Toxiflavin hapten as well as preparation method and application thereof

Abstract

The invention relates to the technical field of immunology, in particular to a toxoflavin hapten as well as a preparation method and application thereof. The toxoflavin hapten provided by the invention is disclosed for the first time, the toxoflavin artificial antigen obtained by coupling the toxoflavin hapten with a carrier protein can be used as a coating antigen or an immunizing antigen, and a specific nano antibody with high titer and high affinity can be obtained after an animal is immunized. The toxoflavin hapten and the toxoflavin nano antibody rapidly prepared through B cell sorting and high-throughput sequencing provide materials for establishing a rapid, simple, sensitive and specific toxoflavin detection method.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology, and in particular to a toxin hapten and a preparation method and application thereof. Background Art

[0002] Small molecule compounds such as antimicrobial drugs, mycotoxins, environmental pollutants, illegal additives, etc. are widely present in biological samples such as the environment and food, and it is very necessary to detect them quickly and accurately. At present, the methods for detecting small molecule compounds in biological samples mainly include thin layer chromatography, spectrophotometry, high performance liquid chromatography, high performance liquid chromatography-mass spectrometry, and biosensor determination. These instrumental analysis methods have high sensitivity and good reproducibility, but they are time-consuming, labor-intensive, and expensive to detect. They are not suitable for on-site detection of a large number of samples. There is an urgent need to establish a rapid, accurate, reliable, and highly sensitive rapid analysis method.

[0003] Immunoassay based on antigen-antibody specific reaction is sensitive, rapid, low-cost and easy to operate, and is suitable for rapid on-site detection. Among them, antibodies as core reagents determine the sensitivity, specificity and stability of the method. Small molecule compounds have small molecular weights and cannot directly induce strong immune responses in the body. They need to be designed as haptens and coupled with carrier proteins to become complete antigens to induce the production of high-performance antibodies. Traditional monoclonal antibodies and polyclonal antibodies derived from mice and rabbits are composed of two heavy chains and two light chains. They have complex structures, poor stability, high production costs, and are not easy to be transformed in vitro, which greatly limits their application in actual sample detection. Heavy chain antibodies (HCAb) that naturally lack light chains exist in camelids. The variable domain of the heavy chain of HCAbs (VHH) can be obtained through genetic engineering technology. Because the size of VHH is at the nanometer level, it is also called nanoantibody. It has high stability, simple structure, easy transformation, and low production cost. It makes up for the shortcomings of traditional antibodies and has important practical value in immunoassay. Phage display technology is currently the most commonly used technology for preparing nanoantibodies, but this technology has many disadvantages. For example, antibody genes need to go through processes such as enzyme ligation and plasmid transformation, and the library capacity and diversity are limited; prokaryotic and eukaryotic codon usage preferences and folding and modification mechanisms are different, and some antibodies cannot be effectively displayed; high-affinity clones may exist at a lower abundance and are easily covered by low-affinity clones; multiple rounds of affinity enrichment and selection are required, which are cumbersome, time-consuming and extremely dependent on the growth status of microorganisms. With the rapid development of biotechnology and sequencing technology, B cell sorting and high-throughput sequencing technology (high throughput sequencing, HTS) are becoming more and more mature. The specific operation of obtaining nanoantibodies based on B cell sorting and high-throughput sequencing methods is: targeting specific B cells, amplifying variable region genes by PCR, and then using high-throughput sequencing technology combined with bioinformatics analysis to quickly obtain nanoantibodies. This process does not involve library capacity loss steps such as plasmid transformation, does not depend on the growth status of microorganisms, and does not require complex affinity enrichment and selection processes. It has the advantages of low cost, simple operation and fast speed.

[0004] Toxoflavin is a cytotoxin produced after the death of Gram-negative bacteria. Its molecular formula is C7H7N5O2 and its molar mass is 193.21g / mo1. It is a small molecule compound. Toxoflavin is a serious food contaminant with extremely strong toxicity and mutagenicity. It can cause pathological damage to animal vascular smooth muscle, kidney, lung, heart and other organs. Toxoflavin can be found in cereals such as wheat, barley, corn, oats, soybeans and rice. When people eat food contaminated with toxoflavin, they will suffer from stomach and abdominal discomfort, and even symptoms such as liver and kidney damage and toxic shock. It will cause damage to the human digestive system, immune system, nervous system and circulatory system, and pose a threat to public health and food safety. Therefore, it is necessary to monitor the pollution of toxins to protect the health of consumers and food safety. At present, there is no research on the preparation of toxoflavin antibodies.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a toxin hapten and a preparation method and application thereof.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a toxin hapten, the structural formula of which is Formula I:

[0009]

[0010] The toxoflavin hapten provided by the invention is disclosed for the first time. The toxoflavin artificial antigen obtained by coupling it with a carrier protein can be used as a coating antigen or an immune antigen. After immunizing animals, specific nano antibodies with high titer and affinity can be obtained.

[0011] In a second aspect, the present invention provides a method for preparing the toxoplasmin hapten described in the first aspect, comprising the following steps: using the compound of formula II and the compound of formula III as raw materials to synthesize the compound of formula IV; using the compound of formula IV and the compound of formula V as raw materials to synthesize the compound of formula VI; reacting the compound of formula VI with nitrite ions to generate the compound of formula VII and the compound of formula I;

[0012]

[0013]

[0014] In a more specific and preferred embodiment, the method for preparing the toxoplasmin hapten comprises the following steps:

[0015] S1. Add 25g of compound 1 (as shown in formula II) and 28g of compound 2 (as shown in formula III) to anhydrous ethanol, stir at 90-95°C for 5-6h, and identify by thin layer chromatography that no compound 1 remains. Cool at room temperature for 1h to precipitate a white solid; wash the mixture twice with 30mL of anhydrous ethanol, and dry at 40-50°C for 1-2h to obtain 20.11g of compound 3 (as shown in formula IV) as a white solid product;

[0016] S2. Add 4.50 g of compound 4 (as shown in formula V) and 5.12 g of compound 3 (as shown in formula IV) to 150 mL of anhydrous ethanol, and stir at 55-60°C for 4-5 h. After identification by thin layer chromatography, no compound 3 remains. Cool the mixture to 30-35°C, wash twice with 30 mL of anhydrous ethanol, and dry at 40-50°C for 2-3 h to obtain 6.55 g of yellow solid compound 5 (as shown in formula VI);

[0017] S3, add 5.40g of compound 5 to 100mL of acetic acid solution, cool to 5-10°C, slowly add 1.85g of NaNO2 and 20mL of H2O. Stir at room temperature overnight to obtain a crude product. Add 50mL of H2O to the crude product, stir for 1h, wash twice with 30mL of anhydrous ethanol, and dry at 40-50°C for 2-3h to obtain a mixture of the compound shown in Formula VII and toxin hapten 1 (shown in Formula I);

[0018] S4. The mixture is purified by high performance liquid chromatography to obtain a yellow solid product which is the hapten of toxin represented by formula I.

[0019] The invention discloses for the first time a method for preparing the toxoflavin hapten, and can stably prepare the toxoflavin hapten.

[0020] In a third aspect, the present invention provides an artificial toxoflavin antigen, which includes the toxoflavin hapten described in the first aspect and a carrier protein; the toxoflavin hapten is coupled to the carrier protein.

[0021] In the present invention, the artificial antigen of toxoflavin can be used as an immunogen or a coating. By immunizing animals with the artificial antigen provided by the present invention, specific nano antibodies with high titer and high affinity can be obtained. The artificial antigen provided by the present invention can be used as a coating to detect toxoflavin.

[0022] Preferably, the carrier protein is selected from bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH).

[0023] In a fourth aspect, the present invention provides a method for synthesizing the toxoflavin artificial antigen described in the third aspect, comprising the following steps: coupling the carrier protein to the carboxyl carbon of the toxoflavin hapten using an active ester method.

[0024] In a fifth aspect, the present invention provides the use of the toxoflavin hapten described in the first aspect or the toxoflavin artificial antigen described in the second aspect in the preparation of toxoflavin antibodies;

[0025] Preferably, the toxin antibody is a polyclonal antibody, a monoclonal antibody or a nanobody.

[0026] In a sixth aspect, the present invention provides a method for preparing a vesin nanobody, comprising the following steps: immunizing an animal with the vesin artificial antigen described in the second aspect as an immunogen; then performing high-throughput sequencing analysis on specific B cells in the peripheral blood of the immunized animal to obtain a nanobody;

[0027] Preferably, the animal is an alpaca.

[0028] The conjugate of the hapten and the carrier protein provided by the present invention is used to immunize alpacas, and the toxin nanobody is quickly prepared by using B cell sorting and high-throughput sequencing technology. The IC 50 The value is 1.21ng / mL, and the linear detection range is 0.27-5.34ng / mL. It has high practical value. It only takes 1 month from immunization to antibody preparation. It has good application prospects in the rapid detection of toxin.

[0029] In a seventh aspect, the present invention provides a toxin nanobody, the amino acid sequence of which is shown in SEQ ID NO.1.

[0030] MQVQLVESGGGSVQAGGSLILSCAASGRTFM IKRMG WFRQAPGKEREFVAAINYYDRTDYADSVKGRFTISGNHAKEAIYLQMNSLKSEDTAIYYCAADQSYVLSGSQYTYWGQGTQVTVSS(SEQ ID NO.1)

[0031] Among them, the amino acid sequence of the CDR1 region is: IKRMG (SEQ ID NO.3); the amino acid sequence of the CDR2 region is: AINYYDRTDYADSVKG (SEQ ID NO.4); and the amino acid sequence of the CDR3 region is: DQSYVLSGSQYTY (SEQ ID NO.5).

[0032] The present invention uses toxin as a model small molecule compound, and prepares a toxin nanoantibody as shown in SEQ ID NO.1 based on B cell sorting and high-throughput sequencing. The toxin nanoantibody has high affinity and strong specificity, and can be used to detect toxin contamination in food, providing core recognition materials for its pollution monitoring.

[0033] In an eighth aspect, the present invention provides a biological material for encoding the cytotoxic Taurantil nanobody described in the seventh aspect, wherein the biological material is a nucleic acid with a sequence as shown in SEQ ID NO.2; or, the biological material contains a nucleic acid with a sequence as shown in SEQ ID NO.2.

[0034] caggtgcagctggtggagtctgggggaggctcggtgcagcctggaggatctctgagactctcctgcacagcctctagatacacctatagtcgcggatgcatgggctggttccgccagcctccagggaagggccgcgagggggtcgcggctatttatggtggtaatggtggcacatactatgccgactcc gtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacagcctgaagcctgaggacactggcatgtactactgtgcggcagaaacccatactggtggttactgttacagttgggctgactttaattattggggccaggggacccaggtcaccgtctcctca(SEQ ID NO.2)

[0035] Preferably, the biological material is recombinant DNA, an expression cassette, a plasmid vector, a viral vector, an engineered bacterium or a transgenic cell line.

[0036] In a ninth aspect, the present invention provides a toxin detection reagent or kit, the active ingredient of which includes the toxin nanobody described in the seventh aspect.

[0037] In the tenth aspect, the present invention provides any of the following uses of the venom nanobody described in the seventh aspect, the biomaterial described in the eighth aspect, or the reagent or kit described in the ninth aspect:

[0038] (1) Used for the detection of toxoplasmin;

[0039] (2) For preparing a toxin detection reagent or kit;

[0040] (3) Used for the enrichment and purification of toxic flavonoids;

[0041] (4) Enrichment and purification reagents for preparing toxin.

[0042] Beneficial effects:

[0043] The present invention discloses for the first time a novel cypermethrin hapten, which is coupled with a carrier protein to obtain a conjugate (i.e., cypermethrin artificial antigen) which can be used as a coating antigen or an immune antigen, and has a good application effect. After immunizing an animal with the cypermethrin artificial antigen provided by the present invention, a specific nano antibody with high titer and high affinity can be obtained. In addition, the present invention immunizes an alpaca with the cypermethrin artificial antigen, collects peripheral blood for cypermethrin-specific B cell sorting, and uses high-throughput sequencing technology to sequence the cypermethrin-specific B cells, and this method can also obtain a nano antibody with high specificity and high affinity. The cypermethrin hapten provided by the present invention and the cypermethrin nano antibody rapidly prepared by B cell sorting and high-throughput sequencing provide materials for establishing a rapid, simple, sensitive, and specific cypermethrin detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0045] Figure 1 This is a flow chart for preparing the toxic flavin hapten represented by Formula I in Example 1 of the present invention.

[0046] Figure 2 This is a titer curve of alpaca serum in Example 3 of the present invention.

[0047] Figure 3 This is the percentage of high-frequency nanobodies in the antibody library obtained by sequencing in Example 5 of the present invention.

[0048] Figure 4 This is a diagram of the cytotoxic toxin nanobody expressed by the prokaryotic expression system in Example 6 of the present invention; wherein lane M is a protein marker, lane 1 is a crude extract before purification, and lane 2 is a purified nanobody.

[0049] Figure 5 This is a standard curve diagram of detecting toxoflavin using toxoflavin nanoantibodies in Example 6 of the present invention. DETAILED DESCRIPTION

[0050] The technical solutions provided by the present invention are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used can be obtained from commercial sources.

[0051] Example 1 Preparation of toxic flavin hapten

[0052] The preparation steps of toxic flavin hapten are as follows Figure 1 As shown, the specific steps include:

[0053] S1. Add 25 g of compound 1 (as shown in formula II) and 28 g of compound 2 (as shown in formula III) to anhydrous ethanol, stir at 90-95°C for 5-6 h, and identify by thin layer chromatography that no compound 1 remains. Cool at room temperature for 1 h to precipitate a white solid; wash the mixture twice with 30 mL of anhydrous ethanol, and dry at 40-50°C for 1-2 h to obtain 20.11 g of compound 3 (as shown in formula IV) as a white solid product.

[0054]

[0055]

[0056] S2. Add 4.50 g of compound 4 (as shown in formula V) and 5.12 g of compound 3 (as shown in formula IV) to 150 mL of anhydrous ethanol, and stir at 55-60° C. for 4-5 h. Thin layer chromatography showed that no compound 3 remained. The mixture was cooled to 30-35° C., washed twice with 30 mL of anhydrous ethanol, and dried at 40-50° C. for 2-3 h to obtain 6.55 g of yellow solid compound 5 (as shown in formula VI).

[0057]

[0058] S3. Add 5.40 g of compound 5 to 100 mL of acetic acid solution, cool to 5-10° C., slowly add 1.85 g of NaNO2 and 20 mL of H2O. Stir overnight at room temperature to obtain a crude product. Add 50 mL of H2O to the crude product, stir for 1 h, wash twice with 30 mL of anhydrous ethanol, and dry at 40-50° C. for 2-3 h to obtain a mixture of the compound shown in Formula VII and toxin hapten 1 (shown in Formula I).

[0059]

[0060] S4. The mixture is purified by high performance liquid chromatography to obtain a yellow solid product, which is the hapten of toxin represented by formula I.

[0061] Example 2 Preparation of artificial toxoplasmin antigen

[0062] This embodiment provides two methods for preparing artificial antigens of cypermethrin (coating agent and immunogen). The only difference between the two preparation methods is the type of carrier protein (KLH is used as the immunogen carrier protein, BSA is used as the coating agent carrier protein, and both coupling methods are active ester method).

[0063] (I) Synthesis of toxic flavonoids coating

[0064] S1, dissolving 29.25 mg of the compound of formula I prepared in Example 1 in 1 mL of DMF, adding 12 mg of NHS and 95.75 mg of EDC, and stirring at room temperature overnight to obtain solution I;

[0065] S2, add 111.70 mg BSA into 3 mL PBS buffer and fully dissolve to obtain solution II;

[0066] S3. Slowly add solution I to solution II, slowly stir at 4°C for 24 hours, then put into a dialysis bag, dialyze in PBS at room temperature for 72 hours (change the solution 6 times in the middle), and obtain the original solution of toxaphene coating, which is stored at -20°C.

[0067] The toxic flavonoids coating material synthesized from the compound shown in formula I is referred to as TXF-BSA.

[0068] (II) Synthesis of toxic flavin immunogen

[0069] Compared with the above-mentioned synthesis method of toxic flavin coating source, the only difference is that KLH is used instead of BSA.

[0070] The synthetic tadalafil immunogen of the compound shown in formula I is referred to as TXF-KLH.

[0071] Example 3 Alpaca immunization and detection of antiserum

[0072] In this example, TXF-KLH prepared in Example 2 was mixed with Freund's adjuvant to immunize alpacas. In the first immunization, 300 μg of the immunogen was mixed with an equal volume of Freund's complete adjuvant, and after sufficient emulsification, multiple injections were made into the alpaca's neck and back, intradermally or subcutaneously. A booster immunization was performed in the second week, with the same immunization dose and the adjuvant replaced with Freund's incomplete adjuvant. Peripheral blood was collected from the alpacas one week after each immunization, and the serum titer was monitored by ELISA (see Figure 2 ).

[0073] The titer of alpaca antiserum was determined by ELISA, and the specific operation was as follows:

[0074] (1) Coating: Dilute the homologous coating source TXF-BSA to 0.1 μg / mL with coating solution, add 100 μL to each well of the ELISA plate, incubate at 37°C for 2 h, pour out the liquid in the well, wash three times with washing solution, 280 μL per well, and pat dry on absorbent paper.

[0075] (2) Blocking: Add 150 μL of blocking solution to each well, incubate at 37°C for 1 h, pour out the liquid in the well, and pat dry on absorbent paper.

[0076] (3) Binding: Add 50 μL of PBS and 50 μL of graded diluted antiserum to each well in sequence, incubate at 37°C for 30 min, pour out the liquid in the well, wash three times with washing solution, and pat dry.

[0077] (4) Adding enzyme label: Add 100 μL of 5000-fold diluted HRP-labeled goat anti-alpaca enzyme-labeled antibody to each well, incubate at 37°C for 30 min, pour out the liquid in the well, wash four times with washing solution, and pat dry.

[0078] (5) Color development: Add 100 μL of freshly prepared TMB color development solution to each well and incubate at 37°C in the dark for 10 min.

[0079] (6) Termination: Add 50 μL of 2M H2SO4 to each well.

[0080] (7) Determination: Use an ELISA reader to read the OD value of each well at 450 nm (dual wavelength: 630 nm is the reference filter wavelength).

[0081] (8) The antiserum titer curve was drawn using OriginPro8.5, with the antiserum dilution factor as the horizontal axis and the average OD value as the vertical axis. The antiserum titer in this study was defined as: when the concentration of the fixed homologous coating original TXF-BSA was 0.1 μg / mL, the antiserum dilution factor corresponding to the final OD value of 1.5 was the antiserum titer.

[0082] Example 4 Isolation of specific B cells from alpaca peripheral blood

[0083] In this example, peripheral blood of the alpaca after the second immunization in Example 3 was collected, lymphocytes were separated by density gradient centrifugation, and the lymphocytes were incubated with fluorescein EDF-labeled toxin hapten at 37°C for 2 hours, and then specific B cells with fluorescein labeled on the surface were sorted by flow cytometry. The specific operation steps are as follows:

[0084] (1) Take fresh anticoagulated alpaca peripheral blood and dilute the whole blood with an equal volume of PBS.

[0085] (2) Take a 50 mL sterile centrifuge tube, add an equal volume of Ficoll separation solution into the centrifuge tube, slowly and steadily spread the diluted peripheral blood on the Ficoll separation solution, and centrifuge at a balanced level.

[0086] (3) After centrifugation, the centrifuge tube is divided into four layers from top to bottom. The first layer is the plasma layer, the second layer is the lymphocyte layer, the third layer is the separation fluid layer, and the fourth layer is the red blood cell layer.

[0087] (4) Aspirate the upper plasma layer and discard it, collect the lymphocyte layer into another 50 mL centrifuge tube, add PBS to 45 mL, mix well, and centrifuge for 10 minutes.

[0088] (5) Quickly discard the supernatant in a clean bench, gently tap the lymphocytes at the bottom of the centrifuge tube, add PBS to 45 mL, mix well, and centrifuge for 10 minutes.

[0089] (6) Add 2 mL of PBS and 10 μL of trypan blue, mix well, and count the cells under a microscope using a cell counting plate; adjust the cell concentration to 2 × 10 6 cells / mL.

[0090] (7) Lymphocytes were incubated with toxic flavin hapten labeled with fluorescein EDF at 37°C for 2 h, and then the cells were sorted using a flow cytometer.

[0091] Example 5 High-throughput sequencing analysis of specific B cells

[0092] In this embodiment, the RNA of specific B cells was extracted by Trizol method and reverse transcribed into cDNA. A DNA library was constructed and sent to a sequencing company for high-throughput sequencing. The amino acid sequence with the highest abundance was shown in SEQ ID NO.1 (the nucleotide sequence was shown in SEQ ID NO.2), which was the toxin-specific nanobody.

[0093] The specific steps are as follows:

[0094] (1) Transfer the cells to a 1.5 mL centrifuge tube, add 1 mL of Trizol, mix well, and let stand at room temperature for 5 min.

[0095] (2) Add 0.2 mL of chloroform, shake for 15 seconds, let stand for 2 minutes, add 0.5 mL of isopropanol, gently mix the liquid in the tube, and let stand at room temperature for 10 minutes.

[0096] (3) Centrifuge at 4°C, 12000 g for 10 min, discard the supernatant, add 1 mL of 75% ethanol, gently wash the precipitate, centrifuge at 4°C, 7500 g for 5 min, discard the supernatant, air-dry naturally, add 50 μL of DEPC H2O to dissolve, and obtain total lymphocyte RNA.

[0097] (4) The RNA was reverse transcribed into cDNA, the resulting cDNA library was sequenced using the Illumina MiSeq platform, and the high-throughput sequencing results were analyzed using bioinformatics.

[0098] The main contents of the analysis of nanobody high-throughput sequencing results include the following two aspects:

[0099] ① Sequence abundance analysis: analyze the proportion of nanobodies with each unique sequence in the antibody library and sort them from high to low according to abundance;

[0100] ② Somatic hypermutation analysis of nanoantibodies, including mutation position and mutation frequency. Generally, nanoantibodies with high mutation frequency have stronger antigen binding activity.

[0101] The results of the proportion of high-frequency nanobodies in the antibody library analyzed by sequencing results are shown in Figure 3 , No. 1 clone sequence accounts for 57.2% of the total nanobody sequences, and other clone sequences account for a smaller proportion. After the above analysis, the possible toxin flavin nanobody No. 1 sequence (amino acid sequence as shown in SEQ ID NO.1) was artificially synthesized and cloned into the corresponding expression vector for activity identification.

[0102] Example 6 Expression, purification and activity identification of cytoxan nanobody

[0103] In this example, the venom nanobody was constructed into the expression vector pET-22b, and after the sequencing was correct, it was induced to express in the expression host BL21.

[0104] The specific steps for inducing expression are as follows:

[0105] Dilute the correct sequence sample bacterial solution with 2YT medium at 1:100, shake the bacteria at 37℃ and 220rpm until the OD value of the bacterial solution at 600nm is 0.6-1.0, add IPTG with a final concentration of 1mmol / L to induce expression for 16h, and collect the bacterial solution. Centrifuge the bacterial solution induced and expressed by IPTG at 6000rpm in a centrifuge for 20min to collect the bacteria, wash it once with PBS, and centrifuge it again to collect the bacteria; use 10mL of PBS to blow and mix the bacterial solution, put it on ice, and ultrasonically disrupt it for 10min according to the program of "ultrasonic oscillation time 5s; interval time 5s; protection temperature 60℃; ultrasonic power 10%". Put the disrupted bacterial solution in a centrifuge at 12000rpm and centrifuge it for 30min. The supernatant is purified by affinity chromatography. The purification experiment results are shown in Figure 4 .

[0106] The activity of the venom nanobody was identified by indirect ELISA method. The specific operation steps are as follows:

[0107] (1) Coating: Dilute the coating original TXF-BSA to the optimal working concentration with coating solution, add 100 μL to each well of the ELISA plate, incubate at 37°C for 2 h, pour out the liquid in the well, wash three times with washing solution, 280 μL per well, and pat dry on absorbent paper.

[0108] (2) Blocking: Add 150 μL of blocking solution to each well, incubate at 37°C for 1 h, pour out the liquid in the well, and pat dry on absorbent paper.

[0109] (3) Competition: Add 50 μL of serially diluted cytochrome P60 standard to each well in sequence, then add 50 μL of antiserum at the optimal working concentration, gently shake the ELISA plate to mix the liquid in the well, incubate at 37°C for 30 min, pour out the liquid in the well, wash three times with washing solution, and pat dry.

[0110] (4) Adding enzyme label: Add 100 μL of 5000-fold diluted HRP-labeled goat anti-alpaca enzyme-labeled antibody to each well, incubate at 37°C for 30 min, pour out the liquid in the well, wash four times with washing solution, and pat dry.

[0111] (5) Color development: Add 100 μL of freshly prepared TMB color development solution to each well and incubate at 37°C in the dark for 10 min.

[0112] (6) Termination: Add 50 μL of 2M H2SO4 to each well.

[0113] (7) Determination: Use an ELISA reader to read the OD value of each well at 450 nm (dual wavelength: 630 nm is the reference filter wavelength). Use OriginPro8.5 to draw a standard curve with the logarithm of the concentration of the cypermethrin standard as the horizontal axis and the average OD value as the vertical axis. Figure 5 The IC50 value of the obtained nanoantibody was 1.21 ng / mL, and the linear detection range was 0.27-5.34 ng / mL.

[0114] The above-described embodiments only express several implementation methods of the present invention, which are convenient for understanding the technical solution of the present invention in detail, but cannot be understood as limiting the scope of protection of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the scope of protection of the present invention.

Claims

1. A hapten of toxin, characterized in that Its structural formula is Formula I:

2. The method for preparing the toxoplasmin hapten according to claim 1, characterized in that: The method comprises the following steps: using the compound shown in formula II and the compound shown in formula III as raw materials to synthesize the compound shown in formula IV; using the compound shown in formula IV and the compound shown in formula V as raw materials to synthesize the compound shown in formula VI; The compound represented by formula VI is reacted with nitrite ions to generate the compound represented by formula VII and the compound represented by formula I; Formula II: Formula III: Formula IV: Formula V: Formula VI: Formula VII:

3. The artificial antigen of cytoxan, characterized in that: The method comprises the toxoflavin hapten according to claim 1, and also comprises a carrier protein; the toxoflavin hapten is coupled to the carrier protein; Preferably, the carrier protein is selected from bovine serum albumin and keyhole limpet hemocyanin.

4. The method for synthesizing the artificial antigen of toxoplasmin according to claim 3, characterized in that: The method comprises the following steps: using an active ester method to couple the carrier protein to the carboxyl carbon of the toxoplasmin hapten.

5. Use of the toxoflavin hapten according to claim 1 or the toxoflavin artificial antigen according to any one of claims 2 to 3 in the preparation of toxoflavin antibodies; Preferably, the toxin antibody is a polyclonal antibody, a monoclonal antibody or a nanobody.

6. A method for preparing a venom nanobody, characterized in that: The method comprises the following steps: immunizing an animal with the artificial antigen of toxin according to any one of claims 2 to 3 as an immunogen; and then performing high-throughput sequencing analysis on specific B cells in the peripheral blood of the immunized animal to obtain nanobodies; Preferably, the animal is an alpaca.

7. A vesin nanobody, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

8. The biomaterial encoding the venom nanobody according to claim 7, characterized in that: The biological material is a nucleic acid whose sequence is shown in SEQ ID NO.2; or, the biological material contains a nucleic acid whose sequence is shown in SEQ ID NO.2; Preferably, the biological material is recombinant DNA, an expression cassette, a plasmid vector, a viral vector, an engineered bacterium or a transgenic cell line.

9. A reagent or kit for detecting cytotoxicity factor, characterized in that: Its effective ingredients include the venom nanobody as claimed in claim 7.

10. Any of the following uses of the vena cava nanobody according to claim 7, the biomaterial according to claim 8, or the reagent or kit according to claim 9: (1) Used for the detection of toxoplasmin; (2) For preparing a toxin detection reagent or kit; (3) Used for the enrichment and purification of toxic flavonoids; (4) Enrichment and purification reagents for preparing toxin.

Images