CN110684080B

CN110684080B - scFv-ELISA kit suitable for thiamethoxam residue analysis

Info

Publication number: CN110684080B
Application number: CN201910791323.XA
Authority: CN
Inventors: 许艇; 何金鑫; 徐波杰; 王楷; 李季
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2019-08-26
Filing date: 2019-08-26
Publication date: 2021-01-15
Anticipated expiration: 2039-08-26
Also published as: CN110684080A

Abstract

The invention provides a scFv-ELISA kit suitable for thiamethoxam residue analysis. The kit comprises a kit body, an ELISA plate and a reagent, wherein the ELISA plate and the reagent are arranged in the kit body; each hole of the ELISA plate is coated with a thiamethoxam coating antigen, and the reagent comprises an anti-thiamethoxam single-chain antibody, a thiamethoxam standard solution, an enzyme-labeled secondary antibody, a buffer solution PBS, a washing solution PBST, a substrate solution, a developing solution, a reaction stop solution and the like. In the detection process, the envelope antigen adsorbed on the pore wall of the ELISA plate and the thiamethoxam to be detected compete with each other to react with the antibody, and the result is observed through a color reaction. The thiamethoxam with known concentration is detected, a standard curve is drawn, and the concentration of the thiamethoxam to be detected can be calculated. The method has the advantages that the thiamethoxam residues in water, vegetables and fruits can be accurately and sensitively detected, the defects that the sample pretreatment process is complicated, the operation is complex and time-consuming and the like in the instrument detection are overcome, and meanwhile, the detection cost is greatly saved.

Description

scFv-ELISA kit suitable for thiamethoxam residue analysis

Technical Field

The invention relates to the technical fields of genetic engineering, phage display technology and ELISA (enzyme-linked immunosorbent assay) detection, in particular to a scFv-ELISA kit suitable for thiamethoxam residue analysis.

Background

Thiamethoxam is a neonicotinoid insecticide, has broad-spectrum insecticidal activity, can be used for preventing and treating various chewing mouthpart pests and piercing-sucking mouthpart pests, and is widely used in the global range. The conventional detection method of the pesticide residue is mainly an instrumental analysis method, and comprises a Gas Chromatography (GC), a High Performance Liquid Chromatography (HPLC), a gas/liquid chromatography-mass spectrometry combined method (GC/LC-MS) and the like. Although the analysis methods have high accuracy, the required instruments are expensive, the pretreatment of samples such as separation, extraction, purification, derivatization and the like is complex, the analysis speed is low, the detection sensitivity is low, and the rapid detection on site is difficult to realize. With the rapid increase of samples to be detected, particularly the amount of samples required to be detected on site rapidly, the traditional pesticide residue analysis means is difficult to adapt to the requirements, so that a high-efficiency and convenient thiamethoxam detection method is urgently required to be developed.

The immunoassay method is a method for detecting microorganisms, proteins and other substances according to specific reaction of antigen and antibody, has the advantages of simple and convenient operation, high sensitivity, strong specificity, capability of realizing on-site rapid detection and the like compared with the traditional instrument detection, and can provide an efficient and convenient detection method for the residual detection of thiamethoxam. The selection of the antibody is the core of an immunoassay method, and compared with the conventional antibody, the single-chain antibody has the characteristics of small molecular weight, easy expression, low production cost and the like, and the poultry-derived single-chain antibody has the advantages of relatively simple preparation process, low antibody cross reaction and the like. The invention provides a rapid, simple and convenient method for detecting thiamethoxam residues in environment and food by taking an anti-thiamethoxam single-chain antibody as a basis and combining an enzyme-linked immunosorbent assay method.

Disclosure of Invention

Aiming at the defects of high cost, complex pretreatment, poor specificity, low sensitivity, difficult realization of field detection and the like of the existing pesticide residue instrument analysis method, the invention provides the ELISA detection kit which has high specificity, high sensitivity, high accuracy, high precision and simple operation method and can be used for rapidly detecting and analyzing thiamethoxam residues on a large scale of samples. The method can be used for quickly determining thiamethoxam residues in samples such as water, vegetables and fruits.

To achieve the object of the present invention, in a first aspect, the present invention provides an isolated polypeptide comprising or consisting of an amino acid sequence as follows:

i) 1, SEQ ID NO; or

ii) an amino acid sequence obtained by connecting a label at the N end and/or the C end of the i); or

iii) the amino acid sequence of i) or ii) is substituted, deleted and/or added with one or more amino acids to obtain the polypeptide with the same function.

The polypeptide can be used as an anti-thiamethoxam single-chain antibody.

The anti-thiamethoxam single-chain antibody can be prepared according to the following method: hapten Thiamphetamine is synthesized by utilizing chemical reaction Thiamphetamine, as the immunogen after hapten and keyhole limpet hemocyanin coupling, immunity experiment animal SPF arrives at a journey chicken, extract the total RNA of peripheral blood lymphocyte, through reverse transcription and overlapping PCR, single chain antibody scFv gene fragment is cloned out, connect through the enzyme digestion, clone gene fragment to phagemid carrier, high-efficient electric transformation to escherichia coli, save through the auxiliary phage, the construction obtains phage single chain antibody library, select specific thiamethoxam phage single chain antibody, express the purification with it, high sensitivity is obtained, the strong anti-thiamethoxam single chain antibody of specificity. The single-chain antibody prepared by the method has small molecules, strong solubility, high temperature resistance, easy purification and easy expression.

The thiamethoxam hapten Thi-5C can be prepared according to the following method:

3.0g of 3-methyl-4-nitroiminotetrahydro-1, 3, 5-oxadiazine, 5.0g of 2-chloro-5-bromomethylthiazole and 6.25g of potassium carbonate were dissolved in 20 ml of Dimethylformamide (DMF) at 30 ℃ overnight and reacted at 50 ℃ for 16 hours. The reaction mixture was filtered through celite and the DMF was removed under vacuum. And purifying the product by flash chromatography to obtain the thiamethoxam.

In a 50 mL round bottom flask, 0.5g thiamethoxam, 0.2g 3-mercaptopropionic acid and 0.2g 85% potassium hydroxide (powder) were heated to 75 ℃ in 15mL DMSO for 3 days. The solution was poured into 100mL of water, adjusted to pH 3.0, and extracted with ethyl acetate (3 times 30mL each). The organic phase is washed with 1mol/L hydrochloric acid and then with anhydrous Na₂SO₄Drying and removing the organic solvent under reduced pressure. Dissolving the residue with methanol, and separating by thin layer chromatography to obtain 3- [2- (2-carboxyethylthio) -5-ethylmethyl]-5-methyl-4-nitrosamine-1, 3, 5-oxadiazinane.

In a second aspect, the invention provides a nucleic acid molecule encoding said polypeptide.

In a third aspect, the invention provides biological materials containing the nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, phage vectors, viral vectors or engineered bacteria.

In a fourth aspect, the invention provides a thiamethoxam detection reagent or kit, wherein the polypeptide is used as an active ingredient.

In a fifth aspect, the invention provides a thiamethoxam hapten which is 3- [2- (2-carboxyethylthio) -5-ethylmethyl ] -5-methyl-4-nitrosamine-1, 3, 5-oxadiazinane. The structural formula is as follows:

in a sixth aspect, the invention provides a thiamethoxam artificial antigen which is obtained by coupling a thiamethoxam hapten and a carrier protein.

Wherein the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroid protein, human serum albumin; bovine serum albumin and keyhole limpet hemocyanin are preferred.

Preferably, the carrier protein is coupled to the carboxyl group of the thiamethoxam hapten by an activated ester method.

In a seventh aspect, the invention provides a scFv-ELISA kit suitable for thiamethoxam residue analysis, which comprises a kit body, a detachable ELISA plate arranged in the kit body, a reagent arranged in the kit body, and the like; each hole of the enzyme label plate is coated with the thiamethoxam artificial antigen, and the reagent comprises an anti-thiamethoxam single-chain antibody and at least one of a thiamethoxam standard solution, an enzyme-labeled secondary antibody, a buffer solution PBS, a washing solution PBST, a color developing solution, a reaction stopping solution and the like.

The preparation method of the antigen coating solution for coating the ELISA plate comprises the following steps:

(1) dissolving 7.788-15.76mg of thiamethoxam hapten as described in claim 5, 2.76-5.52mg of N-hydroxysuccinimide (NHS) and 4.738-9.476mg of Dicyclohexylcarbodiimide (DCC) in 200-400 microliter of anhydrous Dimethylformamide (DMF), placing the mixture on a magnetic stirrer, stirring and reacting at room temperature overnight, and centrifuging the reaction solution at 5000rpm for 10-20min the next day to obtain a supernatant which is an active ester solution;

(2) dissolving 20mg of bovine serum albumin in 2mL of 0.05M carbonate buffer solution with pH of 9.6, slowly stirring the mixture on a magnetic stirrer, dropwise and slowly adding the active ester solution, finishing the addition for about 20min, and then continuously stirring and reacting for 6h at room temperature;

(3) after the reaction is finished, putting the reaction solution into a dialysis bag and dialyzing with PBS; changing the liquid once every 6h, and changing the liquid 5-6 times in total; and centrifuging after dialysis, discarding the precipitate, and collecting supernatant as antigen coating solution.

Preferably, the antigen coating concentration for coating the microplate is 100 ng/mL.

The enzyme-labeled secondary antibody is a His label antibody labeled by horseradish peroxidase, and the concentration is 0.1 mu g/mL.

The color developing solution comprises solution A and solution B, wherein the solution A is prepared from carbamide peroxide 1g, citric acid 10.3g, and Na₂HPO₄·12H₂O35.8 g, Tween-20100 mu L and distilled water 1000mL, and the pH value is 5; the solution B is prepared from 700mg of tetramethylbenzidine, 40mL of DMSO, 10.3g of citric acid and 1000mL of distilled water, and the pH value is 7.4.

The reaction termination solution is 2M sulfuric acid.

In an eighth aspect, the invention provides any one of the following uses of the polypeptide:

1) the method is used for thiamethoxam detection;

2) is used for preparing a thiamethoxam detection reagent or a kit.

In a ninth aspect, the invention provides a thiamethoxam ELISA detection reagent, and the effective component is the antibody.

In a tenth aspect, the invention provides an application of the kit or the reagent in detecting thiamethoxam residues in a sample by an ELISA method.

During actual analysis and detection, a thiamethoxam sample to be detected and the anti-thiamethoxam single-chain antibody are sequentially added into each hole of an enzyme label plate coated with the thiamethoxam coating antigen, the solid-phase coating antigen and the thiamethoxam to be detected compete with each other to react with the single-chain antibody, and because the contents of the solid-phase antigen and the added single-chain antibody in each hole are consistent, when the concentration of the thiamethoxam to be detected is high, the amount of the antibody bound on the solid-phase antigen is small, the binding amount of the added enzyme-labeled secondary antibody and the fixed antibody is small, and finally, a substrate solution and a developing solution are added, so that the developing reaction is light, the OD value detected by an enzyme label is low, and the inhibition rate is high; on the contrary, when the concentration of the thiamethoxam to be detected is low, the detected OD value is high, and the inhibition rate is low. The concentration of the thiamethoxam to be detected can be calculated according to a standard curve drawn by using the standard thiamethoxam solution with known concentration.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the kit provided by the invention can be used for accurately and sensitively detecting thiamethoxam residues in water and vegetables, and has the advantages of simple pretreatment process of samples, less time consumption, good specificity and high sensitivity (minimum detection limit IC)₁₀4ng/mL), can detect a large amount of samples simultaneously, and the sample detection cost is far lower than that of the traditional instrument detection method. The method has important significance for solving the problem of the thiamethoxam residue field detection technology of mass samples.

Drawings

FIG. 1 is a standard inhibition curve of thiamethoxam based on single chain antibodies of example 6 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

EXAMPLE 1 Synthesis of hapten 3- [2- (2-carboxyethylthio) -5-ethylmethyl ] -5-methyl-4-nitrosamino-1, 3, 5-oxadiazine

In a 50 mL round bottom flask, 0.5g thiamethoxam, 0.2g 3-mercaptopropionic acid and 0.2g 85% potassium hydroxide (powder) were heated to 75 ℃ in 15mL DMSO for 3 days. The solution was poured into 100ml of water, the pH was adjusted to 3.0 and extracted with ethyl acetate (3 times 30ml each). The organic phase is washed with 1mol/L hydrochloric acid and then with anhydrous Na₂SO₄Drying and removing the organic solvent under reduced pressure. Dissolving the residue with methanol, and separating by thin layer chromatography to obtain 3- [2- (2-carboxyethylthio) -5-ethylmethyl]-5-methyl-4-nitrosamine-1, 3, 5-oxadiazinane.

Example 2 preparation of thiamethoxam-coated antigen

The thiamethoxam coating antigen used in the invention is a compound coupling thiamethoxam half-antibody Thi-5C and Bovine Serum Albumin (BSA), and the preparation method of the coating antigen is as follows:

(1) respectively weighing 7.788mg of thiamethoxam hapten Thi-5C, 2.76mg of N-hydroxysuccinimide (NHS) and 4.738mg of Dicyclohexylcarbodiimide (DCC), dissolving in 200 mu L of anhydrous Dimethylformamide (DMF), placing on a magnetic stirrer, stirring at room temperature for reacting overnight, and centrifuging the reaction solution at 5000rpm for 15min the next day to obtain a supernatant which is the active ester solution.

(2) Weighing 20mg of bovine serum albumin, dissolving the bovine serum albumin in 2mL0.05M carbonate buffer solution with pH of 9.6, placing the bovine serum albumin in a magnetic stirrer, slowly stirring, dropwise and slowly dropwise adding the active ester solution, finishing the addition for about 20min, and then continuously stirring and reacting for 6h at room temperature.

(3) After the reaction, the reaction solution was filled into a dialysis bag and dialyzed with PBS (0.01mol/L, pH 7.4); the liquid is changed once every 6h, and the liquid is changed for 5-6 times. And centrifuging after dialysis, discarding the precipitate, and collecting supernatant as antigen coating solution.

Example 3 construction of the thiamethoxam phage display Single chain antibody library

The hapten synthesized in example 1 is coupled with keyhole limpet hemocyanin by an active ester method, which comprises the following steps:

equimolar amounts of thiamethoxam hapten Thi-5C, NHS and DCC were dissolved in DMF and stirred overnight at room temperature. The reaction solution is centrifuged and the precipitate is discarded, and the supernatant is the active ester solution. And adding the supernatant into the keyhole limpet hemocyanin solution under the stirring state, and continuously stirring and reacting for 4 hours at room temperature. The reaction solution was filled in a dialysis bag and dialyzed with PBS. Centrifuging, collecting supernatant, and freeze-drying to obtain conjugate of thiamethoxam hapten Thi-5C and keyhole limpet hemocyanin (Thi-5C-KLH).

And (2) dissolving 100 mu L of immunogen (Thi-5C-KLH) in sterile physiological saline, mixing with equal volume of complete adjuvant (priming) or Freund's incomplete adjuvant (secondary and later), fully emulsifying, performing multi-point injection at the muscle of SPF leghorn, and immunizing once every 14 days for 5-6 times. Starting from the third immunization, blood was taken from the vein of the quiet wing one week after each immunization to detect serum titers.

After 6 th immunization, taking spleens of SPF leghorns, extracting total RNA, cloning scFv gene fragments through reverse transcription PCR and overlap PCR, carrying out enzyme digestion on pComb3X plasmid vectors and scFv gene fragments by using restriction enzyme SfiI, connecting the VHH gene fragments to phagemid pComb3x through T4 ligase, and carrying out high-efficiency electric transformation to escherichia coli ER2738 to construct a phage single-chain antibody library of thiamethoxam. The primary reservoir volume is determined to be 10⁸cfu, rescued by adding helper phage (multiplicity of infection 20:1) M13KO7 to obtain phage single chain antibody library (phage display scFv library) with a library capacity of 10¹³pfu/mL, the diversity of the library was good.

Reverse transcription PCR:

the reverse transcription kit was M-MLV first strand cDNA synthesis kit, purchased from OMEGA.

The reverse transcription system is as follows:

reverse transcription at 42 deg.C for 30min

Overlapping PCR:

first round PCR:

the reaction system is as follows:

the reaction procedure was as follows:

second round PCR:

the reaction system is as follows:

the reaction procedure was as follows:

the PCR primer sequences were as follows:

primers for amplification of heavy chain VH gene fragments:

HF：5'-GGCCCAGGCGGCCCCGTGACGTTGGACGAGTCCG-3'

HR：5'-CAGAGCCACCTCCGCCTGAACCGCCTCCACCGGAGGAGACGATGACT-3'

primers for amplification of light chain VL gene fragment:

LF:5'-TTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGCGCGACTCAGC-3'

LR:5'-GGCCGGCCTGGCCACTAGGACGGTCAGGGTTGTC-3'

example 4 screening of specific thiamethoxam phage-displayed Single-chain antibodies

Coating the coated antigen prepared in the example 2 on the 1 st hole of a 96-hole enzyme label plate, wherein the coating concentration is 100ng/mL, and the temperature is 4 ℃ overnight; the next day, the coating solution was poured out, washed 3 times with PBST, 150. mu.L of 1% gelatin was added to the first two wells, 150. mu.L of 1% BSA solution was added to the last four wells, and incubated at room temperature for 1 h; taking 110 mu L of the phage display scFv library of the example 3, adding 110 mu L of 3% BSA solution, carrying out shaking for 1h at 25 ℃ and 220rpm on a shaking table, so that the phage can be fully combined with the BSA, and thus removing the phage antibody specifically combined with the BSA; discarding the liquid, washing the PBST for three times, each time for 1min, respectively adding 100 mu L of premixed library into the first two holes, and oscillating and reacting for 2h at room temperature to ensure that the phage can be fully combined with the coating antigen; discarding the liquid, washing with PBST for three times, each time for 1 min; adding 100 mu L of diluted thiamethoxam standard substance into the first two holes respectively, and carrying out oscillation reaction for 1h at room temperature to enable the thiamethoxam standard substance and the coating antigen to compete and bind with the phage antibody, so that the phage antibody with strong binding capacity with the thiamethoxam standard substance is eluted; transferring the competitive eluent in the first two holes into the last four holes, wherein each hole is 50 mu L, and carrying out oscillation reaction for 1h at room temperature, so as to remove the phage antibody specifically bound with BSA; phage eluates were collected from wells, and 10. mu.L of the eluates were diluted and then titrated, and the remainder was used for amplification.

Adding the phage eluate into fresh Escherichia coli ER2738 bacterial liquid, and standing at 37 deg.C for 15 min; adding carbenicillin and SB culture medium, culturing at 37 deg.C and 220rpm for 2 hr; adding helper phage M13KO7 (MOI 20:1) and kanamycin, and culturing overnight; the next day, the supernatant was centrifuged and purified by adding PEG-NaCl solution.

And (3) carrying out next round of screening on the amplification product to ensure that the addition amount of each round of screening is the same, the antigen coating concentration and the thiamethoxam standard substance competitive elution concentration are decreased progressively according to 2 times, calculating the titer of each round, and selecting a monoclonal for amplification and ELISA identification. Positive monoclone is obtained through 4 rounds of panning.

Example 5 expression of specific thiamethoxam Single-chain antibodies

Extracting positive monoclonal plasmid, transforming to escherichia coli TOP 10F' competent cell, recovering, and coating on solid culture medium for overnight culture. The next day, selecting a single clone to be cultured in an LB-carboxybenzyl culture medium, and adding IPTG to induce overnight expression; the next day, cells are cracked by an ultrasonic crusher, filtered by a filter membrane and purified by a nickel column, and the high-purity anti-thiamethoxam single-chain antibody is obtained. The amino acid sequence of the obtained single-chain antibody is shown in SEQ ID NO. 1 through amino acid sequencing analysis.

Example 6 ELISA detection kit for analyzing thiamethoxam residues and application thereof

The kit comprises a kit body, a detachable 96-hole elisa plate arranged in the kit body and a reagent arranged in the kit body, wherein each hole of the elisa plate is coated with the thiamethoxam coating antigen prepared in the embodiment 2, and the reagent comprises the anti-thiamethoxam single-chain antibody, a thiamethoxam standard solution, an enzyme-labeled secondary antibody, a buffer solution PBS, a washing solution PBST, a substrate solution (A solution), a color development solution (B solution), a reaction termination solution and the like in the embodiment 5.

The enzyme-labeled secondary antibody is an anti-His tag antibody labeled by horseradish peroxidase, and the concentration is 0.1 mu g/mL. Purchased from Abcam.

The solution A comprises carbamide peroxide 1g, citric acid 10.3g and Na₂HPO₄·12H₂35.8g of O, 20100 mu L of Tween-and 1000mL of distilled water, and the pH value is 5.

The solution B is prepared from 700mg of tetramethylbenzidine, 40mL of DMSO, 10.3g of citric acid and 1000mL of distilled water, and the pH value is 2.4.

The reaction termination solution was 2M sulfuric acid solution.

Coating the coated antigen on a 96-well enzyme label plate, wherein the coating concentration of each well is 100ng/mL, and reacting overnight at 4 ℃; the next day, the liquid in the wells was spun off, washed 3 times with PBST containing 0.05% tween, and the microplate was inverted and patted dry on absorbent paper; adding a sealing solution, incubating at 37 ℃ for 30 minutes, throwing the liquid in the hole, washing for 3 times by using 0.05% PBST, and inverting the ELISA plate on water-absorbent paper to dry; preparing thiamethoxam standard solution with the concentration of 0ng/mL, 1ng/mL, 4ng/mL, 12ng/mL, 37ng/mL, 111ng/mL, 333ng/mL and 1000ng/mL, adding 50 mu L of standard sample or processed sample into each well, repeating the standard sample and the sample for 2-4 times, adding 50 mu L of diluted antibody, and incubating for 30 minutes at 37 ℃; throwing the liquid in the hole, washing with PBST for 3 times, and inversely arranging the ELISA plate on water-absorbent paper for patting dry; adding enzyme-labeled secondary antibody, and incubating for 30 minutes at 37 ℃; the liquid in the wells was spun off, washed 3 times with PBST and patted dry; and (3) uniformly mixing the solution A and the solution B in equal volume, adding 100 mu L of solution A into each hole, performing light-shielding color development for 10-15 minutes, adding a stop solution to terminate the reaction, and measuring the OD value of each hole at the wavelength of 450nm on an enzyme-labeling instrument.

The OD value of the standard well containing the maximum concentration subtracted from the OD value of the standard well containing 0ng/mL is determined as B₀The OD values of the other holes corrected by the same method are set as B; with B/B₀Value is longitudinalAnd (3) drawing a standard inhibition curve of thiamethoxam by taking the coordinate and the concentration of the corresponding standard substance as the abscissa (figure 1). The concentration of the corresponding sample can be obtained according to the regression equation of the curve, and the concentration IC in thiamethoxam inhibition can also be obtained₅₀(B/B₀50%) and minimum detection limit IC₁₀(B/B₀90%). By the curve equation (y ═ y)₀+a/[1+(x/x₀)b]) Can calculate the IC₅₀At 37ng/mL, linear range (IC) was measured₂₀-IC₈₀) 11-477ng/mL, minimum detection limit IC₁₀It was 4 ng/mL. In the curve equation, y₀＝-0.0272，a＝1.0300，b＝0.8514，x₀＝32.5027。

In the actual sample detection process, the coating antigen (coating concentration is 100ng/mL) adsorbed on the pore wall of the ELISA plate and the thiamethoxam sample to be detected compete with each other to react with the antibody, and the competition result is obtained through a color reaction. And detecting the thiamethoxam with known concentration and drawing a standard inhibition curve, so that the concentration of the thiamethoxam to be detected can be calculated.

The method has the advantages that the thiamethoxam residues in water, vegetables and fruits can be accurately and sensitively detected, the defects that the sample pretreatment process is complicated, the operation is complex and time-consuming and the like in the instrument detection are overcome, and meanwhile, the detection cost is greatly saved.

Example 7 investigation of specificity of anti-thiamethoxam Single-chain antibody

Evaluating the specificity of the antibody SEQ ID NO. 1 to thiamethoxam by using cross reaction rate, coating the coating antigen on a 96-hole enzyme label plate, wherein the coating concentration of each hole is 100ng/mL, and reacting overnight at 4 ℃; the next day, the liquid in the wells was spun off, washed 3 times with PBST containing 0.05% tween, and the microplate was inverted and patted dry on absorbent paper; adding a sealing solution, incubating at 37 ℃ for 30 minutes, throwing the liquid in the hole, washing for 3 times by using 0.05% PBST, and inverting the ELISA plate on water-absorbent paper to dry; respectively preparing 0ng/mL, 1ng/mL, 4ng/mL, 12ng/mL, 37ng/mL, 111ng/mL, 333ng/mL and 1000ng/mL of standard solutions of thiamethoxam, imidacloprid, imidaclothiz, acetamiprid, clothianidin, dinotefuran, thiacloprid, nitenpyram and pymetrozine, adding 50 mu L of standard samples into each hole, repeating for 2-4 times, adding 50 mu L of diluted antibody, and incubating for 30 minutes at 37 ℃; get rid of in the holeThe liquid is washed for 3 times by PBST, and the ELISA plate is inverted on absorbent paper and is patted dry; adding enzyme-labeled secondary antibody, and incubating for 30 minutes at 37 ℃; the liquid in the wells was spun off, washed 3 times with PBST and patted dry; and (3) uniformly mixing the solution A and the solution B in equal volume, adding 100 mu L of solution A into each hole, performing light-shielding color development for 10-15 minutes, adding a stop solution to terminate the reaction, and measuring the OD value of each hole at the wavelength of 450nm on an enzyme-labeling instrument. Separately calculating IC of each analog to be measured₅₀Value, using the formula, cross-reactivity ═ IC₅₀(thiamethoxam)/IC₅₀(analogues)]The cross-reactivity can be calculated at 100%. The experimental result shows that the cross reaction rate of the SEQ ID NO. 1 and the thiamethoxam structural analogue is less than 0.5%, which indicates that the antibody has good specificity to thiamethoxam (Table 1).

TABLE 1 specificity examination of anti-thiamethoxam single-chain antibody

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> university of agriculture in China

<120> scFv-ELISA kit applicable to thiamethoxam residue analysis

<130> KHP191114321.3

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 239

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly Gly

1 5 10 15

Leu Ser Leu Ile Cys Lys Ala Ser Gly Phe Asp Phe Ser Thr Tyr Ala

20 25 30

Val Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

35 40 45

Ser Ile Tyr Ser Gly Ser Tyr Thr Trp Tyr Ala Ala Val Lys Gly Arg

50 55 60

Ala Thr Ile Ser Lys Asp Asn Gly Gln Ser Thr Val Arg Leu Gln Leu

65 70 75 80

Asn Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Thr Lys Cys

85 90 95

Ala Tyr Ser Gly Cys Val His Arg Ile Asp Ala Trp Gly His Gly Thr

100 105 110

Glu Val Ile Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn

130 135 140

Pro Gly Glu Thr Val Lys Ile Thr Cys Ser Gly Ser Ser Ser Gly Asn

145 150 155 160

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

165 170 175

Val Ile Tyr Arg Asn Asn Gln Arg Pro Ser Asn Ile Pro Ser Arg Phe

180 185 190

Ser Gly Ser Leu Ser Gly Ser Thr Asn Thr Leu Thr Ile Thr Gly Val

195 200 205

Gln Val Glu Asp Glu Ala Val Tyr Tyr Cys Gly Ser Ile Asp Asn Phe

210 215 220

Gly Ala Gly Thr Thr Leu Thr Val Leu His His His His His His

225 230 235

Claims

1. An isolated polypeptide, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO. 1.

2. A nucleic acid molecule encoding the polypeptide of claim 1.

3. Biological material comprising a nucleic acid molecule according to claim 2, wherein said biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector or an engineered bacterium.

4. A thiamethoxam detection reagent or kit, wherein the effective component is the polypeptide of claim 1.

5. The thiamethoxam hapten is 3- [2- (2-carboxyethylthio) -5-ethylmethyl ] -5-methyl-4-nitrosamine-1, 3, 5-oxadiazine and has the following structural formula:

6. the thiamethoxam artificial antigen, which is obtained by coupling the thiamethoxam hapten and carrier protein according to claim 5;

wherein the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroid protein, and human serum albumin.

7. The thiamethoxam artificial antigen according to claim 6, wherein the carrier protein is bovine serum albumin or keyhole limpet hemocyanin.

8. An scFv-ELISA kit suitable for thiamethoxam residue analysis is characterized by comprising a kit body, a detachable ELISA plate arranged in the kit body and a reagent arranged in the kit body; each hole of the ELISA plate is coated with the thiamethoxam artificial antigen of claim 6 or 7, and the reagent comprises an anti-thiamethoxam single-chain antibody and at least one of a thiamethoxam standard solution, an enzyme-labeled secondary antibody, a buffer solution PBS, a washing solution PBST, a developing solution and a reaction stop solution;

wherein the anti-thiamethoxam single-chain antibody is the polypeptide of claim 1.

9. The kit of claim 8, wherein the preparation method of the antigen coating solution for coating the ELISA plate comprises:

(1) dissolving 7.788-15.76mg of thiamethoxam hapten as described in claim 5, 2.76-5.52mg of N-hydroxysuccinimide and 4.738-9.476mg of dicyclohexylcarbodiimide in 400 microliter of anhydrous dimethylformamide, then placing the mixture on a magnetic stirrer, stirring at room temperature for reaction overnight, and centrifuging the reaction solution at 5000rpm for 10-20min the next day to obtain a supernatant which is an active ester solution;

(2) dissolving 20-40mg bovine serum albumin in 2-4mL of 0.05M carbonate buffer solution with pH of 9.6, placing the mixture on a magnetic stirrer for stirring, dropwise adding the active ester solution, completing the addition for 20-30min, and then continuously stirring at room temperature for reaction for 6 h;

10. The kit of claim 8, wherein the antigen coating concentration for coating the microplate is 100 ng/mL.

11. The kit of any one of claims 8 to 10, wherein the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled His-tag antibody at a concentration of 0.1 μ g/mL; and/or

The color developing solution comprises solution A and solution B, wherein the solution A is prepared from carbamide peroxide 1g, citric acid 10.3g, and Na₂HPO₄·12H₂O35.8 g, Tween-20100 mu L and distilled water 1000mL, and the pH value is 5; the solution B is prepared from 700mg of tetramethylbenzidine, 40mL of DMSO, 10.3g of citric acid and 1000mL of distilled water, and the pH value is 7.4; and/or

The reaction termination solution is 2M sulfuric acid.

12. The polypeptide of claim 1 for use in any one of:

1) the method is used for thiamethoxam detection;

2) is used for preparing a thiamethoxam detection reagent or a kit.