CN114702592A

CN114702592A - Nano antibody for identifying quinalphos pesticide and enzyme-linked immunoassay method

Info

Publication number: CN114702592A
Application number: CN202210271306.5A
Authority: CN
Inventors: 王弘; 李家冬; 吴广培; 徐振林; 孙远明
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2022-03-18
Filing date: 2022-03-18
Publication date: 2022-07-05
Anticipated expiration: 2042-03-18
Also published as: CN114702592B

Abstract

The invention discloses a nano antibody for identifying quinalphos pesticide and an enzyme-linked immunoassay method. The invention firstly constructs a phage display nano antibody library, and obtains a nano antibody aiming at quinalphos pesticide by screening from the phage display nano antibody library, wherein the amino acid sequence of the nano antibody is shown as SEQ ID NO.1, and the nucleotide sequence for coding the nano antibody is shown as SEQ ID NO. 2. The nano antibody can identify quinalphos pesticide, and has low cross reaction rate to other organophosphorus pesticides; has high thermal stability and organic tolerance. The enzyme-linked immunoassay method provided by the invention has the advantages that the lowest detection limit is 2.57ng/mL, the linear range is 5.84-88.34ng/mL, the detection result is accurate, the stability is good, and the enzyme-linked immunoassay method can be widely applied to detection of quinalphos pesticide residues in agricultural products.

Description

Nano antibody for identifying quinalphos pesticide and enzyme-linked immunoassay method

Technical Field

The invention belongs to the technical field of organophosphorus pesticide residue detection. More particularly, relates to a nano antibody for identifying quinalphos pesticide and an enzyme-linked immunoassay method.

Background

Quinalphos (Quinalphos), also known as Episca, quinovos, etc., and its molecular structural formula is C₁₂H₁₅N₂O₃PS, an industrial product is white tasteless crystal, and has high solubility in common organic solvents such as benzene, acetone, diethyl ether, acetonitrile, ethyl acetate and the like. Quinalphos is a broad-spectrum organophosphorus insecticide, belongs to moderate toxicity, but has high degradation speed on plants and short residual period, and is allowed to be used for vegetable crops. Is widely used for preventing and controlling diseases and pests of rice, cotton, citrus, tea trees, vegetables and the like. Quinalphos has toxicity to people, causes symptoms such as nausea, vomiting, abdominal pain, diarrhea, coma and the like, and acute poisoning can also endanger life.

At present, the problems of abuse and residue of quinalphos are still outstanding, the degradation of quinalphos in the environment is slow, and once the environment is polluted, the long-time pollution can be caused. The research shows that the degradation rate of quinalphos in cotton fields is 50.9 percent 292 days after the application of the pesticide. Therefore, the abuse of quinalphos brings serious hidden dangers to the environmental safety and the human health, and the enhancement of the monitoring and the detection of the quinalphos pesticide is necessary.

At present, the detection technical methods of quinalphos pesticide residue at home and abroad are more, and mainly comprise an instrument method, an enzyme inhibition method and an immunoassay method. Although the instrument method has high accuracy, the problems of complicated sample pretreatment process, expensive instrument and equipment, complex operation and the like can not meet the requirement of screening a large number of agricultural products in the current market; the enzyme preparation in the enzyme inhibition method has poor stability and is easy to generate false positive; an immunoassay method established based on the monoclonal antibody has the advantages of rapidness, sensitivity and high flux, but the antibody is often poor in stability and easy to inactivate under extreme conditions. For example, in the prior art, a fast detection method of quinalphos pesticide residue is disclosed, which utilizes a chemiluminescence principle to establish a mathematical model of quinalphos concentration and relative luminous intensity and obtains the quinalphos pesticide residue through measurement and analysis of the relative luminous intensity. However, there are few reports of nano-antibodies and detection methods that can be used alone for detection of quinalphos pesticide residues, and therefore, more efficient, convenient and low-cost on-site rapid detection and analysis methods for quinalphos pesticide residues need to be developed.

Disclosure of Invention

The invention aims to overcome the defects of the problems and provide a nano antibody for identifying quinalphos pesticide and an enzyme-linked immunoassay method.

The first purpose of the invention is to provide a nano antibody for specifically recognizing quinalphos pesticide.

The second purpose of the invention is to provide a nucleotide for coding the specific identification quinalphos pesticide nano antibody.

The third purpose of the invention is to provide the application of the nano antibody and the nucleotide.

The fourth purpose of the invention is to provide an enzyme-linked immunoassay method for detecting quinalphos pesticide.

The fifth object of the present invention is to provide a recombinant vector.

The sixth object of the present invention is to provide a recombinant cell.

The seventh purpose of the invention is to provide the application of the recombinant vector and the recombinant cell.

The eighth purpose of the invention is to provide a kit for enzyme-linked immunoassay of quinalphos pesticide.

The above purpose of the invention is realized by the following technical scheme:

the invention utilizes phage display technology to carry out affinity panning in a constructed bactrian camel immune antibody library to obtain a nano antibody VHH 8F for resisting quinalphos, wherein the amino acid sequence of the nano antibody is shown as SEQ ID NO.1, and the nucleotide sequence of the coded nano antibody is shown as SEQ ID NO. 2. Research shows that the cross reaction of the nano antibody VHH 8F provided by the invention to other organophosphorus pesticides is low by carrying out cross reaction to different organophosphorus pesticides; meanwhile, experimental analysis such as thermal stability analysis and organic solvent tolerance shows that the nano antibody VHH 8F has stronger thermal stability, and the antigen binding activity after incubation for 5min at 95 ℃ is still higher than 80%; and the antibody also has high acetonitrile tolerance, and when the concentration of acetonitrile is about 30%, the nano antibody VHH 8F still can keep more than 95% of antigen binding activity.

The invention establishes an enzyme-linked immunoassay method for detecting quinalphos pesticide based on a nano antibody VHH 8F by optimizing parameters such as coating antigen concentration, antibody concentration, ion concentration and the like, and the nano antibody detects IC of the quinalphos pesticide₅₀22.72ng/mL, a minimum detection limit of 2.57ng/mL, and a linear range of 5.84-88.34 ng/mL.

The invention provides application of the nano antibody or the nucleotide in detection of quinalphos pesticide or preparation of an immunoassay kit for detection of quinalphos pesticide.

The invention provides an enzyme-linked immunoassay method for detecting quinalphos pesticide, which adopts a nano antibody VHH 8F to carry out enzyme-linked immunoassay and comprises the following steps:

(1) adding a quinalphos pesticide standard substance or a sample to be detected into micropores of an enzyme label plate coated with a complete antigen containing the quinalphos pesticide, and then adding the nano antibody;

(2) adding enzyme-labeled secondary antibody, and incubating;

(3) adding a color development solution, and incubating;

(4) and finally, adding a stop solution, measuring a light absorption value, and establishing a standard curve to calculate the content of the quinalphos pesticide in the sample.

Preferably, the established standard curve is a log of the concentration of each drug₁₀The value is the abscissa, and the light absorption value B of each concentration of the drug and the light absorption value B of the control hole are taken₀The ratio of (A) to (B) is a longitudinal coordinate, a standard curve is established, and then the B/B of the sample to be detected is determined₀The content of the quinalphos pesticide in the sample is calculated.

The invention provides a recombinant vector, which contains the nucleotide, and the sequence of the nucleotide is shown as SEQ ID NO. 2.

The invention provides a recombinant cell containing the recombinant vector.

The invention provides application of the recombinant vector or the recombinant cell in detection of quinalphos pesticide or preparation of an immunoassay kit for detection of quinalphos pesticide.

The invention also provides a kit for enzyme-linked immunoassay of the quinalphos pesticide, which contains the nano antibody.

The invention has the following beneficial effects:

the invention obtains a nano antibody aiming at quinalphos pesticide by screening from a phage display nano antibody library, and shows that the nano antibody VHH 8F can be specifically combined with the quinalphos pesticide by carrying out cross reaction on different organophosphorus pesticides, and the cross reaction rate on other organophosphorus pesticides is low; meanwhile, experimental analysis such as thermal stability analysis and organic solvent tolerance shows that the nano antibody VHH 8F provided by the invention has stronger thermal stability, and the antigen binding activity after incubation for 5min at 95 ℃ is still higher than 80%; and the antibody also has high acetonitrile tolerance, and when the concentration of acetonitrile is about 30%, the nano antibody VHH 8F still can keep more than 95% of antigen binding activity.

The invention provides an enzyme-linked immunoassay method for detecting quinalphos pesticide, which adopts a nano antibody VHH 8F for detection, and the nano antibody detects IC of the quinalphos pesticide₅₀22.72ng/mL, a minimum detection limit of 2.57ng/mL, and a linear range of 5.84-88.34 ng/mL. The detection result is accurate, the effect is good, the stability is good, and the method can be widely applied to the detection of the quinalphos pesticide residue in agricultural products.

Drawings

FIG. 1 is a bactrian camel antiserum titer;

FIG. 2 is bactrian camel antiserum inhibition;

FIG. 3 is agarose gel electrophoresis to identify total RNA;

FIG. 4 shows purified nano-antibody VHH 8F after SDS-PAGE analysis;

FIG. 5 shows the thermal stability of Nanobody 8F and monoclonal antibody at different temperatures;

FIG. 6 shows the thermal stability of Nanobody 8F and monoclonal antibody at 95 ℃

FIG. 7 is the tolerance analysis of the nanobody 8F and the monoclonal antibody to acetonitrile;

FIG. 8 is a standard curve for detecting quinalphos by using a nano antibody 8F.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 construction of an immune library against quinalphos pesticide Nanobody

1. Test method

(1) The hapten H1 synthesized in the early stage of the research laboratory is adopted to be respectively coupled with ovalbumin OVA (albumin) and keyhole limpet hemocyanin KLH (keyhole limpet hemocyanin) by an active ester method to prepare complete antigens H1-OVA and H1-KLH.

The specific operation method comprises the following steps: 10mg of H1, 3mg of NHS and 5mg of EDC were weighed out and dissolved in 500. mu.L of DMF, and stirred overnight at 4 ℃. 10mg OVA/KLH was dissolved in 3mL PBS and added dropwise to the overnight H1/NHS/EDC solution and stirring was continued at 4 ℃ for 12H. The stirred solution was dialyzed 5 times against PBS, each time at 12h intervals. Centrifuging the dialyzed solution, taking supernatant, and packaging at-20 deg.C for freezing. The chemical formula of the hapten H1 is shown as follows:

0.5mg of H1-KLH was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously into the neck of a healthy 3 year old Bactrian camel at multiple points. The booster immunization was performed every 2 weeks at the same dose as the first immunization, and 10mL of venous blood was collected on day 7 after each immunization.

The results of the indirect competitive ELISA method for determining serum titer and drug inhibition are shown in FIG. 1 and FIG. 2, and the antiserum titer is significantly improved after the 3 rd immunization and is continuously improved along with the increase of the immunization times. The batch (5 th) of peripheral blood with the best serum inhibitory effect was taken for lymphocyte isolation and RNA extraction.

(2) The method comprises the following specific steps of collecting bactrian camel peripheral blood, and then separating lymphocytes as soon as possible: peripheral blood was diluted with sterile saline in an equal volume of a clean 50mL centrifuge tube without rnase. And (3) carrying out centrifugal separation on the diluted peripheral blood by using a commercial lymphocyte separation solution, wherein the reference centrifugal parameters are as follows: room temperature (25 deg.C), 500g, 30 min. Different blood cells were distributed at different depths in the lymphocyte separation fluid after centrifugation due to different densities, wherein the lymphocytes formed a white cell layer at a depth of about 1/3 below the surface of the liquid. The RNase free pipettes carefully collect the lymphocyte layer into RNase free clean 50mL centrifuge tubes, wash the excess lymphocyte separation medium on the cell surface with sterile saline, and centrifuge to collect the cell pellet. Resuspending and lysing the lymphocytes with a lysis solution TRNsol, wherein about 1-2 mL of TRNsol is required for every 10mL of lymphocytes isolated from the blood, and storing at-80 ℃ for at least one year.

(3) Extraction of total RNA was performed according to the Trizol reagent instructions of Invitrogen corporation. After extracting total RNA, taking 1-2 microliter samples to carry out nucleic acid electrophoresis and using a ultramicro spectrophotometer Nanodrop to identify the purity and the concentration of the total RNA.

Clear

28S and 18S bands were observed on the nucleic acid electrophoresis gel, and the results are shown in FIG. 3, without significant degradation and genomic DNA contamination. A260/A280 of the Nanodrop test is about 2.0, which shows that the quality of the extracted total RNA is better.

First strand cDNA synthesis was performed using total RNA extracted as a template according to the TARAKA first strand reverse transcription kit instructions. After the reverse transcription is finished, the products are firstly mixed uniformly, then are packaged in different sterile centrifuge tubes, and are stored in an environment of minus 80 ℃.

(4) Variable region coding genes of camel heavy chain antibodies are obtained by two rounds of PCR amplification by using Taq Mix DNA polymerase, and primers are adopted as shown in the following table 1. The first round of PCR used primers P1 and P2, and the reaction conditions were as follows: 94 ℃, 4min, 94 ℃, 30s, 55 ℃, 1min, 72 ℃, 1min, 30 cycles, and complete extension for 10min at 72 ℃.

The first round PCR product was electrophoresed through agarose gel, and the desired fragment (600-700bp) was recovered by DNA gel recovery kit. Using the recovered target fragment as a template, a second round of amplification was performed using primers P3 and P4 in Table 1. The PCR reaction conditions are as follows: 94 ℃, 4min, 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 1min, 30 cycles, and complete extension for 10min at 72 ℃. The nano antibody gene fragment (about 400 bp) is obtained by cutting and recovering the gel and is stored at the temperature of minus 20 ℃ for later use.

And carrying out enzyme digestion on the obtained nano antibody gene fragment and phagemid vector pComb3xss by using sfi at 50 ℃, and recovering an enzyme digestion product. Then ligated with T4 ligase overnight at 16 ℃ (pComb3xss to nanobody fragment molar ratio 1: 3).

TABLE 1 primer sequences for amplification of VHH genes

(5) The ligation product was recovered by PCR clean recovery kit, dissolved in 20. mu.L of sterile water, 1. mu.L of purified ligation product was added to 25. mu.L of TG1 electroporation competent cells (removed at-80 ℃ C., thawed on ice), and mixed by flicking the EP tube with the hand to avoid the generation of bubbles, and immediately inserted into ice. Transfer to ice-precooled 0.1cm cuvette to avoid bubble formation and shock inversion (1.8 kv). Quickly adding 0.5mL of non-resistant LB culture medium, blowing and beating the bottom of an electric shock cup for several times, uniformly mixing, transferring into a 50mL centrifuge tube, and culturing at 37 ℃ for 1h at 250 rpm. All purified ligation products were electroporated 20 times into TG 1.

The cells were pooled together (about 10mL) after electroporation, and 10. mu.L of the pooled cells was diluted to 10 with LB medium^-4、10^-5、10^-6100. mu.L of each concentration of the bacterial liquid was applied to an LB-Amp plate, and the remaining electrotransformation bacterial liquid was applied to an LB-Amp plate having a diameter of 13cm in an amount of 0.5mL, to which 20 cells were applied in total, and was subjected to inverted culture at 37 ℃ overnight. The next day the transformation library size was 1.43X 10⁷cfu/mL, and randomly picking 25 single clones, sending them to the company for sequencing, and identifying the antibody libraryAnd (4) diversity. The library capacity was calculated based on the number and diversity of clones.

(6) Scraping the monoclonal on the culture medium by using an LB culture medium, adding glycerol to adjust the concentration to 20%, then subpackaging the obtained product into 1.5mL centrifuge tubes, and placing the centrifuge tubes at the temperature of minus 80 ℃ for freezing storage to obtain the antibody gene bacterial bank of the VHH pesticide resisting quinalphos.

(7) 1mL of the anti-quinalphos pesticide VHH antibody gene bacterial bank is inoculated into 200mL of LB culture medium, 200 mug/mL of ampicillin is added, the temperature is 37 ℃, the rpm is 250/min, and the culture is carried out until the OD600 is about 0.5. The helper phage M13K07 was added and left to infect for 30min (multiplicity of infection 20:1), 37 ℃ at 250rpm/min for 1h, after which 50. mu.g/mL kanamycin was added and cultured overnight. The following day, the supernatant was collected (12000rpm/min, 20min), 1/5 in 20% PEG-NaCl was added, and the mixture was incubated on ice for 2 h. Then, collecting the phage at 12000rpm/min for 20min, and resuspending with PBS to obtain the anti-quinalphos pesticide phage display nano antibody library, and determining the titer of the antibody library to be 5 multiplied by 10¹¹pfu/mL, the remainder was stored at-80 ℃ for use.

Example 2 screening and identification of Quinalphos pesticide Nanobody

The nanobody library established in example 1 was subjected to 4 rounds of affinity panning using H1-OVA of example 1, which was conjugated with OVA protein at carboxyl group of hapten H1, as a coating source, and the panning protocol is shown in table 2.

TABLE 2 Nanobody library panning protocol

(1) Wrapping a plate: and selecting an enzyme label plate with strong adsorbability for elutriation. Each round was coated with 3 background-free wells (KLH, BSA and OVA, 1mg/mL, 100. mu.L/well) and coated wells (100. mu.L/well) and incubated at 37 ℃ for 12-14 h. The next day, wash the plate 2 times with PBST buffer, add 150. mu.L of blocking solution per well, and incubate for 3h at 37 ℃. Removing the blocking solution, oven drying at 37 deg.C for 1h, and storing in 4 deg.C refrigerator.

(2) And (4) elutriation: 100 μ L of the Nanobody library was added to the background-free wells and incubated at 37 ℃ for 1 h. The liquid was then transferred to coated wells and incubated for 1h at 37 ℃. The liquid in the coated wells was discarded, washed 5 times with PBST buffer and 15 times with PBS buffer. The first round was eluted with acid, 100. mu.L of 0.1M Gly-HCl (pH 2.2) was added, incubated at 37 ℃ for 15min, the liquid was aspirated, and neutralized immediately with 50. mu.L of 1M Tris-HCl (pH 8.0). The remaining three rounds were performed by competitive elution, 100. mu.L of the solution of quinalphos diluted in a gradient was added and incubated at 37 ℃ for 1 h. And (3) taking 10 mu L of elution products, calculating the titer through the colony number of a plate, and using the rest elution products for next round of elutriation and screening after the auxiliary phage rescue amplification.

(3) Selection and identification of specific phage clones: 96 phage clones were randomly picked and inoculated into a deep-well plate containing 500. mu.L/well LB medium (Amp), and shake-cultured overnight at 37 ℃ and 180 rpm. mu.L of overnight strain was inoculated into a 1 mL/well medium (Amp) deep well plate, shake-cultured at 37 ℃ and 180rpm for 3h, a final concentration of 1mM IPTG was added to each well, and shake-cultured at 28 ℃ and 180rpm overnight. The next day, centrifuging at 4500rpm for 20min, discarding supernatant, and placing in-80 deg.C ultra-low temperature refrigerator for 3 h. After thawing at room temperature, the pellet was resuspended in 200. mu.L PBS buffer and the deep-well plate was shaken for 1h at 4 ℃. Centrifuging at 4500rpm for 20min, collecting supernatant, and performing ic-ELISA detection, which comprises the following steps:

1) wrapping a plate: the coating antigen H1-OVA was diluted to 1. mu.g/mL with the coating solution, and 100. mu.L of the diluted coating antigen was added to each well, followed by incubation at 37 ℃ for 12-14H in an incubator.

2) And (3) sealing: the next day, each well was washed 2 times with PBST buffer, the liquid in the well was patted dry, 120. mu.L of 2% nonfat dry milk was added to each well, and the wells were closed in a 37 ℃ incubator for 2 h. Washed 2 times with PBST buffer and patted dry for use.

3) Incubating the primary antibody: each well was added 50. mu.L periplasmic protein and 50. mu.L PBS buffer, which was the titer well. Each well was filled with 50. mu.L of the supernatant albumin and 50. mu.L of quinalphos solution at a concentration of 1. mu.g/mL, which was a suppression well. After incubation in a 37 ℃ incubator for 40min, the cells were washed 5 times with PBST buffer and patted dry.

4) Incubation of secondary antibody: mu.L of rabbit anti-VHH-HRP secondary antibody (5000 fold dilution) was added to each well, incubated in a 37 ℃ incubator for 40min, washed 5 times with PBST buffer and patted dry.

5) Color development and termination: mu.L of TMB two-component color developing solution was added to each well, and after incubation at 37 ℃ for 10min, 50. mu.L of 10% H2SO4 was added to each well to terminate the reaction.

6) Reading: and reading the light absorption value at 450nm by using a microplate reader.

From the ic-ELISA assay results, the Inhibition rate (Inhibition rate, I) was calculated as follows:

I(％)＝(1-B/B₀)×100

in the formula, B₀The absorbance value corresponding to the titer hole, and the absorbance value corresponding to the inhibition hole.

The clone with the inhibition rate of more than 50 percent is sent to Rui sequencing company for gene sequencing, and the amino acid sequence is contrasted and analyzed to obtain the nano antibody VHH 8F, wherein the amino acid sequence is shown as SEQ ID NO.1, and the nucleotide sequence for coding the nano antibody VHH 8F is shown as SEQ ID NO. 2.

Example 3 soluble expression and identification of anti-quinalphos Nanobodies

The pComb3xss-VHH 8F plasmid of example 1 was extracted and transformed into E.coli BL21(DE3) competent by chemical transformation. BL21(DE3) recombinant bacteria containing pComb3xss-VHH 8F plasmid were cultured to OD₆₀₀The value is 0.6-0.8, 1mM IPTG is added, and the expression is induced at 37 ℃ for 20 h. The next day, the cells were collected by centrifugation. And then extracting soluble protein in the periplasmic cavity by a Tris-sucrose low-temperature osmotic pressure method, and performing affinity purification on the extracted protein by a Ni column to obtain a soluble nano antibody VHH 8F.

The molecular weight of VHH 8F is about 17-18kDa and the purity is more than 90% as shown in figure 4 by SDS-PAGE analysis and identification. The expression level of VHH 8F was 3mg/L as determined by microspectrophotometer. Example 4 detection of the specificity and sensitivity of quinalphos and similar pesticides by the indirect competitive ELISA of the Nanobody VHH 8F

Selecting other 9 organophosphorus pesticides with relatively serious banning or abuse conditions such as parathion: quinalphos, triazophos, coumaphos, chlorpyrifos, vozapyr, fenitrothion, methyl parathion, fenthion and disulfoton as structural analogs, respectively drawing standard curves by adopting IC-ELISA, and calculating respective IC₅₀And (3) calculating the cross reaction rate of each organophosphorus pesticide and the quinalphos-resistant nano antibody by adopting a formula, wherein the formula is calculated as follows: CR (%) - < 100 × IC₅₀(Quinathion)/IC₅₀(analogs) the results are shown in table 3 below.

TABLE 3 sensitivity and specificity of detection of quinalphos analogue by nano antibody VHH 8F ic-ELISA method

As can be seen from Table 3, the nano antibody VHH 8F can be specifically combined with quinalphos pesticide, has low cross reaction rate to other organophosphorus pesticides, and can be used for detecting quinalphos drugs in practice.

Example 5 thermal stability analysis of Nanobody VHH 8F

The nano antibody VHH 8F and the anti-quinalphos monoclonal antibody prepared in the early stage of the laboratory are diluted to working concentration and respectively placed in 20, 37, 50, 65, 80 and 95 ℃ for incubation for 5 min. After the antibody was returned to room temperature, the binding capacity of the antibody to the antigen was measured by ic-ELISA, and the stability of different antibodies heated at different temperatures for 5min was evaluated using the binding capacity of the unheated antibody to the antigen as 100%.

The nanobody VHH 8F and the monoclonal antibody were diluted to the same working concentration and incubated at 95 ℃ for 10, 20, 30, 40, 50 and 60 min. After the antibody was returned to room temperature, the binding capacity of the antibody to the antigen was determined by ic-ELISA, and the stability of the different antibodies under extreme high temperature conditions was evaluated using the binding capacity of the unheated antibody to the antigen as 100%.

The results are shown in fig. 5, as the temperature is gradually increased from 4 ℃ to 95 ℃, the nanobody still maintains higher activity, and the antigen binding activity of VHH 8F after 5min incubation at 95 ℃ is still higher than 80%, while the monoclonal antibody loses the ability to bind antigen after 5min incubation at 80 ℃. As can be seen from FIG. 6, VHH 8F still has over 95% antigen binding activity after 1h incubation at 95 ℃ whereas the monoclonal antibody has lost its ability to bind antigen under equivalent conditions. Combining the above results, the nanobody VHH 8F has stronger thermal stability compared to the monoclonal antibody.

Example 6 organic solvent tolerance of Nanobody VHH 8F

The nanobody VHH 8F and the monoclonal antibody were diluted to the same working concentration using acetonitrile of different concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70%, and 80%) as antibody diluents, the binding ability of the antibody to the antigen was measured by the ic-ELISA method, and the tolerance of different antibodies to acetonitrile was evaluated using PBS buffer as the antibody diluent with the antibody-antigen binding ability as 100%.

As shown in fig. 7, when the concentration of acetonitrile is about 30%, the antigen binding activity of nanobody 8F can still be maintained at 95% or more, while at the same concentration of acetonitrile, the antigen binding activity of monoclonal antibody can only be maintained at about 40%, indicating that the acetonitrile tolerance of nanobody 8F is better than that of monoclonal antibody.

Example 7 enzyme-linked immunoassay method for detecting quinalphos pesticide

An ic-ELISA method for detecting quinalphos based on VHH 8F is established by optimizing parameters such as coating antigen concentration, antibody concentration, ion concentration and the like, and comprises the following specific steps:

mu.L of the

Nanobody VHH

8F and 50. mu.L of the quinalphos pesticide diluted in a gradient (starting at 4. mu.g/mL, 2-fold dilution, 15 gradients in total) were added to the wells previously coated with H1-OVA and incubated at 37 ℃ for 30 min. Plates were washed 5 times with 200 μ L PBST and patted dry. Add 100. mu.L rabbit anti-HA polyclonal anti-HRP, incubate 30min at 37 ℃. Plates were washed 5 times with 200 μ L PBST and patted dry. Then, 100. mu.L of TMB developing solution was added thereto, and the mixture was incubated at 37 ℃ for 10 min. Finally, 50. mu.L of stop solution (10% H) was added₂SO₄) The absorbance at 450nm was read.

The standard curve for detecting quinalphos pesticide is shown in FIG. 8, IC₅₀22.72ng/mL, a minimum detection limit of 2.57ng/mL, and a linear range of 5.84-88.34 ng/mL.

In conclusion, the nano antibody VHH 8F aiming at quinalphos pesticide is obtained by screening from the phage display nano antibody library and can be matched with the quinalphos pesticideThe combination of different properties, the cross reaction rate to other organophosphorus pesticides is low; VHH 8F has stronger thermal stability and acetonitrile tolerance. In addition, the invention provides an IC-ELISA method for detecting quinalphos based on VHH 8F, and IC thereof₅₀22.72ng/mL, a minimum detection limit of 2.57ng/mL, and a linear range of 5.84-88.34 ng/mL. The detection result is accurate, the effect is good, and the stability is good. The method can be widely applied to detection of quinalphos pesticide residues in agricultural products.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> southern China university of agriculture

<120> nano antibody for identifying quinalphos pesticide and enzyme-linked immunoassay method

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 119

<212> PRT

<213> Nanobody VHH 8F (SIPOS sequencing Listing 1.0)

<400> 1

Glu Val Gln Leu Gln Gln Ser Gly Gly Asp Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Ala Tyr Cys Ile Tyr

20 25 30

Asp Val Thr Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ser Phe Ile Asp Thr Asn Asp Arg Lys Thr Tyr Ala Asp Ser Val Glu

50 55 60

Gly Arg Phe Thr Ile Ser Gln Asp Lys Pro Ser Ala Thr Val His Leu

65 70 75 80

Gln Met Asn Thr Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Lys

85 90 95

Ala Ser Val Thr Trp Pro Cys Arg Pro Ala Thr Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 2

<211> 357

<212> DNA

<213> nucleotide sequence of Nanobody VHH 8F (SIPOS sequencing Listing 1.0)

<400> 2

gaggtgcagc tgcagcagtc tgggggagac tcggtgcagg ctggagggtc tctgagactc 60

tcctgtgtag cctctggacg cgcctactgt atctacgacg tgacctggta ccgccaggct 120

ccaggcaagg agcgcgagtt cgtctcgttt attgatacaa atgaccggaa aacatacgca 180

gactctgttg agggccgatt caccatctcc caagacaaac ccagtgctac ggtgcatctg 240

caaatgaaca ccctgaaacc tgaagacacg gccatgtatt actgcaaagc gagtgtgacc 300

tggccctgtc ggccggctac gtactggggc caggggaccc tggtcaccgt ctcctca 357

Claims

1. The nano antibody for specifically recognizing quinalphos pesticide is characterized in that the amino acid sequence of the nano antibody is shown as SEQ ID No. 1.

2. The nucleotide for coding the specific recognition quinalphos pesticide nano antibody of claim 1 is characterized in that the nucleotide sequence is shown as SEQ ID No. 2.

3. The application of the nano-antibody of claim 1 or the nucleotide of claim 2 in the detection of quinalphos pesticide or the preparation of an immunoassay kit for the detection of quinalphos pesticide.

4. An enzyme-linked immunoassay method for detecting quinalphos pesticide, which is characterized in that the nano antibody of claim 1 is adopted for enzyme-linked immunoassay.

5. The ELISA method of claim 4 comprising the steps of:

(1) adding a quinalphos pesticide standard substance or a sample to be detected into micropores of an enzyme label plate coated with a complete antigen containing the quinalphos pesticide, and then adding the nano antibody of claim 1;

(2) adding enzyme-labeled secondary antibody, and incubating;

(3) adding a color development solution, and incubating;

6. The ELISA method of claim 5 wherein the standard curve established in step (4) is the log of the concentration of each drug₁₀The value is the abscissa, and the light absorption value B of each concentration of the drug and the light absorption value B of the control hole are taken₀The ratio of (A) to (B) is a longitudinal coordinate, a standard curve is established, and then the B/B of the sample to be detected is determined₀The content of the quinalphos pesticide in the sample is calculated.

7. A recombinant vector comprising the nucleotide of claim 2.

8. A recombinant cell comprising the recombinant vector of claim 7.

9. The use of the recombinant vector of claim 7 or the recombinant cell of claim 8 in the detection of quinalphos pesticide or in the preparation of an immunoassay kit for the detection of quinalphos pesticide.

10. An enzyme-linked immunoassay kit for detecting quinalphos pesticide, which is characterized by comprising the nano antibody of claim 1.