CN114702592B

CN114702592B - Nanobody for recognizing quetiapine pesticide and enzyme-linked immunoassay method

Info

Publication number: CN114702592B
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: 2023-04-25
Anticipated expiration: 2042-03-18
Also published as: CN114702592A

Abstract

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

Description

Nanobody for recognizing quetiapine 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 recognizing quetiapine pesticides and an enzyme-linked immunoassay method.

Background

Quinalphos (also known as Icass, quioxaphos, etc.), of which the molecular structural formula is C ₁₂ H ₁₅ N ₂ O ₃ PS, industrial products are white odorless crystals, and have higher solubility in several common organic solvents such as benzene, acetone, diethyl ether, acetonitrile, ethyl acetate and the like. The quinfos is a broad-spectrum organophosphorus insecticide, belongs to moderate toxicity, 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 insect pests of rice, cotton, citrus, tea trees, vegetables and the like. The quetiapine has toxicity to people, causes symptoms such as nausea, vomiting, abdominal pain, diarrhea, coma and the like, and can also endanger life when acute poisoning.

At present, the problems of misuse and residue of the quinalphos are still outstanding, the degradation of the quinalphos in the environment is slow, and once the environment is polluted, the long-time pollution is caused. Studies have shown that the degradation rate of quetiapine in cotton fields after 292 days of application is 50.9%. Therefore, the misuse of the quetiapine brings serious hidden danger to the safety of the environment and the health of human beings, and the enhancement of the monitoring and the detection of the quetiapine pesticide is necessary.

At present, the method for detecting the quinfos pesticide residue at home and abroad is more in technology and mainly comprises an instrument method, an enzyme inhibition method and an immunoassay method. Although the instrument method has high accuracy, the pretreatment process of the sample is complicated, the price of the instrument and the equipment is high, the operation is complex and the like, and the method can not meet the requirement of screening a large amount 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; the 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. The method comprises the steps of determining the relative luminous intensity of the quetiapine pesticide residue, and determining the relative luminous intensity of the quetiapine pesticide residue. However, at present, there are few reports on nanobodies and detection methods which can be used for detecting the quinfos pesticide residue independently, so that more efficient, convenient and low-cost on-site rapid detection and analysis methods for the quinfos pesticide residue need to be developed.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the problems and provide a nano antibody for identifying the quetiapine pesticide and an ELISA method.

The first object of the invention is to provide a nanobody which specifically recognizes the quetiapine pesticide.

The second object of the invention is to provide a nucleotide for encoding the specific recognition quinalphos pesticide nano antibody.

It is a third object of the present invention to provide the use of said nanobodies and nucleotides.

The fourth object of the invention is to provide an ELISA method for detecting quetiapine pesticides.

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

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

It is a seventh object of the present invention to provide the use of said recombinant vector and recombinant cells.

The eighth object of the invention is to provide a kit for enzyme-linked immunoassay for detecting the quinfos pesticide.

The above object of the present invention is achieved 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 of anti-quetiapine, the amino acid sequence of which is shown as SEQ ID NO.1, and the nucleotide sequence of which is coded as SEQ ID NO. 2. Research shows that the nano antibody VHH 8F provided by the invention has low cross reaction rate on other organophosphorus pesticides by carrying out cross reaction on 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%; the nanometer antibody VHH 8F still can maintain more than 95% of antigen binding activity when the acetonitrile concentration is about 30%.

The invention establishes an ELISA method for detecting the quetiapine pesticide based on the nano antibody VHH 8F by optimizing parameters such as the concentration of the coating antigen, the concentration of the antibody, the concentration of the ion and the like, and the nano antibody detects the IC of the quetiapine pesticide ₅₀ 22.72ng/mL, a minimum detection limit of 2.57ng/mL, and a linear range of 5.84-88.34ng/mL.

The invention provides application of the nano antibody or the nucleotide in detecting quetiapine pesticides or preparing an immune kit for detecting quetiapine pesticides.

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

(1) Adding a quetiapine pesticide standard substance or a sample to be detected into a micropore of an ELISA plate coated with a quetiapine pesticide complete antigen, and then adding the nano antibody;

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

(3) Adding a color development liquid and incubating;

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

Preferably, the established standard curve is in log of each drug concentration ₁₀ Values are on the abscissa, and the absorbance value B of each concentration of drug is compared with the value B of the control Kong Xiguang ₀ The ratio of (2) is the ordinate, a standard curve is established, and then according to the B/B of the sample to be tested ₀ The values were used to calculate the amount of quinfos pesticide in the samples.

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

The invention provides a recombinant cell containing the recombinant vector.

The invention provides an application of the recombinant vector or the recombinant cell in detecting a quetiapine pesticide or preparing an immune kit for detecting the quetiapine pesticide.

The invention also provides an enzyme-linked immunoassay kit for detecting the quinfos pesticide, which contains the nano antibody.

The invention has the following beneficial effects:

according to the invention, the nano antibody aiming at the quetiapine pesticide is obtained by screening a phage display nano antibody library, and through cross reaction on different organophosphorus pesticides, the nano antibody VHH 8F can be specifically combined with the quetiapine pesticide, and has low cross reaction rate on other organophosphorus pesticides; 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%; the nanometer antibody VHH 8F still can maintain more than 95% of antigen binding activity when the acetonitrile concentration is about 30%.

The invention provides an ELISA method for detecting quetiapine pesticides, which adopts a nano antibody VHH 8F for detection, and the nano antibody detects IC of the quetiapine pesticides ₅₀ 22.72ng/mL, a minimum detection limit of 2.57ng/mL, and a linear range of 5.84-88.34ng/mL. The method has the advantages of accurate detection result, good effect and good stability, and can be widely applied to detection of the quinfos pesticide residue in agricultural products.

Drawings

FIG. 1 shows the serum titers of Bactrian camels;

FIG. 2 shows the serum inhibition of Bactrian camels;

FIG. 3 is an agarose gel electrophoresis for identifying total RNA;

FIG. 4 shows the purified nanobody VHH 8F by 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 an analysis of the tolerance of nanobody 8F and monoclonal antibody to acetonitrile;

FIG. 8 is a standard curve of the detection of quetiapine by nanobody 8F.

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 construction of anti-quetiapine pesticide nanobody immune library

1. Test method

(1) The hapten H1 synthesized in the early stage of a research laboratory, ovalbumin (OVA) and keyhole limpet hemocyanin KLH (keyhole limpet heocyanin) are respectively coupled by an active ester method to prepare complete antigens H1-OVA and H1-KLH.

The specific operation method is as follows: 10mg of H1,3mg of NHS and 5mg of EDC were weighed out respectively, dissolved in 500. Mu.L of DMF and stirred at 4℃overnight. 10mg of OVA/KLH was weighed, dissolved in 3mL of PBS, and added dropwise to the H1/NHS/EDC solution overnight, followed by stirring at 4℃for 12 hours. The stirred solution was dialyzed 5 times against PBS, each time for 12h. Centrifuging the dialyzed solution, taking supernatant, and then packaging at-20 ℃ 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. Boosting was performed every 2 weeks at the same dose as the first immunization, and 10mL venous blood was collected on day 7 after each immunization.

The serum titers and drug inhibition were determined by indirect competition ELISA, and the results are shown in fig. 1 and 2, where the antiserum titers increased significantly after the 3 rd immunization and increased continuously with increasing number of immunizations. The peripheral blood of the best lot (5 th time) for serum inhibition was taken for lymphocyte separation and RNA extraction.

(2) After the peripheral blood of the Bactrian camel is collected, lymphocyte separation needs to be carried out as soon as possible, and the specific operation method comprises the following steps: peripheral blood was diluted by mixing with sterile saline in equal volumes in clean 50mL centrifuge tubes without rnase. Centrifuging the diluted peripheral blood by using commercial lymphocyte separating liquid, wherein reference centrifugation parameters are as follows: room temperature (25 ℃), 500g,30min. Different blood cell densities are centrifuged and distributed at different depths in the lymphocyte separation medium, wherein the lymphocytes are at about 1/3 depth below the liquid surface to form a white cell layer. The RNase-free pipettor carefully collected the lymphocyte layer into a clean 50mL centrifuge tube free of RNase, washed the cell surface with sterile physiological saline and centrifuged to collect the cell pellet. The lymphocytes are resuspended and lysed with a lysate TRNSol, and about 1-2 mL TRNSol per 10mL of blood isolated lymphocytes are required, and stored at-80℃for at least one year.

(3) Total RNA extraction was performed according to Trizol reagent Specification from Invitrogen. After total RNA extraction, 1-2. Mu.L of the sample was taken for nucleic acid electrophoresis and the purity and concentration were identified by Nanodrop using an ultra-micro spectrophotometer.

Clear

28S and 18S bands were seen on the nucleic acid electrophoresis gel, and the results are shown in FIG. 3, without significant degradation and no hybridization of genomic DNA. The Nanodrop measurement shows that the A260/A280 is about 2.0, which indicates that the total RNA quality is better.

The first strand cDNA was synthesized using the total RNA extracted as a template according to the TARAKA first strand reverse transcription kit. After the reverse transcription is completed, the products are uniformly mixed, and then are subpackaged in different sterile centrifuge tubes and stored in an environment of-80 ℃.

(4) The variable region encoding gene of the camel heavy chain antibody was obtained by two rounds of PCR amplification using Taq Mix DNA polymerase, and the primers were used as shown in Table 1 below. 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, 72 ℃ and 10min of thorough extension.

The PCR product of the first round is subjected to agarose gel electrophoresis, and the target fragment (600-700 bp) is recovered by a DNA gel cutting recovery kit. The second round of amplification was performed using the recovered target fragment as a template and primers P3 and P4 in Table 1. The PCR reaction conditions were: 94 ℃,4min,94 ℃,30s,55 ℃,30s,72 ℃,1min,30 cycles, 72 ℃ and thoroughly extending for 10min. The nanometer antibody gene fragment (about 400 bp) is obtained by cutting and recycling, and is preserved at the temperature of minus 20 ℃ for standby.

The nanobody gene fragment and phagemid vector pComb3xss obtained above were digested with sfi at 50 ℃ and the digested product was recovered. Then ligated overnight (pComb 3xss and nanobody fragment molar ratio 1:3) with T4 ligase at 16 ℃.

TABLE 1 primer sequences for amplifying VHH genes

(5) After the above-mentioned connection product is recovered by means of PCR cleaning recovery kit, it is dissolved in 20 mul of sterile water, 1 mul of purified connection product is added into 25 mul of TG1 electrotransformation competent cells (-80 deg.C, taken out from ice and melted), and then the above-mentioned connection product is uniformly mixed by using light-flicked EP tube so as to prevent air bubble from being produced, and immediately inserted into ice. Transfer to an ice-chilled 0.1cm electric stun cup to avoid air bubbles and shock conversion (1.8 kv). 0.5mL of the LB medium without resistance is rapidly added, the bottom of the electric shock cup is blown and evenly mixed for a plurality of times, and then the mixture is transferred into a 50mL centrifuge tube for culture at 37 ℃ for 1h at 250 rpm. All purified ligation products were electrotransferred to TG1 by 20 times.

The 20 bacterial liquids after electrotransformation are put together (about 10 mL), 10 mu L is taken and diluted into 10 by LB culture medium ^-4 、10 ^-5 、10 ^-6 100. Mu.L of each concentration of bacterial liquid was spread on an LB-Amp plate, and the remaining electrotransfer bacterial liquid was spread on a 13cm diameter LB-Amp plate every 0.5mL, and 20 pieces were spread together, and cultured upside down at 37℃overnight. The next day the transformation library size was calculated to be 1.43×10 ⁷ cfu/mL, 25 monoclonal were randomly picked, sent to company for sequencing, and the diversity of the antibody library was identified. The pool capacity was calculated based on clone number and diversity.

(6) Scraping the monoclonal on the culture medium by using an LB culture medium, adding glycerol to adjust the concentration to 20%, subpackaging the obtained product into a 1.5mL centrifuge tube, and placing the obtained product at-80 ℃ for freezing storage to obtain the quinalphos-resistant pesticide VHH antibody gene bacteria library.

(7) 1mL of the anti-quinalphos pesticide VHH antibody gene bacteria library 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. Helper phage M13K07 was added and allowed to stand for infection for 30min (multiplicity of infection ratio 20:1), 37℃at 250rpm/min for 1h, followed by addition of 50. Mu.g/mL kanamycin for overnight incubation. The next day, the supernatant was collected (12000 rpm/min, 20 min), 1/5 of 20% PEG-NaCl solution was added, and incubated on ice for 2h. Subsequently, collecting phage at 12000rpm/min for 20min, and re-suspending with PBS to obtain the anti-quetiapine pesticide phage display nanometer antibody library, and determining the titer of the antibody library to be 5×10 ¹¹ pfu/mL, the rest is stored at-80℃for later use.

Example 2 screening and identification of quetiapine pesticide nanobodies

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

TABLE 2 nanobody library panning protocol

(1) And (3) wrapping the plate: and selecting an ELISA plate with stronger adsorptivity for washing and screening. Each round was coated with 3 background-removed wells (KLH, BSA and OVA,1mg/mL, 100. Mu.L/well) and coated wells (100. Mu.L/well), and incubated at 37℃for 12-14h. The next day, plates were washed 2 times with PBST buffer, 150 μl of blocking solution was added to each well, and incubated 3h at 37 ℃. Discarding the sealing solution, oven drying at 37deg.C for 1 hr, and storing in a refrigerator at 4deg.C.

(2) And (3) washing and screening: 100. Mu.L of nanobody library was added to the background-removed wells and incubated at 37℃for 1h. The liquid was then transferred to the coated primary wells and incubated at 37℃for 1h. The liquid in the original wells of the coating 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 50. Mu.L of 1M Tris-HCl (pH 8.0) was immediately added for neutralization. The remaining three rounds were eluted by competition, 100. Mu.L of a gradient diluted quetiapine solution was added and incubated at 37℃for 1h. And (3) taking 10 mu L of the eluted product, calculating the titer through the colony count of the flat plate, and amplifying the rest eluted product through the rescue of the auxiliary phage for the next round of elutriation.

(3) Selection and identification of specific phage clones: 96 phage clones were randomly picked and inoculated into deep well plates containing 500. Mu.L/well LB medium (Amp), and shake-cultured overnight at 37℃and 180 rpm. mu.L of overnight bacteria were inoculated into 1 mL/Kong Peiyang-base (Amp) deep well plates, shake-cultured at 37℃and 180rpm for 3 hours, 1mM IPTG was added to each well, and shake-cultured at 28℃and 180rpm for overnight. The next day, centrifugation was carried out at 4500rpm for 20min, the supernatant was discarded, and the mixture was placed in an ultra-low temperature refrigerator at-80℃for 3h. After thawing at room temperature, the pellet was resuspended in 200. Mu.L PBS buffer and the deep well plate was placed in 4℃and shaken for 1h. Centrifuging at 4500rpm for 20min, collecting supernatant, and performing ic-ELISA detection, which comprises the following steps:

1) And (3) wrapping the plate: the coating antigen H1-OVA was diluted to 1. Mu.g/mL with the coating solution, 100. Mu.L of the diluted coating antigen was added to each well, and incubated for 12-14H in an incubator at 37 ℃.

2) Closing: the next day, each well was washed 2 times with PBST buffer, the wells were drained, 120. Mu.L of 2% nonfat dry milk was added to each well, and the wells were closed in a 37℃incubator for 2 hours. Wash 2 times with PBST buffer and dry for use.

3) Incubating primary antibodies: mu.L of periplasmic protein and 50. Mu.L of PBS buffer were added to each well, which was the titer well. 50. Mu.L of the supernatant and 50. Mu.L of the quetiapine solution at a concentration of 1. Mu.g/mL were added to each well, which was the inhibition well. After incubation for 40min in an incubator at 37 ℃, the incubation was performed 5 times with PBST buffer and the incubation was performed.

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

5) Color development and termination: after adding 100. Mu.L of TMB two-component color development solution to each well and incubating for 10min at 37℃in an incubator, 50. Mu.L of 10% H2SO4 was added to each well to terminate the reaction.

6) Reading: the absorbance at 450nm was read with a microplate reader.

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

I(％)＝(1-B/B ₀ )×100

wherein B is ₀ And B is the absorbance corresponding to the inhibition hole.

And (3) sending the clone with the inhibition rate of more than 50% to a Rui sequencing company for gene sequencing, and comparing and analyzing the amino acid sequence to obtain the nano antibody VHH 8F, wherein the amino acid sequence of the nano antibody VHH 8F is shown as SEQ ID NO.1, and the nucleotide sequence of the nano antibody VHH 8F is shown as SEQ ID NO. 2.

Example 3 soluble expression and identification of anti-quetiapine nanobodies

The pComb3xss-VHH 8F plasmid of example 1 was extracted and transformed by chemical transformationThe method is transferred into escherichia coli BL21 (DE 3) competent. BL21 (DE 3) recombinant bacteria containing pComb3xss-VHH 8F plasmid are cultivated to OD ₆₀₀ The value is 0.6-0.8, 1mM IPTG is added, and the induction of expression is carried out for 20h at 37 ℃. The following day, the cells were harvested by centrifugation. And extracting periplasmic cavity soluble protein by a Tris-sucrose low-temperature osmotic pressure method, and carrying out Ni column affinity purification to obtain the soluble nano antibody VHH 8F.

As shown in FIG. 4, the molecular weight of VHH 8F is about 17-18kDa, and the purity is over 90%. The expression level of VHH 8F was 3mg/L as determined by a micro-spectrophotometer. Example 4 specificity and sensitivity of nanobody VHH 8F Indirect competitive ELISA for detecting quetiapine and similar pesticides

Selecting 9 other organophosphorus pesticides with serious forbidden or abusive conditions, such as parathion and the like: quinfos, triazophos, coumaphos, chlorpyrifos, phoxim, fenitrothion, methyl parathion, bendrophos and etoxyphos are used as structural analogues, IC-ELISA is adopted to respectively draw standard curves, and respective IC is calculated ₅₀ And calculating the cross reaction rate of each organophosphorus pesticide and the anti-quetiapine nano antibody by adopting a formula, wherein the formula is as follows: CR (%) =100×ic ₅₀ (quetiapine)/IC ₅₀ (analog) and the results are shown in Table 3 below.

TABLE 3 sensitivity and specificity of detection of quetiapine analogs by nanobody VHH 8F ic-ELISA method

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From Table 3, it is clear that the nanobody VHH 8F can specifically bind with the quetiapine pesticide, has low cross reaction rate to other organophosphorus pesticides, and can be used for detecting the quetiapine pesticide in practice.

Example 5 thermal stability analysis of nanobody VHH 8F

The nanobody VHH 8F and the anti-quetiapine monoclonal antibody prepared in the earlier stage of the laboratory are diluted to the working concentration and respectively placed in 20, 37, 50, 65, 80 and 95 ℃ for incubation for 5min. After the antibody was returned to room temperature, the binding capacity of the antibody to the antigen was measured by an ic-ELISA method, and the stability of different antibodies heated at different temperatures for 5min was evaluated with the binding capacity of unheated antibodies 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 60min. 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 the different antibodies under extremely high temperature conditions was evaluated with the unheated binding capacity of the antibody to the antigen as 100%.

As shown in fig. 5, the nanobody remained highly active as the temperature was gradually increased from 4 ℃ to 95 ℃, and the antigen binding activity of VHH 8F after 5min incubation at 95 ℃ was still higher than 80%, whereas the monoclonal antibody had lost the ability to bind antigen after 5min incubation at 80 ℃. As can be seen from fig. 6, VHH 8F still had more than 95% antigen binding activity after incubation for 1h at 95 ℃, whereas monoclonal antibodies had lost the ability to bind antigen under comparable conditions. Taken together, nanobody VHH 8F has a greater thermal stability than monoclonal antibodies.

Example 6 organic solvent tolerance of nanobody VHH 8F

Nanobody VHH 8F was diluted with monoclonal antibody to the same working concentration using acetonitrile at different concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70% and 80%) as antibody dilutions, the binding capacity of the antibody to antigen was measured using ic-ELISA, and the tolerance of the different antibodies to acetonitrile was evaluated using PBS buffer as antibody dilution with the binding capacity of the antibody to antigen as 100%.

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

Example 7 ELISA method for detecting quetiapine pesticide

By optimizing parameters such as the concentration of the coating antigen, the concentration of the antibody, the concentration of the ions and the like, an ic-ELISA method for detecting the quinfos based on the VHH 8F is established, and the specific steps are as follows:

50. Mu.L of

nanobody VHH

8F and 50. Mu.L of a gradient diluted (starting from 4. Mu.g/mL, 2-fold dilution, 15 gradients total) of quetiapine pesticide were added to wells previously coated with H1-OVA and incubated for 30min at 37 ℃. Plates were washed 5 times with 200 μl PBST and dried by pipetting. mu.L of rabbit anti-HA polyclonal-HRP was added and incubated at 37℃for 30min. Plates were washed 5 times with 200 μl PBST and dried by pipetting. Then 100. Mu.L of TMB developing solution was added thereto and incubated at 37℃for 10min. Finally, 50. Mu.L of stop solution (10% H) was added ₂ SO ₄ ) The absorbance at 450nm was read.

The standard curve of the quinfos pesticide detection is shown in figure 8, and the IC ₅₀ 22.72ng/mL, a minimum detection limit of 2.57ng/mL, and a linear range of 5.84-88.34ng/mL.

In conclusion, the nano antibody VHH 8F aiming at the quetiapine pesticide is screened from a phage display nano antibody library, can be specifically combined with the quetiapine pesticide, and has low cross reaction rate on other organophosphorus pesticides; VHH 8F has a strong thermal stability and acetonitrile tolerance. In addition, the invention provides an IC-ELISA method for detecting quetiapine based on VHH 8F, which comprises the IC ₅₀ 22.72ng/mL, a minimum detection limit of 2.57ng/mL, and a linear range of 5.84-88.34ng/mL. Accurate detection result, good effect and good stability. The method can be widely applied to detection of the quinfos pesticide residue in agricultural products.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Sequence listing

<110> agricultural university of south China

<120> a nanobody for recognizing quetiapine pesticide and enzyme-linked immunoassay method

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 119

<212> PRT

<213> nanobody VHH 8F (SIPOSEQUENCELISTER 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 (SIPOSEQUENECLISTERING 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. A nano antibody for specifically recognizing quetiapine pesticide is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. A nucleotide for coding the nano antibody for specifically recognizing the quinfos pesticide according to claim 1, which is characterized in that the nucleotide sequence is shown as SEQ ID NO. 2.

3. Use of the nanobody of claim 1 or the nucleotide of claim 2 in detecting quetiapine pesticides or preparing immunoassay kit for detecting quetiapine pesticides.

4. An enzyme-linked immunoassay method for detecting a quetiapine pesticide, which is characterized in that the nanobody of claim 1 is adopted for enzyme-linked immunoassay.

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

(1) Adding a quetiapine pesticide standard substance or a sample to be detected into a micropore of an ELISA plate coated with a quetiapine pesticide complete antigen, and then adding the nanobody of claim 1;

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

(3) Adding a color development liquid and incubating;

6. The method of claim 5, wherein the standard curve is established in step (4) as log of each drug concentration ₁₀ Values are on the abscissa, and the absorbance value B of each concentration of drug is compared with the value B of the control Kong Xiguang ₀ The ratio of (2) is the ordinate, a standard curve is established, and then according to the B/B of the sample to be tested ₀ The values were used to calculate the amount of quinfos pesticide in the samples.

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

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

9. Use of the recombinant vector of claim 7 or the recombinant cell of claim 8 in detecting a quetiapine pesticide or preparing an immunoassay kit for detecting a quetiapine pesticide.

10. An enzyme-linked immunoassay kit for detecting a quetiapine pesticide, which is characterized by comprising the nanobody of claim 1.