CN113637080B

CN113637080B - VHH-ELISA method based on AFM1 anti-idiotype nano antibody substituted antigen

Info

Publication number: CN113637080B
Application number: CN202110327861.0A
Authority: CN
Inventors: 张奇; 唐晓倩; 李培武; 白艺珍; 姜俊
Original assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Current assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Priority date: 2021-03-26
Filing date: 2021-03-26
Publication date: 2023-08-08
Anticipated expiration: 2041-03-26
Also published as: CN113637080A

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 coating antigen to establish VHH-ELISA for sample addition and recovery experiments, which shows that aflatoxin M ₁ The anti-idiotype nano antibody replaces 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

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

Technical Field

The invention belongs to the field of molecular biology, and in particular relates to aflatoxin M ₁ Anti-idiotype nano antibody as aflatoxin M ₁ Use of antigen substitutes.

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 causative agents of human hepatic and extrahepatic carcinogenesis. Aflatoxin B ₁ (AFB ₁ ) The highest toxicity is classified by the international cancer research institute as a class 1 human carcinogen. AFM (atomic force microscope) ₁ Is AFB ₁ Is a hydroxylation metabolite of (a) in the host cell. Ingest quilt AFB ₁ The lactating animals of the contaminated feed will be AFM ₁ Is excreted into milk and can therefore be found in a wide variety of milk products. AFM (atomic force microscope) ₁ Also toxic and carcinogenic, have been classified as class 1 carcinogens. Due to AFM ₁ Contamination is a hazard to human health, particularly to infants and children, and has become a global concern. AFM in EU vs milk ₁ Is limited to 0.05ng/mL. However, the limiting criteria for China, the United states and Brazil were all 0.5ng/mL.

Many have been previously established for AFM ₁ And (5) a detection method. High performance liquid chromatographyHPLC) and Fluorescence Detection (FD) 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 costs, well-equipped laboratories and hours, thereby weakening them in AFM ₁ Application in detection. On the other hand, enzyme-linked immunosorbent assays (ELISA) are becoming more and more popular due to their low cost and ease of application. The immunological method is suitable for rapid detection of aflatoxin. The ELISA detection method has small sample amount and is a high-flux AFM detection method ₁ Is a conventional analytical method. Due to AFM ₁ Since small molecule substances cannot be used alone as antigens, they are often used as complete antigens after coupling with Bovine Serum Albumin (BSA), ovalbumin (OVA), or the like. However, the complete antigen synthesis process is complex and expensive, requiring the use of a large number of AFMs ₁ The standard substance not only causes great threat to operators, but also can cause secondary pollution to the environment. Thus, the proposal of Anti-Idiotype antibodies (Anti-Idiotype) can provide a solution for this, anti-Idiotype antibodies recognizing the variable region of Idiotype antibodies mimic the three-dimensional structure of antigens that express the immunological "internal image" described by Jerne and mimic the configuration of the antigen, so that Anti-Idiotype antibodies can be used as AFMs ₁ Antigen substitutes were developed to develop a safe form of ELISA.

Disclosure of Invention

Aiming at overcoming the defects of the prior art, the invention aims to provide an AFM 1-based anti-idiotype nano antibody for replacing aflatoxin M ₁ ELISA immunoassay method for standard.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

providing aflatoxin M ₁ Anti-idiotype nano antibody as aflatoxin M ₁ Use of an antigen substitute, aflatoxin M ₁ The anti-idiotype nanometer antibody is aflatoxin M ₁ The amino acid sequence of the anti-idiotype nano antibody VHH C4 is shown as SEQ ID NO. 1.

According to the scheme, the aflatoxin M is encoded ₁ The nucleotide sequence of the amino acid of the anti-idiotype nano antibody is shown as SEQ ID NO. 2.

AFM1 anti-idiotype nano antibody substituted aflatoxin M ₁ The ELISA immunoassay method of the standard comprises the following steps: aflatoxin M ₁ Anti-idiotype nanobody VHH C4 substituted aflatoxin M ₁ Antigen coated ELISA plate, sample to be detected (containing aflatoxin M) ₁ The sample of (2) as a competitor to perform a competitive inhibition ELISA reaction, and detecting to obtain a binding rate B/B ₀ Value based on aflatoxin M ₁ Concentration-binding ratio B/B ₀ Obtaining aflatoxin M by a value standard curve ₁ Is a concentration of (3).

According to the scheme, the competition inhibition ELISA reaction specifically comprises the following steps: aflatoxin M ₁ The anti-idiotype nano antibody VHH C4 is coated on an ELISA plate after being diluted, and is coated overnight at 4 ℃; the next day, sealing with sealing liquid, and incubating for 1h at 37 ℃; adding methanol/PBS buffer solution into the enzyme-labeled well, and adding aflatoxin M ₁ The monoclonal antibody and the competitor are diluted by PBS7.4 buffer solution and added into an enzyme-labeled hole for incubation for 1h at 37 ℃; adding horseradish peroxidase-labeled goat anti-mouse antibody, and incubating for 1h at 37 ℃; adding color developing solution, reacting at 37deg.C for 15min, and measuring OD ₄₅₀ Obtaining the binding ratio B/B ₀ Values.

According to the scheme, the aflatoxin M ₁ The coating concentration of the anti-idiotype nano antibody is 20 mug/mL-1.25 mug/mL.

According to the above 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 above scheme, the sample to be detected (containing aflatoxin M ₁ Is a milk product, including yoghurt, milk or milk powder, etc.

The invention provides aflatoxin M ₁ Anti-idiotype nano-meterAntibody VHH C4 as aflatoxin M ₁ Use of antigen substitutes. Provides a VHH-ELISA method based on the nanobody substituted antigen, and simultaneously optimizes the coating concentration and aflatoxin M used in the VHH-ELISA method based on the nanobody substituted antigen ₁ Monoclonal antibody working concentration, blocking reagent, pH, methanol concentration.

The beneficial effects of the invention are as follows:

the invention provides aflatoxin M ₁ Anti-idiotype nanobody VHH C4 as aflatoxin M ₁ Application of antigen substitute and aflatoxin M establishment ₁ Anti-idiotype is based on VHH-ELISA method of nanobody substituted antigen. The invention selects the sample to measure the aflatoxin M ₁ The addition recovery rate of the anti-idiotype nano antibody VELISA is between 84.1% and 119.1%, and the accuracy is good, thus proving that the aflatoxin M ₁ The anti-idiotype nano antibody replaces antigen VHH-ELISA method, has accurate and reliable result, and is an effective and feasible immunoassay method.

Drawings

FIG. 1 is a test result for judging the diversity of phage display nanobody immune libraries.

FIG. 2 shows the identification of positive clones by phase-ELISA.

FIG. 3 shows VHH C4 and anti-aflatoxin B ₁ (abbreviated as AFB) ₁ ) Binding of ochratoxin A (OTA) and T-2 toxin (T-2) monoclonal antibodies.

FIG. 4 is a checkerboard optimization of VHH C4 coating concentration and aflatoxin M ₁ Monoclonal antibody working concentration.

FIG. 5 is the effect of blocking reagent on VELISA.

FIG. 6 shows the effect of phosphate buffer pH on VELISA.

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

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

FIG. 9 shows VELISA vs. aflatoxin B based on VHH C4 ₁ Aflatoxin B ₂ Aflatoxin G ₁ Aflatoxin G ₁ And aflatoxin M ₂ Cross-reaction conditions of (a) are defined.

FIG. 10 shows VHH C4 vs. AFM in different sample matrices ₁ Is a competition curve of (2).

Detailed Description

For a better understanding of the present invention, the following examples are further illustrated, but are not limited to the following examples.

In the following examples, the aflatoxin M ₁ The monoclonal antibody is secreted by hybridoma cell line 2C9 with the preservation number of CCTCC No. C201018, the hybridoma cell line 2C9 has been preserved in China Center for Type Culture Collection (CCTCC) at 7 months 13 of 2010, and the monoclonal antibody has been produced by anti-aflatoxin M in the patent hybridoma cell line 2C9 ₁ Monoclonal antibodies and uses thereof, application number: 201110108230.6.

EXAMPLE 1 construction of phage display nanobody immune library

1. Alpaca immunity

Taking aflatoxin resistant M ₁ 200 μg of monoclonal antibody (dissolved in PBS 7.4) was emulsified with an equal volume of Freund's incomplete adjuvant, followed by subcutaneous multipoint injection of three year old male Alpaca (Alpaca), followed by immunization once every two weeks for 8 total immunizations, and blood sampling detection was initiated after four-immunization. After 7-10 days of the fourth immunization, 10mL of EDTA was used for vacuum blood collection from the jugular vein, the blood collection tube was gently inverted to avoid blood clotting, the blood was treated with the filter in the Leukolock kit, the filter was then rinsed with 3mL of RNAlater buffer and 3mL of 3mLPBS buffer sequentially, the desired white blood cells were trapped in the filter, and finally the filter was stored in a sealed condition at-80℃for RNA extraction.

2. Total RNA extraction

Total RNA in alpaca blood was isolated according to the operating manual of Life Technology company LeukoLOCK total RNA extraction kit, and the specific procedure is as follows:

(1) Adding 70 mu LpH adjusting buffer solution into 2.5mL of lysis/binding solution for preparation; (2) The sealed filter was left to stand at room temperature, the filter cap was opened, and the filter was left with a 2mL syringeIs washed out by RNAlater buffer; (3) The filter was rinsed once with 2.5mL of freshly prepared lysis/binding buffer, aspirated with a 2.5mL syringe, and the effluent was collected using a 15mLRNAse-free centrifuge tube; (4) Adding nuclease-free ddH ₂ O2.5 mL, vortex mixing, adding 25 μL proteinase K, shaking the centrifuge tube at room temperature of 250g for 5min; (5) Taking out the RNA-combined magnetic beads stored at 4 ℃, sucking 50 mu L into the centrifuge tube after vortex mixing, vortex the centrifuge tube, adding 2.5mL of isopropanol into the centrifuge tube, and vibrating for 5min at room temperature; (6) Centrifuging the centrifuge tube at 3200g for 3min, carefully sucking out supernatant, discarding, and taking care not to suck the precipitated magnetic beads; (7) Adding 600 mu L of washing liquid I, repeatedly blowing 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 by using 600 mu L of washing liquid I, and transferring the suspension to the same 1.5mL centrifuge tube; (8) Centrifuge 1.5mL centrifuge tube at 16000g for 30s, carefully discard supernatant; (9) Adding 750 mu L of washing liquid 2/3, shaking with intense vortex for 30s to disperse the magnetic beads which are aggregated and precipitated, centrifuging for 30s at 16000g to collect the magnetic beads, and carefully discarding the supernatant; (10) Standing the centrifuge tube with the cover open in an ultra-clean bench for 2min, volatilizing alcohol remained in 2/3 of the washing liquid, during the period, preparing TURBODNase master mixed liquid, adding 4 mu LTURBO DNase (20U/. Mu.L) into 296 mu L LeukoLOCK DNase buffer solution, uniformly mixing, transferring into the centrifuge tube, repeatedly blowing the precipitated magnetic beads by using a pipetting gun to uniformly disperse the magnetic beads, shaking for 10min at room temperature by 1000g, and gently inverting and mixing for several times in the middle; (11) Sequentially adding 300 mu L of lysis/binding solution (without adding pH adjusting buffer) and isopropanol into the centrifuge tube, uniformly mixing, performing inching centrifugation for 2s, and then incubating for 3min at room temperature; (12) Centrifuging at 16000g for 30s, discarding supernatant, adding 750 μl of lotion 2/3, vigorously swirling for 30s, centrifuging at 16000g for 30s, discarding supernatant, adding 750 μl of lotion 2/3 again, vigorously swirling for 30s, centrifuging at 16000g for 1min, discarding supernatant as dry as possible, standing at room temperature for 3min to volatilize residual lotion, and taking care not to stand for too long to avoid excessive drying of magnetic beads; (13) Adding 60 μL of eluent, vortex shaking 30s, centrifuging 16000g for 2min, transferring the eluted RNA solution into another 1.5mL centrifuge tube without nuclease, collecting 1 μLRNA solution, measuring concentration with nanodrop, collecting about 3 μL of RNA solution, performing agarose gel electrophoresis analysis, standing the rest RNA solutionI.e., all of the cDNA is inverted to prevent degradation.

Synthesis of cDNA

The first strand of 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 dNTPM ix, 3. Mu.L of 50. Mu. Moligo (dT) 20, 24. Mu. LRNA were added respectively, and gently mixed;

(2) Heating at 65deg.C for 5min to denature the template, and opening the secondary structure;

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

(4) The premix solution of cDNA is prepared by the following steps:

(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 ℃ for 50min, and heating at 85 ℃ for 5min to terminate the reaction;

(7) Adding 3 mu LRNAseH into the reaction mixture, mixing, heating at 37 ℃ for 20min, and decomposing RNA which is not completely reacted;

(8) The synthesis of the first strand cDNA is completed, and the split charging is carried out, and the split charging is preserved at the temperature of minus 20 ℃ for standby.

4. Amplification of heavy chain antibody variable region VHH genes

Taking degenerate primer pairs of VHH genes of IgG2 and IgG3 variable regions, 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 the 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 libraries

Carrying out enzyme on a carrier pComb3X and a carrier VHH respectively by adopting SfiI, connecting the cut VHH fragment with the carrier pComb3X by using T4 ligase, taking 3 mu L of a connecting product into 25 mu L of E.coli ER2738 competent cells, sucking out all the products after light mixing, transferring the products into a precooled electrorotating cup (with the inner diameter of 1 mm), and rapidly placing the products into an electrorotating instrument for electrotransformation; immediately adding 1mL of SOC culture medium preheated at 37 ℃ into an electric rotating cup after electric shock, gently sucking and beating by a pipetting gun, uniformly mixing, transferring into a fungus shaking tube, and shaking and resuscitating for culturing for 1h in 250g in a shaking table at 37 ℃; the electrotransformation was repeated 10 times, 3. Mu.L of the ligation product each time, 10 transformed bacterial solutions were pooled and 1. Mu.L of the solution was diluted 10-fold with sterile water and spread on LB-Amp plates, and incubated overnight at 37℃in an incubator for estimating the reservoir capacity. Transferring all the transformed bacteria to a 200mLSB culture medium, and adding carbenicillin to 50 mug/mL and tetracycline to 20 mug/mL; 250g, cultured at 37℃to OD ₆₀₀ 0.6; add 1mL helper phage (1X 10) ¹³ pfu/mL), standing at 37 ℃ for infection for 30min;250g, incubated at 37℃for 2h, kanamycin was added to a concentration of 70. Mu.g/mL, and incubation was continued overnight; the next day, the bacterial liquid is centrifuged at 4 ℃ and 10000g for 15min; adding the supernatant into sterile centrifuge tube, adding 1/4 volume of PEG/NaCl solution, ice-bathing for 2 hr, centrifuging, discarding supernatant, and adding 10mL heavy suspension (containing 1×protease inhibitor, 0.02% NaN) ₃ PBS buffer of 0.5% bsa) was used to 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 phage displayThe nanobody library was shown, split-packed, labeled, and stored at-70 ℃.

6. Identification of phage display nanobody immune libraries

Randomly picking 30 monoclonals from a flat plate to 1mLSB culture medium, and culturing at 37 ℃ until bacterial liquid OD ₆₀₀ About 0.8, the bacterial solution was taken out, gBack was used as a primer (see Table 1), and the sequence of the clone was analyzed by sequencing the sample by a company, and the degree of diversity of the library was determined. As shown in FIG. 1, it can be seen from the amino acid sequences of the selected 30 monoclonal inserts that the conserved regions FR1, FR2, FR3 and FR4 of the nanobody have a high degree of similarity, while the variable regions CDR1, CDR2 and CDR3 are not identical. In particular, the high diversity of the CDR3 region (region 100 to 120 amino acids) allows the phage display library to be rich in diversity, indicating that the constructed phage display nanobody library is well-diversified and can be used for subsequent screening work.

Example 2 aflatoxin M ₁ Panning and identification of anti-idiotype nanobodies

Aflatoxin M ₁ Panning of anti-idiotype nanobodies

With aflatoxin M ₁ Monoclonal antibodies as targets (aflatoxin M) ₁ The monoclonal antibody is secreted by hybridoma cell line 2C9 with the preservation number of CCTCC No. C201018, the hybridoma cell line 2C9 has been preserved in China Center for Type Culture Collection (CCTCC) at 7 months 13 of 2010, and the monoclonal antibody has been produced by anti-aflatoxin M in the patent hybridoma cell line 2C9 ₁ Monoclonal antibodies and uses thereof, application number: 201110108230.6), by decreasing the concentration of coating antigen round by round, competition eluting aflatoxin M ₁ Standard concentration, alternately using blocking reagent, performing affinity enrichment panning to obtain aflatoxin M ₁ Antibodies, i.e., anti-antibodies, also known as anti-idiotype antibodies, to monoclonal antibodies were panning as follows:

(1) Coating: coating aflatoxin M ₁ The monoclonal antibody, 50 mug/mL, 100 mug/well, 6 wells coated, diluted with coating buffer; desorbing the wells, coating 3% BSA/PBS, 300. Mu.LHole, pack 6 holes; the effect of the overnight coating at 4 ℃ is better than that of incubation at 37 ℃ for 2 hours;

(2) Closing: 3% PBSTM, 300. Mu.L/well, after incubation at 37℃for 2h, the plates were washed 3 times by hand;

(3) Adding the mixture into a coating antigen hole: 100 mu L/hole, reacting for 1h at 37 ℃, then reacting for 1h in a shaking table at room temperature, then washing the 6 holes by hands with a gun head with a filter element, and washing the plate for 10 times;

(4) Eluting: preparation of 100ng/mL aflatoxin M ₁ Standard substance, shake table reaction for 30min at room temperature;

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

(6) mu.L of eluate, termed "1st output", was pooled and the first round of panning was completed; 1st output was amplified for the second round of panning;

(7) The "1st output" titer was determined and 10. Mu.L of "1st output" was taken and diluted to 10 in gradient ³ 、10 ⁴ 、10 ⁵ The titer was estimated by taking 10. Mu.L of each of these 3 dilutions, 90. Mu.L of ER2738 (OD 0.8) infected, standing at 37℃for 30min, coating LB-Amp (ampicillin) plates, overnight incubation at 37℃and the number of monoclonal on the next day plates.

In subsequent panning, the coated antibody concentration was gradually reduced and aflatoxin M in the eluate ₁ The concentration was gradually decreased and three rounds of panning were performed, the panning process is shown in table 2 and the panning enrichment results are shown in table 3.

Table 2 panning of phage nanobody libraries

TABLE 3 phage enrichment results per 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 simultaneously inoculating 3mL of SB culture medium;

(2) Shaking culture at 37deg.C for 4-5 hr until OD value is 0.8, adding 30 μ L M KO7 helper phage, standing at 37deg.C for 30min;

(3) Shake culturing at 37deg.C for 1 hr, adding Kana with final concentration of 70 μg/mL, and culturing at 37deg.C;

(4) The next day, 500 mu L of each bacterial liquid is centrifuged, 5000g and 10min are respectively taken, and the supernatant is directly used for Phage-ELISA;

(5) ELISA plates were blocked, 3% PBSTM, 300. Mu.L/well, incubated at 37℃for 1h, and machine washed 3 times;

(6) Adding the phage supernatant of each clone into 3 corresponding holes, adding 50 mu L of standard substances into 50 mu L of 1,4,7 and 10 columns of each hole, adding 50 mu L of 10% methanol/PBS buffer solution into 2,3,5,6,8,9,11,12 columns, and vibrating and uniformly mixing by an enzyme-labeled instrument after adding;

(7) Standing for 1h at 37 ℃ and washing the plate by hand for 10 times;

(8) Secondary antibody, anti-M13 horseradish peroxidase, 1:5000 dilution, incubation at 37 ℃ for 1h;

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

The 30 clones were subjected to phase-ELISA and found to be associated with aflatoxin M ₁ Monoclonal antibodies bind with a large difference in reaction. In the Phage-ELISA assay, the antibody was reacted with OD ₄₅₀ Higher value, at the same time add AFM ₁ Rear OD ₄₅₀ Phage clones with significantly reduced values were judged positive, and as a result, 21 positive clones (clones 1, 2,3, 4, 6, 7, 8, 11,12, 14, 16, 17, 20, 21, 23, 24, 25, 27, 28, 29, 30 in this order) were obtained in total by four rounds of panning as shown in FIG. 2. According to the ELISA result, positive clones are picked out from a bacteria-retaining plate, activated and cultured and sent to Shanghai worker company for sequence analysis, a sequencing primer is gcack, and gene sequencing is carried out on 21 positive clones, and the result shows that 21 monoclonal clones have the same amino acid sequence, and the result shows that 1 strain of nano antibody capable of being specifically combined with the aflatoxin M1 monoclonal antibody is successfully enriched in the screening process, and is named as aflatoxin M ₁ The amino acid sequence of the anti-idiotype nanometer antibody VHH C4 is shown as SEQ ID NO. 1, and the nucleotide sequence is shown as SEQ ID NO. 2。

EXAMPLE 3 Aflatoxin M ₁ Expression and purification of anti-idiotype nanobodies

Preparation of Top10F' competent cells:

(1) Streaking a proper amount of Top10F' on an LB-tetracycline plate, and culturing at 37 ℃ overnight;

(2) Top10F' was selected and cultured in 5mL LB medium at 37℃and 250rpm to OD ₆₀₀ 0.6 to 0.8;

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

(4) Centrifuging at 4deg.C for 5min at 5000g, rapidly returning to ice, discarding supernatant, adding 1mL of pre-cooled 0.1M CaCl ₂ Re-suspending thallus in the solution, sucking, beating, mixing, and ice-bathing for 7min;

(5) Repeating the above operation once;

(6) Centrifuging at 4deg.C for 5min, rapidly returning to ice, thoroughly sucking out liquid, and precipitating with 100 μl of precooled 0.1MCaCl ₂ The solution was resuspended to give Top10F' competent cells.

(II) extraction and transformation of positive phage clone plasmid:

(1) From the glycerol bacteria of VHH C4/ER2738, a proper amount of streaks are selected and cultured at the temperature of 37 ℃ for overnight in LB-ammonia Pingban;

(2) Single colonies of VHH C4/ER2738 were picked up in 2mL SB medium and incubated at 37℃to OD ₆₀₀ Extracting a plasmid of VHH C4 at 0.8;

(3) Adding 1.5 mu LVHH C4 plasmid into 100 mu L Top10F' competent cells prepared above on ice, gently mixing, and standing on ice for 30min;

(4) Heat-shocking at 42 ℃ for 90s, quickly putting back on ice, and standing for 5min;

(5) In an ultra-clean workbench, 700 mu L of LB culture medium is added into each centrifuge tube, and recovery culture is carried out for 45min at 37 ℃ and 200 rpm;

(6) At 4℃12000g was centrifuged for 4min, the supernatant was aspirated, 100. Mu.L of LB medium was added to the tube to resuspend the pellet, and the pellet was spread on LB-ampicillin plates after mixing, and incubated overnight at 37 ℃.

Induced expression and purification of nanobodies

In VHHOn the plate of C4/Top10F', selecting single colony to 3mLSB culture medium, shaking at 37deg.C and 250rpm overnight, transferring 200 μL of overnight bacteria to 200mLSB culture medium, and continuously culturing to OD ₆₀₀ 200. Mu.L of 1M IPTG was added and induced overnight at 37 ℃. Centrifuging at 4deg.C for 8000g and 15min for collecting bacteria, adding lysate B-PER according to thallus precipitation quality of 20mL/g, dispersing and precipitating thoroughly, slowly shaking at room temperature for 10min, centrifuging for 20min at 12000g, and collecting supernatant as crude extractive solution of nanometer antibody. The crude extract was dialyzed against 0.01M PBS7.4 and then passed through a 0.22 μm aqueous filter for purification on a nickel column.

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

(1) Sterile ddH with 10 column volumes in sequence ₂ O, 0.01mol/L PBS7.4 balance column;

(2) Adding the crude extract after filtration and sterilization into a chromatographic column, mixing with a filler, and shaking for 1h at room temperature to fully combine the nano antibody and the filler;

(3) Loading the mixture into a column, and balancing the column with 10 times of 0.01mol/L PBS 7.4;

(4) Preparing 20mM, 40mM and 300mM imidazole-PBS 7.4 respectively by 1M imidazole in 10mL, passing through 0.22 μm water phase filter membrane, performing gradient elution by using the water phase filter membrane as eluent, and collecting effluent by using a 2mL centrifuge tube;

(5) The effluent was not collected by passing 10mL of 20mM imidazole-PBS 7.4 through the column;

(6) The column was run with 10mL of 40mM imidazole-PBS 7.4 and the first 3mL of effluent was collected

(7) Passing through the column with 10mL of 300mM imidazole-PBS 7.4, and collecting the whole effluent;

(8) Washing the column with 10 times of 1M imidazole-PBS 7.4, and collecting no effluent liquid to completely elute nonspecifically bound hybrid protein;

(9) Sequentially using 10 times of PBS and 10 times of ddH ₂ And (3) washing the column with 20% ethanol with the volume of O being 10 times that of the column, and finally preserving the column with an equal volume of 20% ethanol water seal column.

Performing SDS-PAGE electrophoresis analysis on the effluent of each tube, mixing the effluent with obvious nano antibody bands, dialyzing overnight at 4 ℃ in PBS buffer solution, concentrating by using an ultrafiltration tube to obtain VHH C4 nano antibody solution, and sub-packaging at-20 ℃ for preservation.

EXAMPLE 4 specificity analysis of anti-idiotype nanobody VHH C4

(1) Specificity analysis of anti-idiotype nanobody VHH C4: aflatoxin B with the concentration of 0.2 mug/mL is prepared by using coating buffer solution ₁ Antigen, AFB ₁ BSA, ochratoxin A antigen, i.e.OTA-BSA, T-2 toxin antigen, i.e.T-2-BSA and aflatoxin M ₁ Antigen, AFM ₁ -BSA, coated elisa plate overnight at 4 ℃; the next day, the wells were blocked with 3% nonfat milk powder/conventional phosphate buffer conventional phosphate Tween buffer solution, 300. Mu.L/well, incubated at 37deg.C for 1h, and anti-aflatoxin B was then assayed ₁ Aflatoxin M ₁ Four monoclonal antibodies to ochratoxin OTA and T-2 toxin were diluted in a conventional phosphate buffer at a doubling ratio starting at 10. Mu.g/mL and incubated for 1h at 37 ℃; adding goat anti-mouse monoclonal antibody marked by horseradish peroxidase, and incubating for 1h at 37 ℃; adding the color development solution, developing at 37deg.C for 15min, adding stop solution, and measuring OD ₄₅₀ 。

Coating and sealing the mixture according to the method, sequentially diluting VHH C4 by three times by using a conventional phosphate buffer solution, and preparing the aflatoxin B according to the optimized concentration ₁ Aflatoxin M ₁ Four monoclonal antibodies of ochratoxin OTA and T-2 toxin are respectively added into each hole by 50 mu LVHH C4 diluent and 50 mu L of corresponding monoclonal antibody, and the mixture is uniformly mixed by shaking by an enzyme-labeling instrument and incubated for 1h at 37 ℃; then, diluting according to a ratio of 1:5000, adding horseradish peroxidase-labeled goat anti-mouse antibody, and reacting for 1h at 37 ℃; color development was performed in the same manner as above, and OD was measured ₄₅₀ Values.

The test results are shown in FIG. 3, and FIG. 3 shows that VHH C4 can inhibit aflatoxin M ₁ Monoclonal antibodies and antigen AFM ₁ Binding of BSA, with increasing VHH C4 concentration, inhibits more and more markedly against aflatoxin B ₁ No inhibition of binding of the OTA and T-2 toxin monoclonal antibodies to the corresponding antigen indicates that VHH C4 has good selectivity and is only compatible with aflatoxin M ₁ Monoclonal antibodiesThe variable region binds specifically.

EXAMPLE 5 nanobody VHH C4 as aflatoxin M ₁ ELISA (VELISA) method establishment of substituted antigen

Nanobody VHH C4 as aflatoxin M ₁ The course of the competition-inhibiting ELISA reaction for the surrogate antigen was as follows: (1) Aflatoxin M ₁ Substitution of aflatoxin M after dilution of anti-idiotype nanobody ₁ Coating antigen on an ELISA plate, and coating at 4 ℃ overnight; the next day, sealing with sealing liquid, and incubating for 1h at 37 ℃; (2) Adding methanol/PBS 7.4 buffer solution into the enzyme-labeled well, and adding aflatoxin M ₁ The monoclonal antibody and the competitor are diluted by PBS7.4 buffer solution and added into an enzyme-labeled hole for incubation for 1h at 37 ℃; (3) Adding horseradish peroxidase-labeled goat anti-mouse antibody, and incubating for 1h at 37 ℃; (4) Adding color developing solution, reacting at 37deg.C for 15min, and measuring OD ₄₅₀ 。

VELISA Condition optimization

(1) Optimal VHH coating concentration and aflatoxin M ₁ Monoclonal antibody working concentration: sequentially diluting VHH C4 to the concentration of 20, 10, 5, 0.06, 2.5 and 1.25 mug/mL respectively, coating an ELISA plate, and coating the VHH C4 nanobody and aflatoxin M respectively by using a chessboard method ₁ Optimizing working concentration of monoclonal antibody, and taking OD ₄₅₀ Nanobody concentration and aflatoxin M with a value of about 1.0 ₁ The monoclonal antibody concentration is the optimal working concentration, aflatoxin M ₁ Monoclonal antibodies were 9-fold diluted to concentrations of 100, 11.11, 1.23, 0.14, and 0.02. Mu.g/mL, respectively, and indirect ELISA assays were performed at these concentrations to establish standard curves (FIG. 4). According to different color values of each combination, the working concentration of VHH C4 is finally selected to be 5 mug/mL, which corresponds to aflatoxin M ₁ The monoclonal antibody concentration was 2. Mu.g/mL.

(2) Optimal blocking reagent: at optimal VHH C4 coating concentration and aflatoxin M ₁ The effect of different blocking reagents on the VELISA response was investigated at the working concentration of monoclonal antibodies. Standard curves were established with 3% bsa, 1.5% ova and 3% skim milk powder, respectively (fig. 5), and 3% skim milk powder was determined to be the optimal blocking solution.

(3) Optimum pH: diluting aflatoxin M with phosphate buffer solution with pH of 5,6, 7, 8 ₁ Monoclonal antibody, aflatoxin M was diluted in multiple ratios with each buffer containing 10% methanol ₁ Standard, developed VELISA standard curve as in fig. 6, the response sensitivity was highest when ph=7.

(4) Optimal salt ion concentration: respectively add ddH ₂ O,10mmol/LPBS,20mmol/L PBS,40mmol/L PBS four solutions for diluting aflatoxin M ₁ Monoclonal antibody, aflatoxin M was diluted in multiple ratios with each buffer containing 10% methanol ₁ Standard, set up VELISA to detect aflatoxin M ₁ Is a standard curve of (2). As shown in FIG. 7,10 mmol/L PBS was the optimal reaction buffer.

(5) Optimal methanol concentration: the optimal nanometer antibody coating concentration and aflatoxin M are optimized ₁ Under the conditions of monoclonal antibody working concentration, blocking reagent and optimal phosphate buffer, the influence of different methanol concentrations on 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%, respectively ₁ And aflatoxin M ₁ Monoclonal antibodies, inhibition curves were established (FIG. 8), where IC was derived from the curves at methanol concentrations of 5% and 10% ₅₀ IC very close to, but at 10% methanol concentration ₅₀ Smaller and the trend of the competition fit curve is relatively better.

(6) Cross-reactivity of VELISA: VHH C4 is taken as a substitute antigen, and AFB is selected according to the optimization condition ₁ 、AFB ₂ 、AFG ₁ 、AFG ₁ 、AFM ₁ And AFM ₂ Six mycotoxins were used as competitors to determine the cross-reactivity of VELISA. As can be seen from FIG. 9, the ELISA was applied to AFM ₁ Higher sensitivity, VHH C4 IC ₅₀ The value reaches 0.25ng/mL, and the AFB is used for the preparation of the medicine ₁ 、AFB ₂ 、AFG ₁ 、AFG ₁ And AFM ₂ No cross reaction exists, the VELISA has good specificity, and can detect aflatoxin M in dairy products in a targeted manner ₁ 。

EXAMPLE 6 establishment of VHH-ELISA sample analysis method

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

Directly taking 10mL of blank sample of milk and yoghurt, centrifuging at 4deg.C for 10min at 5000g, and directly diluting aflatoxin M with the supernatant of the removed milk fat layer ₁ Standard substances, establishing a VELISA competition inhibition curve of the matrix extracting solution; for a blank sample of milk powder, accurately weighing 10g on an analytical balance, dissolving in 50 ℃ preheated pure water, fixing the volume to 100mL, fully and uniformly mixing, taking 10mL, centrifuging at 4 ℃ for 10min with 5000g, and directly diluting aflatoxin M with the lower supernatant of the milk fat layer removed ₁ Standard, a veliosa competition inhibition curve for matrix extract was established and compared with the standard curve established in the reaction system without matrix, and the results showed that all three samples had less matrix effect on veliosa, making the matrix curve closer to that without matrix, as shown in fig. 10.

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

In order to evaluate the accuracy of the VELISA detection system, an addition recovery test of milk, yoghurt and milk powder samples was performed using a newly established VELISA method. Aflatoxin M of 400 ng/L, 600 ng/L and 800ng/L are added to blank milk, yoghurt and milk powder samples respectively ₁ Standard solution, and AFM in sample extract liquid is detected by using newly established VELISA method ₁ The content and the result are shown in Table 4.AFM (atomic force microscope) ₁ The addition recovery test of (2) shows that the sample addition recovery rate is between 84.1 and 119.1 percent, which indicates that the nano antibody VELISA-based method established in the research can be applied to aflatoxin M ₁ Is used for pollution monitoring.

TABLE 4 nanobody VHH-EILSA based sample addition recovery assay

Note that: each result is an average of three replicates

It is apparent that the above examples are only examples given for clarity of illustration and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. And thus obvious variations or modifications to the disclosure are within the scope of the invention.

< 110 > institute of oil crop and oil crop of national academy of agricultural sciences

< 120 > an aflatoxin M1 anti-idiotype nano-antibody and preparation method thereof

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Claims

1. Aflatoxin M ₁ Aflatoxin M as non-disease diagnostic therapeutic target by anti-idiotype nano antibody ₁ Use of an antigen substitute, aflatoxin M ₁ The anti-idiotype nanometer antibody is aflatoxin M ₁ The amino acid sequence of the anti-idiotype nano antibody VHH C4 is shown as SEQ ID NO. 2.

2. Based on aflatoxin M ₁ Anti-idiotype nano antibodyThe VHH-ELISA method for the purpose of non-disease diagnosis and treatment of replacement antigens comprises the following steps: aflatoxin M ₁ Anti-idiotype nano antibody for replacing aflatoxin M ₁ Antigen coated ELISA plate, sample to be detected as competitive substance is used for competitive inhibition ELISA reaction, and the binding rate B/B is obtained by detection ₀ Value based on aflatoxin M ₁ Concentration-binding ratio B/B ₀ Obtaining aflatoxin M by a value standard curve ₁ Concentration of the aflatoxin M ₁ The anti-idiotype nanometer antibody is aflatoxin M ₁ The amino acid sequence of the anti-idiotype nano antibody VHH C4 is shown as SEQ ID NO. 2.

3. The VHH-ELISA method according to claim 2, characterized in that: the competition inhibition ELISA reaction specifically comprises the following steps: aflatoxin M ₁ The anti-idiotype nano antibody VHH C4 is coated on an ELISA plate after being diluted, and is coated overnight at 4 ℃; the next day, sealing with sealing liquid, and incubating for 1h at 37 ℃; adding methanol/PBS buffer solution into the enzyme-labeled well, and adding aflatoxin M ₁ The monoclonal antibody and the competitor are diluted by PBS7.4 buffer solution and added into an enzyme-labeled hole for incubation for 1h at 37 ℃; adding horseradish peroxidase-labeled goat anti-mouse antibody, and incubating for 1h at 37 ℃; adding color developing solution, reacting at 37deg.C for 15min, and measuring OD ₄₅₀ Obtaining the binding ratio B/B ₀ Values.

4. The VHH-ELISA method according to claim 2 or 3, characterized in that: the aflatoxin M ₁ The coating concentration of the anti-idiotype nano antibody is 1.25 mu g/mL-20 mu g/mL.

5. The VHH-ELISA method according to claim 3, characterized in that: the blocking solution was 3% BSA, 1.5% OVA or 3% skim 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 2, characterized in that: the sample to be detected is a milk product, including yoghurt, milk or milk powder.