effect on the VELISA, so that the matrix curve is close to that without the matrix, and the results are shown in FIG. 10.

(2) Application of VHH-ELISA in actual sample detection

In order to evaluate the accuracy of the VELISA detection system, the newly established VELISA method is adopted to carry out the addition and recovery tests of milk, yoghourt and milk powder samples. Adding into blank milk, yogurt and milk powder samples respectively400. 600 ng/L and 800ng/L aflatoxin M₁Standard solution, and detection of AFM in sample extractive solution by new-established method-VELISA method₁The results are shown in Table 4. AFM₁The addition recovery test shows that the sample addition recovery rate is 84.1-119.1%, which indicates that the nano-antibody-based VELISA method established in the research can be applied to aflatoxin M₁In the monitoring of pollution.

TABLE 4 Nanobody VHH-EILSA-based sample addition recovery assay

Note: each result is an average of triplicates

It is apparent that the above embodiments are only examples for clearly illustrating and do not limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications are therefore intended to be included within the scope of the invention as claimed.

< 110 > institute of oil crops of Chinese academy of agricultural sciences

< 120 > VHH-ELISA method based on AFM1 anti-idiotype nanobody replacement antigen

＜160＞ 2

＜210＞ 1

＜211＞ 402bp

＜212＞ DNA

< 213 > alpaca

＜400＞ 1

gcccaggcgg cccaggtgca gctcgtggag tctgggggag gcttggtgca 50

ggctgggggg tctctgagac tctcctgcgc agcctctgga agaaccttca 100

gtcccaatga catcggctgg taccgccagg ctccaggaaa ggagcgcgac 150

ttggtcgcaa gtattactag tgatgatact aaaaactatg cagactccgt 200

gaagggccga ttcaccatct ccagagacaa cgccaagaac acggtgtatc 250

ttgaaatgaa cagcctgaaa cgtgaggaca Cggccgtcta ttactgttac 300

tacagggtcg cgccgggtta cccctcgtac tggggccagg ggacccaggt 350

caccgtctcc tcagcgcacc acagcgaaga cccccatggc caggccggcc 400

ag 402

＜210＞ 1

＜211＞ 134

＜212＞ PRT

< 213 > alpaca

＜400＞ 2

Ala Gln Ala Ala Gln Leu Gln Leu Val Glu Ser GLy GLy GLy Leu

1 5 10 15

Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

16 20 25 30

Arg Thr Phe Ser Pro Asn Asp Ile Gly Trp Tyr Arg Gln Ala Pro

31 35 40 45

Gly Lys Glu Arg Asp Leu Lal Ala Ser Ile Thr Ser Asp Asp Thr

46 50 55 60

Lys Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

61 65 70 75

Asp Asn Ala Lys Asn Thr Val Tyr Leu Glu Met Asn Ser Leu Lys

76 80 85 90

Arg Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Tyr Arg Val Ala Pro

91 95 100 105

Gly Tyr Pro Ser Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser

106 110 115 120

Ser Ala His His Ser Glu Asp Pro His Gly Gln Ala Gly Gln

121 125 130 134

Claims (8)

1. Aflatoxin M₁Anti-idiotype nano antibody as aflatoxin M₁Application of antigen substitute, aflatoxin M₁The anti-idiotype nano antibody is aflatoxin M₁The amino acid sequence of the anti-idiotype nano antibody VHH C4 is shown in SEQ ID NO. 1.

2. Based on aflatoxin M₁The VHH-ELISA method of anti-idiotype nano antibody replacing antigen comprises the following steps: subjecting aflatoxin M₁Anti-idiotype nano antibody for replacing aflatoxin M₁Antigen coated enzyme label plate, using the sample to be detected as competitor to make competitive inhibition ELISA reaction, detecting and obtaining binding rate B/B₀Value based on aflatoxin M₁Concentration-binding ratio B/B₀Obtaining the aflatoxin M by a value standard curve₁The concentration of the aflatoxin M₁The anti-idiotype nano antibody is aflatoxin M₁The amino acid sequence of the anti-idiotype nano antibody VHH C4 is shown in SEQ ID NO. 1.

3. The VHH-ELISA method according to claim 2, characterized in that: the competitive inhibition ELISA reaction is specifically as follows: subjecting aflatoxin M₁Diluting the anti-idiotype nano antibody VHH C4, coating the diluted anti-idiotype nano antibody on an enzyme label plate, and coating the enzyme label plate overnight at 4 ℃; the next day, sealing with sealing solution, and incubating at 37 deg.C for 1 h; adding methanol/PBS buffer solution into the enzyme labeling hole, and adding aflatoxin M into the enzyme labeling hole₁Diluting the monoclonal antibody and the competitor by PBS7.4 buffer solution, adding the diluted monoclonal antibody and the competitor into an enzyme-labeled hole, and incubating for 1h at 37 ℃; adding a horse radish peroxidase-labeled goat anti-mouse antibody, and incubating for 1h at 37 ℃; then adding color developing solution at 37 DEG CAfter 15min of reaction, OD was measured₄₅₀Obtaining a binding rate B/B₀The value is obtained.

4. The VHH-ELISA method according to claim 2 or 3, characterized in that: the aflatoxin M₁The coating concentration of the anti-idiotype nano antibody is 20 mu g/mL-1.25 mu g/mL.

5. The VHH-ELISA method according to claim 3, characterized in that: the confining liquid is 3% BSA, 1.5% OVA or 3% skimmed milk powder.

6. The VHH-ELISA method according to claim 3, characterized in that: the pH value of the methanol/PBS buffer solution is 7.0-7.4.

7. The VHH-ELISA method according to claim 3, characterized in that: the concentration of methanol in the methanol/PBS buffer solution is 5-10%.

8. The VHH-ELISA method according to claim 3, characterized in that: the sample to be detected is a milk product, including yoghurt, milk or milk powder.

Images