Toxoflavin hapten and methods of making and using same

Abstract

The present application relates to the technical field of immunology, and particularly relates to a toxoflavin hapten, a preparation method and application thereof. The toxoflavin hapten provided by the present application is disclosed for the first time, and 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 the specific nanobody with high potency and high affinity can be obtained after immunizing animals. The toxoflavin hapten provided by the present application and the toxoflavin nanobody 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 application relates to the technical field of immunology, in particular to a toxoflavin hapten as well as a preparation method and application thereof. BACKGROUND

[0002] Small molecule compounds such as antibacterial drugs, mycotoxins, environmental pollutants and illegal additives exist widely in biological samples such as environment and food, and it is 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 instrument analysis methods have high sensitivity and good reproducibility, but are time-consuming, laborious and expensive, and are not suitable for on-site detection of a large number of samples. Therefore, it is urgent to establish a rapid, accurate and reliable and high-sensitivity rapid analysis method.

[0003] Immunoassay based on antigen-antibody specific reaction has the characteristics of sensitivity, rapidity, low cost and simple operation, which is suitable for on-site rapid detection. Antibody as the core reagent determines the sensitivity, specificity and stability of the method. Small molecule compounds cannot directly induce strong immune response due to their small molecular weight. Therefore, 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, which have complex structure, poor stability, high production cost and are not easy to be modified in vitro, greatly limiting their application in actual sample detection. Heavy chain antibodies (HCAbs) naturally lack light chains in camelids. Through genetic engineering technology, the variable domain of the heavy chain of HCAbs (VHH) can be obtained, which retains the antigen binding activity. Due to the nanoscale size of VHH, it is also called nanobody. It has high stability, simple structure, easy modification and low production cost, which makes up for the shortcomings of traditional antibodies and has important practical value in immunoassay. Phage display technology is the most commonly used nanobody preparation technology at present, but it has many shortcomings. For example, antibody genes need to be cut and linked, plasmid transformation and other processes, and the library capacity and diversity are limited; the use of prokaryotic and eukaryotic codons, folding and modification mechanisms are different, and some antibodies cannot be effectively displayed; high-affinity clones may exist in low abundance and be easily covered by low-affinity clones; multiple rounds of affinity enrichment selection are required, which is complicated, time-consuming and extremely dependent on microbial growth conditions. With the rapid development of biotechnology and sequencing technology, B cell sorting and high-throughput sequencing technology (HTS) are becoming mature. Based on B cell sorting and high-throughput sequencing method, the specific operation of obtaining nanobody is as follows: taking specific B cells as the target, amplifying the variable region gene by PCR, and then using high-throughput sequencing technology combined with bioinformatics analysis to quickly obtain nanobody. This process does not involve library capacity loss steps such as plasmid transformation, does not depend on microbial growth state, and does not require complex affinity enrichment selection process, which has the advantages of low cost, simple operation and fast speed.

[0004] The toxoflavin is a cell toxin produced after the death of gram-negative bacteria, and has a molecular formula of C7H7N5O2, a molar mass of 193.21 g / mol, and belongs to a small molecule compound. The toxoflavin is a serious food contaminant, has extremely strong toxicity and mutagenic effect, and can cause pathological damage to animal vascular smooth muscle, kidneys, lungs, hearts and other organs. The toxoflavin can exist in cereals such as wheat, barley, corn, oat, soybean and rice, and eating the food contaminated by the toxoflavin can cause stomach discomfort, even liver and kidney damage, toxic shock and other symptoms, and cause damage to the digestive system, immune system, nervous system and circulatory system of the human body, and pose a threat to public health and food safety. Therefore, it is necessary to monitor the toxoflavin pollution to protect the health of consumers and food safety. At present, there is no research on the preparation of toxoflavin antibody.

[0005] In view of this, the present application is provided. SUMMARY

[0006] To solve the above technical problems, the present application provides a toxoflavin hapten, a preparation method and application thereof.

[0007] Specifically, the technical scheme of the present application is as follows:

[0008] In the first aspect, the present application provides a toxoflavin hapten, and the structural formula is formula I:

[0009]

[0010] The toxoflavin hapten provided by the present application is disclosed for the first time, and 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 the specific nanobody with high titer and high affinity can be obtained after immunizing animals.

[0011] In the second aspect, the present application provides a preparation method of the toxoflavin hapten in the first aspect, and the preparation method comprises the following steps: synthesizing a compound shown in formula IV by taking a compound shown in formula II and a compound shown in formula III as raw materials; synthesizing a compound shown in formula VI by taking the compound shown in formula IV and a compound shown in formula V as raw materials; and generating a compound shown in formula VII and a compound shown in formula I by reacting the compound shown in formula VI with nitrite ions.

[0012]

[0013]

[0014] In a more specific and preferred embodiment, the preparation method of the toxoflavin hapten comprises the following steps:

[0015] S1, 25 g of compound 1 (as shown in formula II) and 28 g of compound 2 (as shown in formula III) were added in anhydrous ethanol, stirred at 90-95 °C for 5-6 h, identified by thin layer chromatography, no compound 1 residue. Cooled to room temperature for 1 h, white solid precipitated; the mixture was washed with 30 mL of anhydrous ethanol for 2 times, dried at 40-50 °C for 1-2 h, to obtain 20.11 g of compound 3 (as shown in formula IV) white solid product;

[0016] S2, 4.50 g of compound 4 (as shown in formula V) and 5.12 g of compound 3 (as shown in formula IV) were added in 150 mL of anhydrous ethanol, stirred at 55-60 °C for 4-5 h. Identified by thin layer chromatography, no compound 3 residue. The mixture was cooled to 30-35 °C, washed with 30 mL of anhydrous ethanol for 2 times, dried at 40-50 °C for 2-3 h, to obtain 6.55 g of compound 5 (as shown in formula VI) yellow solid;

[0017] S3, 5.40 g of compound 5 was added in 100 mL of acetic acid solution, cooled to 5-10 °C, slowly added 1.85 g of NaNO2 and 20 mL of H2O. Stirred at room temperature overnight to obtain a crude product. The crude product was added in 50 mL of H2O, stirred for 1 h, washed with 30 mL of anhydrous ethanol for 2 times, dried at 40-50 °C for 2-3 h, to obtain a mixture of compound as shown in formula VII and toxoflavin hapten 1 (as shown in formula I);

[0018] S4, the mixture was purified by high performance liquid chromatography, and the obtained yellow solid product was toxoflavin hapten as shown in formula I.

[0019] The present application first discloses a preparation method of the toxoflavin hapten, and the toxoflavin hapten can be stably prepared.

[0020] In a third aspect, the present application provides a toxoflavin artificial antigen, which comprises the toxoflavin hapten as described in the first aspect, and further comprises a carrier protein; the toxoflavin hapten is coupled to the carrier protein.

[0021] In the present application, the toxoflavin artificial antigen can be used as an immunogen or a coating agent. The artificial antigen provided by the present application can be used to immunize animals, and specific nanobodies with high titer and high affinity can be obtained. The artificial antigen provided by the present application can be used as a coating agent for detecting 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 application provides a synthesis method of the toxoflavin artificial antigen as described in the third aspect, which comprises the following steps: coupling the carrier protein to the carboxyl carbon of the toxoflavin hapten by using active ester method.

[0024] In a fifth aspect, the present application provides a use of the toxoflavin hapten of the first aspect or the toxoflavin artificial antigen of the second aspect in the preparation of a toxoflavin antibody.

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

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

[0027] Preferably, the animal is a llama.

[0028] The toxoflavin nanobody is prepared by immunizing a llama with the hapten and carrier protein conjugate provided by the present application, using B cell sorting and high-throughput sequencing technology, and the IC50 value of the obtained nanobody is 1.21 ng / mL, the linear detection range is 0.27-5.34 ng / mL, and the nanobody has high practical value. 50 The time required for immunization to antibody preparation is only one month, and the nanobody has good application prospects in rapid detection of toxoflavin.

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

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

[0031] Preferably, 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 application uses toxoflavin as a model small molecule compound, and based on B cell sorting and high-throughput sequencing, a toxoflavin nanobody as shown in SEQ ID NO. 1 is prepared, which has high affinity and strong specificity, and can be used for detecting toxoflavin pollution in food, and provides a core recognition material for pollution monitoring.

[0033] In an eighth aspect, the present application provides a biomaterial for encoding the toxoflavin nanobody of the seventh aspect, wherein the biomaterial is a nucleic acid with a sequence as set forth in SEQ ID NO. 2; or the biomaterial contains a nucleic acid with a sequence as set forth in SEQ ID NO. 2.

[0034] caggtgcagctggtggagtctgggggaggctcggtgcagcctggaggatctctgagactctcctgcacagcctctagatacacctatagtcgcggatgcatgggctggttccgccagcctccagggaagggccgcgagggggtcgcggctatttatggtggtaatggtggcacatactatgccgactccgtgaagggccgattcaccatctcccaagacaacgccaagaacacggtgtatctgcaaatgaacagcctgaagcctgaggacactggcatgtactactgtgcggcagaaacccatactggtggttactgttacagttgggctgactttaattattggggccaggggacccaggtcaccgtctcctca(SEQ ID NO. 2)

[0035] Preferably, the biomaterial is a 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 application provides a toxoflavin detection reagent or kit, wherein the effective component comprises the toxoflavin nanobody of the seventh aspect.

[0037] In a tenth aspect, the present application provides any one of the following applications of the toxoflavin nanobody of the seventh aspect, the biomaterial of the eighth aspect or the reagent or kit of the ninth aspect:

[0038] (1) for toxoflavin detection;

[0039] (2) for preparing a toxoflavin detection reagent or kit;

[0040] (3) for enriching and purifying toxoflavin;

[0041] (4) for preparing an enriching and purifying reagent for toxoflavin.

[0042] Beneficial effects:

[0043] The present application discloses a new hapten of toxoflavin for the first time, and the conjugate (i.e. toxoflavin artificial antigen) obtained by coupling the hapten with a carrier protein can be used as a coating antigen or an immunizing antigen, and has good application effect. After an animal is immunized with the toxoflavin artificial antigen provided by the present application, a specific nanobody with high titer and high affinity can be obtained. In addition, the present application immunizes a llama with the toxoflavin artificial antigen, collects peripheral blood to sort toxoflavin-specific B cells, and uses high-throughput sequencing technology to sequence the toxoflavin-specific B cells, so that a nanobody with high specificity and high affinity can also be obtained by this method. The toxoflavin hapten and the toxoflavin nanobody prepared by B cell sorting and high-throughput sequencing provided by the present application provide materials for establishing a rapid, simple, sensitive and specific toxoflavin detection method. BRIEF DESCRIPTION OF DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the present application or prior art, the drawings needed to be used in the embodiments or prior art description will be described below.

[0045] Figure 1 The preparation flow chart of the toxoflavin hapten shown in formula I in Example 1 of the present application.

[0046] Figure 2 The llama serum titer curve in Example 3 of the present application.

[0047] Figure 3 The proportion of high-frequency nanobodies in the antibody repertoire obtained by sequencing in Example 5 of the present application.

[0048] Figure 4 The toxoflavin nanobody diagram expressed by the prokaryotic expression system in Example 6 of the present application; wherein M lane is protein Marker, 1 lane is crude extract before purification, and 2 lane is nanobody after purification.

[0049] Figure 5 The standard curve diagram for detecting toxoflavin by using the toxoflavin nanobody in Example 6 of the present application. DETAILED DESCRIPTION

[0050] The technical solutions provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application. If not specifically indicated, the experimental methods used in the examples are conventional methods; the materials, reagents, etc. used can be obtained from commercial channels.

[0051] Preparation of toxoflavin hapten in Example 1

[0052] The preparation steps of the toxoflavin hapten are as shown in Figure 1 and specifically include the following steps:

[0053] S1. Add 25 g of compound 1 (as shown in formula II) and 28 g of compound 2 (as shown in formula III) in anhydrous ethanol, stir at 90-95 °C for 5-6 h, identify by thin layer chromatography, no compound 1 residue. Cool to room temperature for 1 h, white solid precipitates; wash the mixture with 30 mL of anhydrous ethanol for 2 times, dry at 40-50 °C for 1-2 h, to obtain 20.11 g of compound 3 (as shown in formula IV) 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) in 150 mL of anhydrous ethanol, stir at 55-60 °C for 4-5 h. Identify by thin layer chromatography, no compound 3 residue. Cool the mixture to 30-35 °C, wash with 30 mL of anhydrous ethanol for 2 times, dry at 40-50 °C for 2-3 h, to obtain 6.55 g of compound 5 (as shown in formula VI) yellow solid.

[0057]

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

[0059]

[0060] S4. Purify the mixture by high performance liquid chromatography, to obtain the yellow solid product of toxoflavin hapten as shown in formula I.

[0061] Example 2. Preparation of toxoflavin artificial antigen

[0062] This example provides two preparation methods of toxoflavin artificial antigen (coating antigen and immunogen), the difference between the two preparation methods is only the type of carrier protein (KLH is used as the carrier protein of immunogen, BSA is used as the carrier protein of coating antigen, and the coupling method is active ester method).

[0063] (I) Synthesis of toxoflavin coating antigen

[0064] S1, 29.25 mg of the compound of formula I prepared in Example 1 was dissolved in 1 mL of DMF, 12 mg of NHS and 95.75 mg of EDC were added, and the mixture was stirred at room temperature overnight to obtain solution I;

[0065] S2, 111.70 mg of BSA was added to 3 mL of PBS buffer and dissolved to obtain solution II;

[0066] S3, solution I was slowly added to solution II, and the mixture was stirred at 4°C for 24 h, then was put into a dialysis bag and dialyzed in PBS at room temperature for 72 h (with 6 times of liquid change), to obtain a toxoflavin-coated original solution, which was stored at -20°C.

[0067] The toxoflavin-coated original solution prepared from the compound of formula I is referred to as TXF-BSA.

[0068] (II) Synthesis of toxoflavin immunogen

[0069] Compared with the synthesis method of the toxoflavin-coated original solution described above, the only difference is that KLH is used instead of BSA.

[0070] The toxoflavin immunogen prepared from the compound of formula I is referred to as TXF-KLH.

[0071] Example 3: Immunization of alpaca and detection of antisera

[0072] In this example, the TXF-KLH prepared in Example 2 was mixed with Freund's adjuvant to immunize an alpaca. For the first immunization, 300 μg of the immunogen was mixed with an equal volume of Freund's complete adjuvant, emulsified, and injected into the alpaca in multiple points on the neck and back. One week later, the alpaca was boosted once, and the immunization dose was unchanged, but the adjuvant was changed to Freund's incomplete adjuvant. One week after each immunization, the peripheral blood of the alpaca was collected, and the titer of the serum was detected by ELISA (see Figure 2 ).

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

[0074] (1) Coating: the homologous coating agent TXF-BSA was diluted to 0.1 μg / mL with the coating solution, and 100 μL of the solution was added to each well of an enzyme-labeled plate, which was incubated at 37°C for 2 h. The liquid in the wells was poured out, and the wells were washed 3 times with 280 μL of washing solution and dried on absorbent paper.

[0075] (2) Blocking: 150 μL of blocking solution was added to each well, which was incubated at 37°C for 1 h. The liquid in the wells was poured out and dried on absorbent paper.

[0076] (3) Binding: 50 μL of PBS and 50 μL of gradient-diluted antisera were sequentially added to each well, which was incubated at 37°C for 30 min. The liquid in the wells was poured out, washed 3 times with washing solution, and dried.

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

[0078] (5) Color development: add 100 μL of freshly prepared TMB color developing liquid to each well, and react at 37°C for 10 min in the dark.

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

[0080] (7) Measurement: read the OD value of each well at 450 nm (reference filter wavelength: 630 nm) using an enzyme-labeled instrument.

[0081] (8) Plot the antiserum titer curve using OriginPro8.5 with the antiserum dilution factor as the abscissa and the average OD value as the ordinate. The definition of the antiserum titer in this study is as follows: when the concentration of the homologous coating agent TXF-BSA is fixed at 0.1 μg / mL, the antiserum dilution factor corresponding to the final OD value of 1.5 is the antiserum titer.

[0082] Example 4 Isolation of Specific B Cells in the Peripheral Blood of Llamas

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

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

[0085] (2) Take a 50 mL sterile centrifuge tube, and add an equal volume of Ficoll separation medium in the centrifuge tube. Slowly and smoothly lay the diluted peripheral blood on the Ficoll separation medium, and balance the centrifugation.

[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 medium layer, and the fourth layer is the red blood cell layer.

[0087] (4) Absorb 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 min.

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

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

[0090] (7) Use fluorescein EDF-labeled toxoflavin hapten to incubate with lymphocytes at 37°C for 2 h, and use a flow cytometer for cell sorting.

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

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

[0093] The specific operation steps are as follows:

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

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

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

[0097] (4) Reverse transcribe the RNA into cDNA, sequence the obtained cDNA library through the Illumina MiSeq platform, and analyze the high-throughput sequencing results by bioinformatics.

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

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

[0100] ② Analysis of somatic hypermutation of nanobodies, including mutation position and mutation frequency, usually nanobodies with high mutation frequency have strong antigen binding activity.

[0101] The proportion of high-frequency nanobodies in the antibody library analyzed by sequencing results is shown in Table 1 Figure 3 , and the sequence of clone No. 1 accounted for 57.2% of the total nanobody sequences, and the proportion of other clone sequences was small. Through the above analysis, the possible toxoflavin nanobody No. 1 sequence (amino acid sequence as shown in SEQ ID NO. 1) was artificially synthesized, cloned into the corresponding expression vector, and prepared for activity identification.

[0102] Example 6 Expression, purification and activity identification of toxoflavin nanobodies

[0103] In this embodiment, the toxoflavin nanobody is constructed into the expression vector pET-22b, and after sequencing, it is induced to express in the expression host BL21.

[0104] The specific operation steps of induction expression are as follows:

[0105] Dilute the correct sequence sample bacterial liquid with 2YT medium at a ratio of 1:100, shake the bacteria at 37°C and 220 rpm, and when the OD value of the bacterial liquid at 600 nm is 0.6-1.0, add IPTG with a final concentration of 1 mmol / L to induce expression for 16 h, and collect the bacterial liquid. After IPTG induction and expression, the bacterial liquid is centrifuged at 6000 rpm for 20 min to collect the bacterial body, washed once with PBS, and centrifuged again to collect the bacterial body; the bacterial liquid is mixed and blown on ice with 10 mL of PBS, and the program is ultrasonicated for 10 min according to the program of "ultrasonic vibration time 5 s; interval time 5 s; protection temperature 60°C; ultrasonic power 10%", and the broken bacterial liquid is centrifuged at 12000 rpm for 30 min. The supernatant is purified by affinity chromatography, and the purification experiment results are shown in Table 2 Figure 4 .

[0106] The activity of toxoflavin nanobodies is identified by indirect ELISA method, and the specific operation steps are as follows:

[0107] (1) Coating: dilute the coating liquid to the optimal working concentration, add 100 μL per well, and incubate at 37°C for 2 h. Pour out the liquid in the well, wash with washing solution for 3 times, 280 μL per well, and dry on the absorbent paper.

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

[0109] (3) Competition: sequentially add 50 μL of gradient-diluted toxoflavin standard to each well, then add 50 μL of the optimal working concentration of antiserum, gently shake the enzyme-labeled plate to mix the liquid in the well, incubate at 37°C for 30 min, pour out the liquid in the well, wash with washing solution for 3 times, and dry.

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

[0111] (5) Color development: add 100 μL of freshly prepared TMB color developing liquid to each well, and react at 37°C for 10 min in the dark.

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

[0113] (7) Determination: read the OD value of each well at 450 nm (double wavelength: 630 nm as the reference filter wavelength) by using an enzyme-labeled instrument. The standard curve is plotted by using the logarithm of the concentration of toxoflavin standard as the abscissa and the average OD value as the ordinate by using OriginPro8.5, as shown in Figure 5 The IC50 value of the obtained nanobody is 1.21 ng / mL, and the linear detection range is 0.27-5.34 ng / mL.

[0114] The above-described embodiments only express several embodiments of the present application, facilitate specific and detailed understanding of the technical solutions of the present application, but cannot be understood as a limitation on the protection scope of the present application. It should be noted that, for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which all belong to the protection scope of the present application.

Claims

1. A toxoflavin artificial antigen, characterized in that, The toxoflavin hapten further comprises a carrier protein; the toxoflavin hapten is coupled to the carrier protein; The toxoflavin hapten has a structural formula of Formula I: 。 2. The toxoflavin artificial antigen of claim 1, wherein, The carrier protein is selected from bovine serum albumin and keyhole limpet hemocyanin.

3. The method for synthesizing a toxoflavin artificial antigen according to claim 1 or 2, characterized in that, The toxoflavin hapten is coupled to the carrier protein on a carboxyl carbon of the toxoflavin hapten by an active ester method.

4. The toxoflavin artificial antigen of claim 1 or 2, or the toxoflavin hapten thereof for use in the preparation of a toxoflavin antibody.

5. Use according to claim 4, characterized in that, The toxoflavin antibody is a polyclonal antibody, a monoclonal antibody, or a nanobody.

6. A method for the preparation of a toxoflavin nanobody, characterized in that, The toxoflavin artificial antigen of any one of claims 1-2 is used as an immunogen to immunize an animal, and then specific B cells in peripheral blood of the immunized animal are subjected to high-throughput sequencing analysis to obtain a nanobody.

7. The production method according to claim 6, characterized by, The animal is a llama.

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