CN107417790B

CN107417790B - VHH-E L ISA kit suitable for triazophos residue analysis

Info

Publication number: CN107417790B
Application number: CN201710641172.0A
Authority: CN
Inventors: 许艇; 王楷; 刘志平; 林优优; 李季; 布鲁斯·杜普里·哈莫克
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2017-07-31
Filing date: 2017-07-31
Publication date: 2020-07-31
Anticipated expiration: 2037-07-31
Also published as: CN107417790A

Abstract

The invention discloses an E L ISA kit for detecting triazophos residue based on a nano antibody and application thereof, wherein 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 triazophos coating antigen, and the reagent comprises an anti-triazophos nano antibody, a triazophos standard solution, an enzyme-labeled secondary antibody, a buffer solution PBS (phosphate buffered saline), a washing solution (PBST), a substrate solution, a color development solution, a reaction termination solution and the like.

Description

VHH-E L ISA kit suitable for triazophos residue analysis

Technical Field

The invention relates to the technical fields of genetic engineering, phage display technology and E L ISA detection, in particular to an E L ISA kit for detecting triazophos residue based on a nano antibody and application thereof.

Background

Triazophos (Triazophos) is an organic phosphate insecticide widely used and has good control effect on pests harmful to grains, cotton, oil, fruits, vegetables, tea leaves and other main crops. Triazophos is highly toxic to fish, bees and silkworms, and is moderately toxic to mammals, and can cause a large amount of acetylcholine to accumulate at the junction of neuroeffector after entering human body, so that symptoms of muscarine, nicotine and central nervous system can occur.

After the use of high-toxicity organophosphorus insecticides such as methamidophos, parathion and the like is limited or forbidden, the use amount of triazophos is rapidly increased in recent years, and the problem of overproof residues in the environment and food is gradually paid attention to due to the fact that the half-life period is long and residues are easy to remain. Therefore, the method has important significance for enhancing the detection of the residual triazophos, ensuring the food safety and human health, reducing the environmental pollution, enhancing the international competitiveness of agricultural and sideline products in China, maintaining the sustainable development of agriculture in China and the like by scientifically and reasonably using the triazophos.

The currently reported triazophos residue detection methods mainly comprise high performance liquid chromatography (HP L C), Gas Chromatography (GC) and chromatography-mass spectrometry (GC/L C-MS), professional laboratories, special instruments and equipment and professional operators are needed to apply the methods, sample pretreatment is complex, analysis cost is high, and the requirement of field rapid monitoring of a large number of samples is difficult to meet, and since the 90 s of the 20 th century, rapidly developed immunoassay technologies have been applied to pesticide residue analysis and environmental monitoring, and the method has the advantages of simplicity, rapidness, low price, high efficiency, strong specificity, high sensitivity and the like.

The enzyme-linked immunosorbent assay method for triazophos residue based on the conventional antibodies (polyclonal antibody and monoclonal antibody) has relatively low stability, and the enzyme-linked immunosorbent assay method is based on the anti-triazophos nano antibody and is adopted, so that the thermal stability of the enzyme-linked immunosorbent assay method is superior to that of the polyclonal antibody immunoassay method.

Disclosure of Invention

The invention aims to provide an E L ISA detection kit for analyzing triazophos residues, which has high specificity, high sensitivity, high accuracy, high precision and simple operation method, can be used for rapidly detecting mass samples and is suitable for rapidly detecting the triazophos residues in samples such as water, vegetables and the like, aiming at the defects of high cost, complex pretreatment, poor specificity, low sensitivity, difficulty in field detection of experiments and the like of the existing pesticide residue instrument analysis method.

In order to realize the purpose of the invention, the invention firstly provides a C-terminal universal PCR primer for constructing a camelid phage display nano antibody library, and the nucleotide sequence of the primer is shown as SEQ ID NO. 7.

The invention further provides an anti-triazophos nano antibody, wherein the amino acid sequence of the antibody is shown as SEQ ID NO. 1, or the amino acid sequence with the same function is formed by replacing, deleting or adding one or more amino acids in the sequence. For example, the amino acid sequence of the sequence shown in SEQ ID NO. 1 with the terminal 6 histidine tags removed.

The nanometer antibody of triazophos can be prepared by the following method, a chemical reaction is utilized to synthesize a hapten O-ethyl-O-3- (1-phenyl-1, 2, 4-triazolyl) -N- (3-carboxymethyl) thiophosphoramide ester of triazophos, the hapten is coupled with keyhole limpet hemocyanin K L H to be used as an immunogen, an immune experimental animal camel is immunized, total RNA of peripheral blood lymphocytes is extracted, a nanometer antibody heavy chain (VHH) gene segment is cloned through reverse transcription and nested PCR, the gene segment is cloned to a phagemid carrier through enzyme digestion connection, the gene segment is efficiently and electrically converted to escherichia coli, a phage nano antibody library is constructed through assisted phage rescue, a specific triazophos phage nano antibody is screened out and expressed and purified to obtain the nanometer antibody of triazophos with high sensitivity and strong specificity, and the prepared nanometer antibody has small molecule, strong solubility, high temperature resistance, easy purification and easy expression.

The E L ISA detection kit for analyzing triazophos residues comprises a kit body, a detachable 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 a triazophos coating antigen, and the reagent comprises the anti-triazophos nano antibody, a triazophos standard solution, an enzyme-labeled secondary antibody, a buffer solution PBS, a washing solution PBST, a developing solution (A solution), a developing solution (B solution), a reaction stop solution and the like.

The triazophos coating antigen is a coupling compound of hapten O-ethyl-O-3- (1-phenyl-1, 2, 4-triazolyl) -N- (3-carboxymethyl) thiophosphoramide ester and bovine serum albumin, and the preparation method of the coating antigen is as follows:

(1) 7.4mg (0.02mmol) O-ethyl-O-3- (1-phenyl-1, 2, 4-triazolyl) -N- (3-carboxymethyl) thiophosphoramide ester (MW 370), 2.65mg (0.024mmol) NHS (N-hydroxysuccinimide, MW 115), 4.8mg (0.023mmol) DCC (dicyclohexylcarbodiimide, MW 206) were dissolved in 200. mu. L anhydrous DMF (N-formylmorpholine), the reaction was stirred overnight at room temperature, the reaction solution was centrifuged (5000rpm, 10min), the precipitate was discarded, and the supernatant was the active ester.

(2) 20mg of BSA (bovine serum albumin, MW 67000) is weighed and dissolved in 2m L carbonate buffer solution (0.05mol/m L, pH9.5), 150 mu L active ester solution is added dropwise with stirring, the dropwise addition is slow and is finished within 20-30 min (preferably about 20 min), and then the reaction is continued for 4-6 h (preferably about 4 h) at room temperature.

(3) And (3) putting the reaction solution into a dialysis bag, dialyzing with PBS (0.01 mol/L pH7.4), changing the solution once every 6-8 h (preferably about 6 h), changing the solution for 5-6 times, centrifuging after dialysis, discarding the precipitate, taking the supernatant as antigen coating solution, wherein the enzyme label plate is a 96-hole enzyme label plate, and the coating concentration of the coated antigen is 285ng/m L.

The concentration of the nano antibody is about 142ng/m L, the enzyme-labeled secondary antibody is an anti-HA label antibody labeled by horseradish peroxidase, the concentration is 0.1 mu g/m L, and the antibody is purchased from Abcam company and HAs the commercial number of ab 1265.

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

The color developing solution B is prepared from 700mg of tetramethylbenzidine, 40m of DMSO L, 10.3g of citric acid and 1000m of distilled water L, and has a pH value of 2.4.

The reaction termination solution is 2M sulfuric acid solution.

The invention also provides a triazophos E L ISA detection reagent, the effective component of which is the anti-triazophos nano antibody.

The invention further provides application of the kit or reagent in detecting triazophos residues in a sample by an E L ISA method, during analysis and detection, a to-be-detected triazophos sample and the anti-triazophos nano antibody are sequentially added into each hole of an ELISA plate coated with the triazophos coated antigen, the solid phase coated antigen and the to-be-detected triazophos compete with each other to react with the nano antibody, because the contents of the solid phase antigen and the added nano antibody in each hole are consistent, when the concentration of the to-be-detected triazophos is high, the antibody bound on the solid phase antigen is less, the binding amount of the added enzyme-labeled secondary antibody and the fixed antibody is less, finally, a substrate solution and a developing solution are added, the developing reaction is light, the OD value detected by an ELISA reader is low, the inhibition rate is high, on the contrary, when the concentration of the to-be-detected, the measured OD value is high, the inhibition rate is low, and the concentration of the to-be-detected triazophos can be calculated according to a standard curve detected by the standard solution.

The method can accurately and sensitively detect the triazophos residues in water and vegetables, the pretreatment process of the sample is simple, the water sample is only required to be filtered and then detected, and the pretreatment of the vegetable sample refers to 'development and application of a triazophos residue detection direct competition E L ISA kit, Lianghuazhou and the like, Chinese food bulletin, No. 8, No. 6 and No. 2008 in 12 months'.

Drawings

FIG. 1 is a standard inhibition curve of triazophos based on nanobodies in example 5 of the present invention. The regression equation of the curve is-0.1591 +1.1293/[1+ (x/106.4572) ^0.8473 ^ y ═ 0.1591+1.1293/[1+ (x/106.4572) ]](R²0.989) inhibitory medium concentration IC₅₀71.46ng/m L, lowest detection limit IC₁₀＝4.33ng/mL。

Detailed Description

Unless otherwise indicated, the examples follow conventional experimental conditions, such as, for example, the Molecular Cloning handbook of Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a L laboratory Manual,2001), or conditions as recommended by the manufacturer's instructions.

EXAMPLE 1 preparation of triazophos-coated antigen

The conjugate compound is prepared from hapten and bovine serum albumin and is used as an envelope antigen. The preparation method comprises the following steps:

(1) 7.4mg of O-ethyl-O-3- (1-phenyl-1, 2, 4-triazolyl) -N- (3-carboxymethyl) thiophosphoramide ester (MW 370) (0.02mmol), 2.65mg of NHS (MW 115) (0.024mmol), 4.8mg of DCC (MW 206) (0.023mmol) were dissolved in 200. mu. L of anhydrous DMF and stirred at room temperature overnight reaction, the reaction solution was centrifuged (5000rpm, 10min), the precipitate was discarded, and the supernatant was the active ester.

(2) 20mg BSA (MW 67000) was dissolved in 2m L carbonate buffer (0.05mol/m L, pH9.5), 150. mu. L active ester solution was added dropwise with stirring, slowly over about 20min, and the reaction was then continued at room temperature for 4 h.

(3) Putting the reaction solution into a dialysis bag, dialyzing with PBS (0.01 mol/L pH7.4), changing the solution once every 6h, changing the solution for 5-6 times in total, centrifuging after dialysis, removing the precipitate, taking the supernatant, and storing at-20 ℃.

Example 2 construction of triazophos phage display Nanobody library

The active ester method is adopted to couple hapten and keyhole limpet hemocyanin, and the specific method is as follows:

(1) 59.7mg of O-ethyl-O-3- (1-phenyl-1, 2, 4-triazolyl) -N- (5-carboxypentyl) thiophosphoramide ester (MW 398) (0.15mmol), 17.825mg of NHS (MW 115) (0.155mmol), 31.518mg of DCC (MW 206) (0.153mmol) were dissolved in 1500. mu. L of anhydrous DMF and the reaction was stirred overnight at room temperature, the reaction mixture was centrifuged (5000rpm, 10min), the precipitate was discarded, and the supernatant was used as the active ester.

(2) A6 m L K L H solution (6.8mg/m L) was added dropwise to a 1200. mu. L active ester solution with stirring, slowly added over about 20min, and then the reaction was continued at room temperature for 4H.

(3) Putting the reaction solution into a dialysis bag, dialyzing with PBS (0.01 mol/L pH7.4), changing the solution every 6h for 5-6 times, dialyzing, centrifuging, removing the precipitate, collecting the supernatant, and storing at-20 ℃.

Dissolving 1mg of conjugate in 1m L physiological saline, mixing with 1m L complete Freund's adjuvant, fully emulsifying, injecting camel, enhancing immunity once every two weeks, mixing with incomplete Freund's adjuvant, performing subcutaneous multipoint immunization on neck and back, performing immunization for 5 times in total, and collecting blood from jugular vein one week after each immunization to detect serum titer from the third immunization.

Separating leukocyte from the 5 th immunized peripheral blood, extracting total RNA, cloning VHH gene segment by reverse transcription PCR and nested PCR, modifying viscosity with restriction enzyme SfiIAnd at the tail end, the VHH gene fragment is connected to a phagemid pComb3x through T4 ligase, and is efficiently electrically transformed into escherichia coli ER2738 to construct a triazophos phage nano antibody library. The primary reservoir volume is determined to be 10⁸cfu, adding helper phage (multiplicity of infection is 20:1) M13KO7 for rescue to obtain phage nanobody library with a library capacity of 10¹⁴pfu/m L, the diversity of the library was good.

Reverse transcription PCR:

the reverse transcription kit adopts PrimeScript^TMRT-PCR Kit, purchased from TaKaRa, under the trade designation: AK 2701.

The reverse transcription system is as follows:

the reaction was carried out at 65 ℃ for 5 min. Taking out and placing on ice, loading the sample according to the following system, and carrying out first strand cDNA synthesis.

30℃ 10min；42℃ 1h；72℃ 5min。

Nested PCR:

first round PCR:

the reaction system is as follows:

the reaction procedure was as follows: pre-denaturation at 94 ℃ for 3 min; 30s at 94 ℃, 30s at 55 ℃, 50s at 72 ℃ and 25 cycles; 5min at 72 ℃.

Second round PCR:

the reaction system is as follows:

the reaction procedure was as follows: pre-denaturation at 94 ℃ for 3 min; 30s at 94 ℃, 40s at 62 ℃, 40s at 72 ℃ and 25 cycles; 5min at 72 ℃.

The nested PCR primer sequences are as follows (SEQ ID NOS: 2-7):

GSP-RT：5’-CGCCATCAATRTACCAGTTGA-3’

LP-leader：5’-GTGGTCCTGGCTGCTCTW-3’

F：5’-CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC-3’

R1：5’-CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG-3’

R2：5’-CATGCCATGACTCGCGGCCGGCCTGGCCGTCTTGTGGTTTTGGTGTCTTGGG-3’

R5：5’-CATGCCATGACTCGCGGCCGGCCTGGCCCTTGCATACTTCATTCGTTCCTG-3’

wherein R represents a base A/G, W represents a base A/T, and K represents a base G/T.

Example 3 screening of specific triazophos phage-displayed Nanobodies

Coating the coated antigen of example 2 on the 1 st well of a 96-well enzyme label plate with the coating concentration of 100 mu g/m L at 4 ℃ overnight, pouring out the coating solution on the next day, washing with PBST for 3 times, sealing the 1 st and 2 nd wells of the enzyme label plate with BSA, incubating at 37 ℃ for 1h, pouring out the sealing solution, washing with PBST for 3 times, adding the phage antibody library of example 2 into the 1 st well, reacting for 2h, pouring out the liquid, drying on clean absorbent paper, washing with PBST for 5 times, adding 100 mu L triazophos standard substance into the 1 st well, reacting for 1h, sucking out the liquid in the 1 st well, adding the 2 nd well, reacting for 1h, removing phage bound with BSA, collecting the eluent, taking 5 mu L for titer determination, and using the rest for amplification.

Adding the phage eluate into fresh Escherichia coli ER2738 bacterial liquid, standing at 37 deg.C for 15min, adding carbenicillin (working concentration 50 mg/L) and SB culture medium, culturing at 37 deg.C under shaking at 220rpm for 2h, adding helper phage M13KO7 (multiplicity of infection MOI ═ 20:1) and kanamycin, culturing overnight, centrifuging the next day, collecting supernatant, and adding PEG-NaCl solution to precipitate and purify phage.

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, decreasing the antigen coating concentration and the competitive elution concentration of the triazophos standard product by 2 times, calculating the titer of each round, selecting the monoclonal for amplification and E L ISA identification, and carrying out 3 rounds of elutriation to obtain the positive monoclonal.

Example 4 expression of specific triazophos Nanobodies

Extracting positive monoclonal plasmid, transferring to escherichia coli TOP 10F' competent cell, coating on solid culture medium for overnight culture after recovery, selecting single clone to culture in SB-benzyl carboxyl medium (carbenicillin working concentration is 50 mg/L) the next day, adding IPTG to induce overnight expression, cracking cell with ultrasonic crusher the next day, filtering with filter membrane, purifying with nickel column, separating and purifying with affinity chromatography of histidine tag and nickel chloride in nickel column to obtain high purity nanometer antibody of triazophos, and analyzing with amino acid sequence to obtain nanometer antibody with amino acid sequence shown in SEQ ID NO 1.

Example 5E L ISA detection kit for analyzing triazophos residue and application thereof

The kit comprises a kit body, a detachable 96-hole enzyme label plate arranged in the kit body and a reagent arranged in the kit body. The reagent comprises the anti-triazophos nano antibody of the embodiment 4, a triazophos standard solution, an enzyme-labeled secondary antibody, a buffer solution PBS, a washing solution PBST, a substrate solution (solution A), a color development solution (solution B), a reaction termination solution and the like.

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

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

The reaction termination solution was 2M sulfuric acid solution.

Coating the antigen on a 96-well enzyme label plate, coating each well with a concentration of 100 μ g/m L, reacting overnight at 4 ℃, throwing out the liquid in the wells the next day, washing 3 times with 0.05% Tween-containing PBST, patting the enzyme label plate upside down on absorbent paper, adding a sealing liquid, incubating at 37 ℃ for 30 minutes, throwing out the liquid in the wells, washing 3 times with 0.05% PBST, inverting the enzyme label plate on the absorbent paper, patting dry, preparing 0ng/m L, 1ng/m L0, 4ng/m L1, 12ng/m L, 37ng/m L, 111ng/m L, 865865/m L, 1000ng/m L triazophos standard, adding 50 μ L standard or treated sample into each well, filtering and then detecting a water sample, referring to a "triazophos residue detection directly competing for E L, applying a reagent kit, applying a staining reagent kit, adding a 50 μ L standard sample or treated sample to each well for a sample, diluting the water sample with a dilution liquid for a fifth year, adding a dilution liquid for a fifth wash liquid, diluting the third time, adding a dilution liquid for a dilution of the same as a dilution of a yellow dye solution, performing a dilution of yellow dye, performing a dilution, and a dilution, performing a dilution for a dilution of yellow dye solution at 35 nm, a fifth time, and a fifth time, wherein the pH is 100-10 minutes, the pH of the wash liquid after the wash liquid is 100 mm, the wash liquid is added to the wash of a wash water of a wash paper, the wells of a wash paper, the wash paper is added to detect the wash paper, the wash paper is added for 30.

The OD value of the standard well containing 0ng/m L minus the OD value of the standard well containing the maximum concentration is determined as B₀The OD values of the other holes corrected by the same method are set as B; with B/B₀Values are plotted on the ordinate and corresponding standard concentrations are plotted on the abscissa, and a standard inhibition curve for triazophos is plotted (FIG. 1). The concentration of the corresponding sample can be obtained according to the regression equation of the curve, and the concentration IC in the triazophos inhibition can also be obtained₅₀(B/B₀50%) and minimum detection limit IC₁₀(B/B₀＝90％)。

In the actual sample detection process, the coating antigen (the coating concentration is 285ng/m L) adsorbed on the pore wall of the ELISA plate and the triazophos to be detected compete with each other and react with an antibody, and the competitive result is obtained through a chromogenic reaction.

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.

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Claims

1. The triazophos resisting nano antibody is characterized in that the amino acid sequence of the antibody is shown as SEQ ID NO. 1.

2. The E L ISA detection kit for analyzing triazophos residues comprises a kit body, a detachable ELISA plate arranged in the kit body and a reagent arranged in the kit body, and is characterized in that each hole of the ELISA plate is coated with a triazophos coating antigen, and the reagent comprises the nano antibody, a triazophos standard solution, an enzyme-labeled secondary antibody, a buffer solution PBS, a washing solution PBST, a developing solution and a reaction stopping solution according to claim 1.

3. The kit according to claim 2, wherein the triazophos coating antigen is a conjugate complex of hapten O-ethyl-O-3- (1-phenyl-1, 2, 4-triazolyl) -N- (3-carboxymethyl) thiophosphoramide ester and bovine serum albumin, and the coating antigen is prepared by the following method:

(1) weighing 7.4mg of O-ethyl-O-3- (1-phenyl-1, 2, 4-triazolyl) -N- (3-carboxymethyl) thiophosphoramide ester, 2.65mg of NHS and 4.8mg of DCC, dissolving in 200 mu L of anhydrous DMF, stirring at room temperature for reaction overnight, centrifuging the reaction solution, removing the precipitate, and taking the supernatant as active ester;

(2) weighing 20mg BSA, dissolving in 0.05mol/m L carbonate buffer solution with pH of 9.5 and 2m L, dropwise adding 150 mu L of the active ester under stirring, finishing the addition for 20-30 min, and then continuously stirring and reacting for 4-6 h at room temperature;

(3) putting the reaction solution into a dialysis bag, dialyzing with PBS (0.01 mol/L pH7.4), changing the solution once every 6-8 h for 5-6 times, centrifuging after dialysis, removing the precipitate, and taking the supernatant as antigen coating solution.

4. The kit of claim 3, wherein the ELISA plate is a 96-well ELISA plate, and the coating concentration of the coating antigen is 285ng/m L.

5. The kit of claim 2, wherein the nanobody concentration is 142ng/m L.

6. The kit according to claim 2, wherein the color developing solution comprises solution A and solution B, and the solution A is prepared from carbamide peroxide 1g, citric acid 10.3g and Na₂HPO₄·12H₂35.8g of O, 20100 mu L of Tween-and 1000m L of distilled water, and the pH value is 5, and the solution B is prepared from 700mg of tetramethyl benzidine, 40m L of DMSO, 10.3g of citric acid and 1000m L of distilled water, and the pH value is 2.4.

7. The kit according to any one of claims 2 to 6, wherein the reaction termination solution is a 2M sulfuric acid solution.

8. The triazophos E L ISA detection reagent, its active ingredient is the nanometer antibody of claim 1.

9. Use of a kit according to any one of claims 2 to 7 or a reagent according to claim 8 for detecting triazophos residues in a sample by the ISA method E L.