CN113061188B - AFM 1-based anti-idiotype nano antibody for replacing aflatoxin M1ELISA immunoassay method for standard substance - Google Patents
AFM 1-based anti-idiotype nano antibody for replacing aflatoxin M1ELISA immunoassay method for standard substance Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology, and particularly relates to AFM 1-based anti-idiotypic nanobody for replacing aflatoxin M1ELISA immunoassay method of the standard substance. According to aflatoxin M1Standard substance and aflatoxin M1Establishing respective ELISA standard curves of the anti-idiotype antibodies under the same inhibition rate1Concentration and Aflatoxin M1And (3) carrying out an exponential regression analysis on the result according to a corresponding relation curve of the concentration of the anti-idiotype antibody to obtain a corresponding relation between the anti-idiotype antibody and the result, and detecting the content of the aflatoxin M1. Aflatoxin M1Anti-idiotype nano antibody for replacing aflatoxin M1The standard substance immunoassay method has accurate and reliable result, is an effective and feasible green immunoassay method, and can be applied to aflatoxin M in agricultural products1And (5) detecting and analyzing the content.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to aflatoxin M1Anti-idiotype nano antibody as aflatoxin M1Application of standard substance 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 crops. These metabolites are highly toxic, mutagenic, teratogenic and carcinogenic compounds that have been considered as causative agents of human hepato-and extrahepatic carcinogenesis. 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. Enzyme-linked immunosorbent assay (ELISA) is a high-throughput assay with small sample amount and is mainly used for AFM1Routine analysis of (2). However, AFM must be used for each plate1The curves are calibrated to reduce the difference in the plate-to-plate variation and improve accuracy. Furthermore, pure toxins used as calibrators are harmful to both the operator and the environment. Therefore, we sought to develop a nontoxic alternative calibrator and test AFM1The green method of (1) is a method for solving the problem. In the indirect competition process of ELISA, the analyte competitively inhibits the binding of specific antibodies to the coating antigen. The higher the analyte concentration, the weaker the resulting signal. Finally, a sigmoidal curve is shown. Non-toxic substances may be used as surrogate calibrators if they can also bind to specific antibodies and exhibit a sigmoidal calibration curve.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide the aflatoxin M1Anti-idiotype nano antibody as aflatoxin M1Application of standard substance substitute.
In order to achieve the purpose, the invention adopts the technical scheme that:
aflatoxin M1Anti-idiotype nano antibody as aflatoxin M1Application of standard substance substitute, namely 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.
According to the scheme, the aflatoxin M1Anti-idiotype nano antibody as aflatoxin M1The application of the standard substance substitute in the ELISA immunoassay method comprises the following steps:
(1) aflatoxin M1Establishment of inhibition standard curve: aflatoxin M1The standard substance is used as a competitor to perform competitive inhibition ELISA reaction, and the binding rate B/B is obtained by detection0Value of aflatoxin M1The concentration logarithm value of the standard substance is the abscissa, and the binding rate B/B is taken as0The value is used as the ordinate to establish ELISA aflatoxin M1Inhibition standard curve;
(2) aflatoxin M1Establishing an anti-idiotype nano antibody inhibition standard curve: aflatoxin M1The anti-idiotype nano antibody VHH C4 is used as a competitor to perform competitive inhibition ELISA reaction, and the binding rate B/B is obtained by detection0Value as aflatoxin M1The concentration logarithm of the anti-idiotype nanobody VHH C4 is plotted on the abscissa as the binding ratio B/B0The value is used as the ordinate to establish ELISA aflatoxin M1An anti-idiotype nanobody inhibition standard curve;
(3) aflatoxin M1Anti-idiotype nano antibody and aflatoxin M1The corresponding relation of the standard products is as follows: according to aflatoxin M1Inhibition of Standard Curve and Aflatoxin M1Calculating a certain binding rate B/B by using an anti-idiotype nano antibody inhibition standard curve0Corresponding aflatoxins M1Concentration of Standard and Aflatoxin M1Concentration of anti-idiotype Nanobody VHH C4 at same binding rate B/B0Corresponding aflatoxins M1The concentration of the anti-idiotype nanobody VHH C4 is plotted on the abscissa for the same binding rate B/B0The value of aflatoxin M1The concentration of the standard substance is used as a vertical coordinate to establish aflatoxin M1Anti-idiotype nano antibody and aflatoxin M1Linear relationship curve of standard.
According to the scheme, the combination ratio B/B in the step (3)0In the range of 20% to 80%.
According to the scheme, step (3) aflatoxin M1Anti-idiotype nano antibody and aflatoxin M1B/B in linear relation curve of standard substance0The concentration interval was 10%.
According to the scheme, the aflatoxin M1Anti-idiotype nano antibody as aflatoxin M1The application of the standard substance substitute in sample detection specifically comprises the following steps:
(1) taking a sample to be detected as a competitor to perform competitive inhibition ELLSA reaction, and detecting to obtain the binding rate B/B0Value, substitution into aflatoxin M1Calculating corresponding aflatoxin M in anti-idiotype nano antibody inhibition standard curve1Anti-idiotype nanobody concentration; then the aflatoxin M is added1Substituting concentration of anti-idiotype nano antibody into aflatoxin M1Anti-idiotype nano antibody and aflatoxin M1In the linear relation curve of the standard substance, the corresponding aflatoxin M is obtained by conversion1And (4) concentration.
According to the scheme, the samples to be detected are milk products including milk, milk powder and yoghourt samples.
In the invention, the competitive inhibition ELISA reaction process is as follows: using aflatoxin M1Coating the diluted solution on an enzyme label plate, and coating the solution overnight at 4 ℃; the next day, sealing with sealing solution, and incubating at 37 deg.C for 1 h; adding methanol/PBS 7.4 buffer solution into the enzyme labeling hole, and adding aflatoxin M1Diluting the antibody and the competitor with PBS7.4 buffer solution, adding the diluted antibody and 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 ℃; adding color developing solution, reacting at 37 deg.C for 15min, and measuring OD450Obtaining a binding rate B/B0The value is obtained.
In this application, aflatoxin M is used1The monoclonal antibody immune alpaca is prepared through extracting the total leucocyte RNA to constitute phage display nanometer antibody library, panning to obtain positive phage aflatoxin M with high specificity1The Anti-Idiotype nano-antibody and the Anti-Idiotype antibody (Anti-Idiopype) can be combined with the specific recognition region of the antigen and the antibody and inhibit the combination between the antigen and the antibody. We further use AFM1Anti-idiotype nano-antibody as AFM1Nontoxic substitute standard substance, establishes environment-friendly competitive ELISA reaction for AFM1The detection of (2) can be specifically carried out by subjecting aflatoxin M1Anti-idiotype nano antibody and aflatoxin M1Respectively establishing ELISA standard curves for the standard substances and simultaneously establishing aflatoxin M1Concentration and Aflatoxin M1The corresponding relation curve of the concentration of the anti-idiotype antibody is used for aflatoxin M in a sample based on the corresponding relation between the anti-idiotype antibody and the sample1And (4) detecting and analyzing the content.
The invention has the following beneficial effects:
the invention provides aflatoxins M1Anti-idiotype nano antibody as aflatoxin M1Application of standard substitute. The ELISA method provided by the invention selects samples such as milk, yoghourt, milk powder and the like to carry out aflatoxin M1The recovery rate is between 84.8 and 92.2 percent by adding a recovery experiment, the accuracy is good, and the aflatoxin M is proved1Anti-idiotype nano antibody for replacing aflatoxin M1The standard ELISA method has accurate and reliable result, and is an effective and feasible green immunoassay method. Has great application priceThe value is obtained.
Drawings
FIG. 1 is the test results for determining the diversity of phage display nanobody immune libraries.
FIG. 2 shows the positive clones identified by the 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 shows a standard curve for VHH C4 as competitor.
FIG. 5 shows aflatoxin M1And (5) making a competitive standard curve.
FIG. 6 shows VHH C4 and aflatoxin M1Linear relationship between concentrations.
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, wherein the hybridoma cell strain 2C9 is preserved in China Center for Type Culture Collection (CCTCC) at 7.13.2010 and is 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 M1200 mu g of monoclonal antibody (dissolved in PBS7.4) is emulsified with equal volume of Freund incomplete adjuvant, subcutaneous multi-point injection is carried out on male Alpaca (Alpaca) aged three years old, immunization is carried out once every two weeks for 8 times, and blood sampling detection is started after four-time immunization. Collecting 10mL of blood by using an EDTA vacuum blood collection tube jugular vein 7-10 days after the fourth immunization, simultaneously slightly reversing the blood collection tube to avoid blood coagulation, treating the blood by using a filter in a LeukoLOCK kit, and then sequentially using 3mL of PBS buffer solution and 3mL of RNAlaterThe buffer solution washes the filter, the desired leukocytes are trapped in the filter, and the filter is sealed and stored at-80 ℃ for RNA extraction.
2. Total RNA extraction
The total RNA in the alpaca blood is separated according to the operating manual of the LeukoLOCK total RNA extraction kit of Life Technology company, and the specific operation is 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, discarding, and taking care not to suck 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 at 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, preparing TURBODASE master mixing solution, adding 4 mu LTURBO DNase (20U/. mu.L) into 296 mu L LeukoLOCK DNase buffer solution, mixing uniformly, transferring to the centrifuge tube, repeatedly blowing and beating the precipitated magnetic beads by a pipetting gun to disperse uniformly, shaking for 10min at room temperature with 1000g, and slightly reversing and mixing in the midway for several times; (11) sequentially adding 300 mu L of lysis/binding solution (without pH regulation buffer solution) and isopropanol into the centrifuge tube, mixing uniformly, performing inching centrifugation for 2s, and then incubating at room temperature for 3 min; (12) centrifuge at 16000g 30s, discarding the supernatant, adding 750. mu.L of a washing solution 2/3, violently vortexing for 30s, centrifuging for 16000g for 30s, discarding the supernatant, adding 750. mu.L of a washing solution 2/3 again, violently vortexing for 30s, centrifuging for 16000g for 1min, discarding the supernatant as much as possible, standing for 3min at room temperature in an open manner to volatilize the residual washing solution, and taking care that the magnetic beads cannot be left for a long time to avoid excessive drying; (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) ready-to-synthesize pre-mix of cDNA:
(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 primers R2 and R1 respectively, 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 on a vector pComb3X and a vector VHH by adopting SfiI, connecting the enzyme-cut VHH fragment with a vector pComb3X by using T4 ligase, taking 3 mu L of a connecting product to 25 mu L of E.coli ER2738 competent cells, carrying out light mixing, completely sucking out and 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 ℃; the electroporation was repeated 10 times, each time 3. mu.L of the ligation product, 10 times of the transformed bacteria solution were pooled and 1. mu.L of the solution was diluted 10-fold with sterile water and spread on LB-Amp plates, and cultured 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 at 37 ℃To OD600Is 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, the amino acid sequences of the inserts of the selected 30 monoclonal antibodies show that the conserved regions FR1, FR2, FR3 and FR4 of the nanobody have high similarity, while the regions CDR1, CDR2 and CDR3 of the variable region 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 M1The monoclonal antibody is used as a target (the aflatoxin M)1The monoclonal antibody is secreted and produced by a hybridoma cell strain 2C9 with the preservation number of CCTCC NO. C201018, wherein the hybridoma cell strain 2C9 is preserved in China Center for Type Culture Collection (CCTCC) at 7.13.2010 and is 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, is coated with 6 holes and can be 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 the subsequent panning, the concentration of the coating antibody was gradually reduced and aflatoxin M was present in the eluate1The 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 4-5 hr 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 M1Monoclonal antibody binding reactions vary widely. OD reaction with antibody in Phage-ELISA assay450Higher value with addition of AFM1Rear OD450Phage clones showing a significant decrease in value were judged to be positive, and as a result, as shown in FIG. 2, 21 positive clones (1, 2,3, 4, 6, 7, 8, 11,12, 14, 16, 17, respectively) were obtained by four rounds of panning,20. Clone No. 21, 23, 24, 25, 27, 28, 29, 30). 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 the 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 of 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 ddH2O, washing the column with 10 times of 20% ethanol, and finally sealing the column with equal volume of 20% ethanol water 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 development solution, developing at 37 deg.C for 15min, adding stop solution, and measuring OD450。
After being coated and sealed according to the method, VHH C4 is sequentially diluted three times by using a conventional phosphate buffer solution, and the anti-aflatoxin B is prepared 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 ℃; then adding the mixture according to the dilution ratio of 1:5000Horse 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
This example is based on aflatoxin M1Competitive inhibition ELISA reaction as coating antigen to study aflatoxin M1Anti-idiotype nano antibody VHH C4 as aflatoxin M1When the standard substance is substituted, the standard substance is mixed with aflatoxin M1The corresponding relation of the standard products is as follows:
(1) aflatoxin M1Establishment of inhibition standard curve: aflatoxin M with a concentration of 0.2 mug/mL is prepared by using coating buffer solution1Antigen namely AFM1-BSA, enzyme plates coated overnight at 4 ℃ with aflatoxin M1The standard substance is used as a competitor to perform competitive inhibition ELISA reaction, and the binding rate B/B is obtained by detection0Value as aflatoxin M1The concentration logarithm value of the standard substance is the abscissa, and the binding rate is B/B0The value is used as a vertical coordinate, and an ELISA detection standard curve is established;
(2) aflatoxin M1Establishing an anti-idiotype nano antibody inhibition standard curve: aflatoxin M with a concentration of 0.2 mug/mL is prepared by coating buffer solution1Antigen namely AFM1BSA, 4 ℃ overnight coating of the ELISA plate, diluting the Nanobody VHH C4 in a conventional phosphate buffer solution in a multiple ratio (starting from 10. mu.g/mL and then 2-fold dilution), and adding aflatoxin M1The anti-idiotype nano antibody is used as a competitor to perform competitive inhibition ELISA reaction, and the binding rate B/B is obtained by detection0Value as aflatoxin M1Anti-idiotype nanoThe antibody concentration log value is the abscissa, and the binding rate B/B0The value is used as a vertical coordinate, and an ELISA detection standard curve is established;
(3) according to aflatoxin M1Inhibition of Standard Curve and Aflatoxin M1Calculating the binding rate B/B by using an anti-idiotype nano antibody inhibition standard curve0Corresponding aflatoxin M in the range of 20-80 percent1Anti-idiotype nano antibody concentration and aflatoxin M1The standard concentration was 10% at each binding rate B/B0Corresponding aflatoxins M1The concentration of the anti-idiotype nano antibody is the abscissa and the same binding rate B/B0The value of aflatoxin M1Taking the concentration of the standard substance as the ordinate, and establishing the aflatoxin M1Anti-idiotype nano antibody and aflatoxin M1Linear relationship curve of standard.
The process of competitive inhibition ELISA reaction is as follows: (1) aflatoxin M with a concentration of 0.2 mug/mL is prepared by using coating buffer solution1Antigen namely AFM1BSA was coated on an ELISA plate and 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。
The standard curve for VHH C4 as a competitor is shown in FIG. 4, aflatoxin M1A competitive standard curve is shown in FIG. 5. FIGS. 4 and 5 show that both curves approximate an "S" shape with VHH C4 or aflatoxin M1Increasing concentration of aflatoxin M1The inhibition effect of the monoclonal antibody and the coating antigen is gradually enhanced, the curve is relatively gentle when the concentration of the competitor is higher or lower, and the curve is linear when the binding rate is 20-80%, and is a linear interval of detection, and the inhibition effect is relatively obvious.
From the two standard curves, the binding rate B/B020-80% of the VHH C4 concentration and the aflatoxin M concentration are respectively calculated by taking a point every 10%1Concentration, taking VHH C4 concentration corresponding to different binding rates as abscissa, and aflatoxin M corresponding to corresponding binding rates1The concentration is vertical coordinate, and VHH C4 and aflatoxin M are established1The correlation curves of (1) and (6) were analyzed by linear regression to obtain VHH C4 and aflatoxin M1The linear regression equation of (1): 0.01611x +0.11112, coefficient of correlation R20.999, wherein x is the concentration of VHH C4 and y is aflatoxin M1And (4) concentration.
VHH C4 as aflatoxin M1The standard substitute for detecting the actual sample can be divided into two steps: (1) calculate the B/B of each well from the OD measured by ELISA0Value, substitution into aflatoxin M1Calculating the corresponding concentration of VHH C4 in an anti-idiotype nano antibody inhibition standard curve equation; (2) substituting the concentration of VHH C4 into VHH C4 and aflatoxin M1In the quantitative conversion linear relation curve of the concentration, the corresponding aflatoxin M is solved1And (4) concentration.
Example 6 Aflatoxin M1Anti-idiotype nano antibody for replacing aflatoxin M1Application of standard substance in actual sample ELISA detection
Substitution of aflatoxin M based on VHH C41The test for adding and recovering the standard substance specifically comprises the following operations:
(1) 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; and (3) accurately weighing 10g of a blank milk powder sample on an analytical balance, dissolving the blank milk powder sample in preheated pure water at 50 ℃, diluting to 100mL of the solution, fully and uniformly mixing the solution, centrifuging 10mL of the solution at 4 ℃ for 10min at 5000g, and removing the lower layer clear solution from which the milk fat layer is removed to directly use the sample in a subsequent experiment.
(2) Taking the pretreated sample as a competitor to perform competitive inhibition ELLSA reaction, wherein the specific operation of the competitive inhibition ELISA reaction is as follows: a) respectively preparing aspergillus flavus with concentration of 0.2 mug/mL by using coating buffer solutionToxin M1Antigen namely AFM1BSA coating on ELISA plates overnight at 4 ℃; the next day, sealing with sealing solution, and incubating at 37 deg.C for 1 h; b) 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 ℃; c) adding a horse radish peroxidase-labeled goat anti-mouse antibody, and incubating for 1h at 37 ℃; d) adding color developing solution, reacting at 37 deg.C for 15min, and measuring OD450；
(3) Obtaining the binding rate B/B through competitive inhibition ELISA reaction detection0Value, substitution into aflatoxin M1Calculating corresponding aflatoxin M in an anti-idiotype nano antibody VHH C4 inhibition standard curve1The concentration of anti-idiotype nanobody VHH C4; then the aflatoxin M is added1Substituting the concentration of the anti-idiotype nano antibody VHH C4 into the concentration of VHH C4 and the concentration of aflatoxin M1In the quantitative conversion linear relation curve of the concentration, the corresponding aflatoxin M is obtained by conversion1And (4) concentration.
To verify the substitution of the aflatoxin M based on VHH C41Indirect competitive ELISA method for detecting aflatoxin M in sample by standard substance1Accuracy of content, AFM was performed on milk, yogurt and milk powder blank samples in this study1Add recovery experiments. The results are shown in Table 4, with 200, 400, 600ng/L AFM added to each sample, respectively1The detection recovery rate is between 84.8 and 92.2 percent, and the detection proves that the substitute standard substance ELISA method is adopted to detect the aflatoxin M in the sample1The result is reliable.
TABLE 4 detection of AFM in agricultural products based on surrogate standard ELISA1Recovery rate
Note: each result is an average of triplicates
It is apparent that the above embodiments are only examples for clearly illustrating, and are not limiting to 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 > ELISA immunoassay method based on AFM1 anti-idiotype nano antibody to replace aflatoxin M1 standard substance
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＜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
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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 (7)
1. Aflatoxin M1Anti-idiotype nano antibody as aflatoxin M1Application of standard substance substitute, namely aflatoxin M1The anti-idiotype nano antibody is aflatoxin M1The amino acid sequence of the anti-idiotype nano antibody VHHC4 is shown in SEQ ID NO. 1.
2. Use according to claim 1, characterized in that: the method comprises the following steps:
(1) yellow rice cakeMycotoxin M1Establishment of inhibition standard curve: aflatoxin M1The standard substance is used as a competitor to perform competitive inhibition ELISA reaction, and the binding rate B/B is obtained by detection0Value as aflatoxin M1The concentration logarithm value of the standard substance is the abscissa, and the binding rate B/B is taken as0The value is used as the ordinate to establish ELISA aflatoxin M1Inhibition standard curve;
(2) aflatoxin M1Establishing an anti-idiotype nano-antibody inhibition standard curve: aflatoxin M1The anti-idiotype nano antibody VHHC4 is used as a competitor to perform competitive inhibition ELISA reaction, and the binding rate B/B is obtained by detection0Value as aflatoxin M1The concentration logarithm of the anti-idiotype nano-antibody VHHC4 is plotted on the abscissa as the binding ratio B/B0The value is used as the ordinate to establish ELISA aflatoxin M1An anti-idiotype nanobody inhibition standard curve;
(3) aflatoxin M1Anti-idiotype nano antibody and aflatoxin M1The corresponding relation of the standard products is as follows: according to aflatoxin M1Inhibition of Standard Curve and Aflatoxin M1Calculating a certain binding rate B/B by using an anti-idiotype nano antibody inhibition standard curve0Corresponding aflatoxins M1Concentration of Standard and Aflatoxin M1Concentration of anti-idiotype Nanobody VHHC4 at same binding rate B/B0Aflatoxins M of corresponding value1The concentration of the anti-idiotype nanobody VHHC4 is plotted on the abscissa for the same binding rate B/B0The value of aflatoxin M1Taking the concentration of the standard substance as the ordinate, and establishing the aflatoxin M1Anti-idiotype nano antibody and aflatoxin M1Linear relationship curve of standard.
3. Use according to claim 2, characterized in that: binding Rate B/B in step (3)0In the range of 20% to 80%.
4. Use according to claim 2, characterized in that: step (3) Aflatoxin M1Anti-idiotype nano antibody and aflatoxin M1B/B in linear relation curve of standard substance0The concentration interval was 10%.
5. Use according to claim 2, characterized in that: the method comprises the following steps: taking a sample to be detected as a competitor to perform competitive inhibition ELLSA reaction, and detecting to obtain the binding rate B/B0Value, substitution into aflatoxin M1Calculating corresponding aflatoxin M in anti-idiotype nano antibody inhibition standard curve1Anti-idiotype nanobody concentration; then the aflatoxin M is added1Substituting concentration of anti-idiotype nano antibody into aflatoxin M1Anti-idiotype nano antibody and aflatoxin M1In the linear relation curve of the standard substance, the corresponding aflatoxin M is obtained by conversion1And (4) concentration.
6. Use according to claim 5, characterized in that: the sample to be detected is a milk product.
7. Use according to claim 6, characterized in that: the milk products include milk, milk powder and yogurt samples.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102109518A (en) * | 2010-12-21 | 2011-06-29 | 中国农业科学院油料作物研究所 | Aflatoxin M1 immunochromatographic test strip and preparation method thereof |
| CN102746403A (en) * | 2012-04-20 | 2012-10-24 | 中国农业科学院油料作物研究所 | Standard substance universal alternate for aflatoxin detection by using ELISA, preparation method thereof, and ELISA detection method for aflatoxin |
| CN103869065A (en) * | 2014-03-28 | 2014-06-18 | 中国农业科学院油料作物研究所 | Aflatoxin M1 nano antibody immunosorbent, immunoaffinity column, preparation method and application thereof |
| CN103898615A (en) * | 2014-03-28 | 2014-07-02 | 中国农业科学院油料作物研究所 | Aflatoxin nanometer antibody gene bank, construction method thereof and application thereof as well as aflatoxin M1 nanometer antibody 2014AFM-G2 |
| CN109705215A (en) * | 2019-02-21 | 2019-05-03 | 武汉中科兴达技术有限公司 | A kind of nano antibody 2018AFB-N11 and its application with high specific identification aflatoxin B1 |
-
2021
- 2021-03-26 CN CN202110327701.6A patent/CN113061188B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102109518A (en) * | 2010-12-21 | 2011-06-29 | 中国农业科学院油料作物研究所 | Aflatoxin M1 immunochromatographic test strip and preparation method thereof |
| CN102746403A (en) * | 2012-04-20 | 2012-10-24 | 中国农业科学院油料作物研究所 | Standard substance universal alternate for aflatoxin detection by using ELISA, preparation method thereof, and ELISA detection method for aflatoxin |
| CN103869065A (en) * | 2014-03-28 | 2014-06-18 | 中国农业科学院油料作物研究所 | Aflatoxin M1 nano antibody immunosorbent, immunoaffinity column, preparation method and application thereof |
| CN103898615A (en) * | 2014-03-28 | 2014-07-02 | 中国农业科学院油料作物研究所 | Aflatoxin nanometer antibody gene bank, construction method thereof and application thereof as well as aflatoxin M1 nanometer antibody 2014AFM-G2 |
| CN109705215A (en) * | 2019-02-21 | 2019-05-03 | 武汉中科兴达技术有限公司 | A kind of nano antibody 2018AFB-N11 and its application with high specific identification aflatoxin B1 |
Non-Patent Citations (2)
| Title |
|---|
| Development of a specific anti-idiotypic nanobody for monitoring aflatoxin M1 in milk and dairy products;Chong Cai;《Microchemical Journal》;20210426;第167卷;第1-8页 * |
| 农产品典型真菌毒素生物识别材料与快速检测方法研究;唐晓倩;《中国优秀博硕士学位论文全文数据库(博士)》;20210115(第01期);第B024-57页 * |
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