CN114507288B - Affinity matured anti-quetiapine nano antibody and application thereof - Google Patents

Abstract

The invention discloses an affinity matured anti-quetiapine nano antibody and application thereof. According to the invention, affinity maturation is carried out on the quetiapine nanobody by phage display and biopanning technology, so that a nanobody with higher affinity to the quetiapine than that of a wild nanobody is obtained, and the amino acid sequence of the nanobody is shown as SEQ ID NO. 1. Compared with the original nano antibody, the sensitivity of the nano antibody can be improved by 18%, and the minimum detection limit is reduced by 25%; the nano antibody provided by the invention is used in an enzyme-linked immunoassay method for detecting the quetiapine pesticide, and the IC thereof ₅₀ The detection result of the method is accurate, the sensitivity is higher, the effect is good, the stability is good, and the method can be widely applied to the detection of the quinfos pesticide residue.

Description

Affinity matured anti-quetiapine nano antibody and application thereof

Technical Field

The invention belongs to the technical field of antibody engineering. More particularly relates to an affinity matured anti-quetiapine nanobody and application thereof.

Background

Quinfos is an organophosphorus pesticide with chemical formula of C ₁₂ H ₁₅ N ₂ O ₃ PS is colorless crystalline powder, is broad-spectrum insecticidal and acaricidal, has stomach toxicity and contact killing effects, has no systemic and fumigating effects, and has certain effectIs effective in killing ovum. The insecticidal composition is widely applied to crops such as cotton, rice, fruit trees, vegetables and the like for preventing and controlling various pests including gall midge, rice leaf rollers, cotton bollworms, citrus leaf miners, leafhoppers, cabbage caterpillars and the like. At present, research on the degradation characteristics of the quetiapine at home and abroad is mainly focused on hydrolysis and photolysis, and the quetiapine poisoning insecticide is not easy to degrade in the environment. The quinfos is widely used in agricultural production, so that residues with different degrees are generated in crops, the harm to human bodies is mainly acute toxicity, a series of neurotoxic symptoms such as sweating, tremors, confusion and language disorder can occur after large dosage or repeated contact, and serious people can breathe paralyzed and even die.

The immunoassay technology is the most common technology applied to the rapid detection of the residues of the veterinary drugs at present, and is also a research hotspot at home and abroad. However, the existing immunodetection technology cannot meet all practical detection requirements in terms of sensitivity, specificity and the like, and the defects of instability, difficulty in mass production and the like of the monoclonal antibody limit the further development of the related immunoanalysis technology. Nanobodies are a new generation of genetically engineered antibody fragments derived from camelid heavy chain antibodies. Heavy chain antibodies (HcAbs), which are antibodies containing only heavy chains and lacking light chains, were discovered by Hamers-masterman et al in 1993 when separating dromedaries serum. The antigen binding region of the heavy chain antibody is expressed by genetic engineering technology, the molecular weight of the antigen binding region is only about 15kD, and the molecular weight of the antigen binding region is only about one tenth of that of a traditional antibody, and the antigen binding region is the minimum antibody fragment with antigen recognition and binding capacity prepared by genetic engineering means at present, and the size of the antigen binding region is in a nano level, so the antigen binding region is also called as a nano antibody.

In the research process of the nano antibody, the nano antibody has a property different from the traditional antibody, wherein the most typical property is that the nano antibody has strong stability, can still keep higher antigen binding capacity after high-temperature treatment and in an organic solution with a certain concentration, and the property has important significance for practical application of the antibody, so that the immunoassay method based on the nano antibody can meet the requirement of the market for rapid detection. However, the high-sensitivity nano antibody capable of being applied to detecting the quinalphos is not seen at present, so that more sensitive antibodies capable of being used for detecting the quinalphos are necessary to be developed in order to meet the actual detection requirements, and the method has practical significance for actual production and application.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings and provide an affinity matured anti-quetiapine nano antibody and application thereof.

It is a first object of the present invention to provide an affinity matured nanobody against quetiapine.

It is a second object of the present invention to provide nucleotides encoding said affinity matured anti-quetiapine nanobody.

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

It is a fourth object of the present invention to provide a recombinant cell.

It is a fifth object of the present invention to provide the use of said nanobody, said nucleotide, said recombinant vector or said recombinant cell.

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

The seventh object of the invention is to provide a kit for detecting quetiapine.

The above object of the present invention is achieved by the following technical scheme:

on the basis of an anti-quetiapine nano antibody 8F (the amino acid sequence of the anti-quetiapine nano antibody is EVQLQQSGGDSVQAGGSLRLSCVASGRAYCIYDVTWYRQAPGKEREFVSFIDTNDRKTYADSVEGRFTISQDKPSATVHLQMNTLKPEDTAMYYCKASVTWPCRPATYWGQGTLVTVSS), homologous modeling and molecular butt joint are carried out, key recognition sites of the nano antibody and the quetiapine are determined, an antibody mutation library is constructed by adopting a site-directed saturation mutation technology, and further affinity maturation is carried out on the anti-quetiapine nano antibody 8F by a phage display and biopanning technology, so that a nano antibody M1-7E with higher affinity to the quetiapine than that of a wild nano antibody (8F) is obtained, the amino acid sequence of the nano antibody is shown as SEQ ID NO.1, and the nucleotide sequence of the specific recognition quetiapine nano antibody with mature coding affinity is shown as SEQ ID NO.2.

The invention provides a recombinant vector, which contains the nucleotide sequence SEQ ID NO.2 of the anti-quetiapine nano antibody with the coded affinity maturation.

The invention provides a recombinant cell, which contains the recombinant vector.

The invention provides application of the nanobody, the nucleotide, the recombinant vector or the recombinant cell in detection of a quetiapine pesticide or preparation of a kit for detecting the quetiapine pesticide.

The invention provides an enzyme-linked immunoassay method for detecting a quetiapine pesticide, which adopts the affinity matured nano antibody for specifically recognizing quetiapine and takes an artificial antigen of quetiapine as a coating source to carry out enzyme-linked immunoassay.

Preferably, the enzyme-linked immunosorbent assay is an indirect enzyme-linked immunosorbent assay.

More preferably, the quinalphos artificial antigen is H1-OVA, and the structural formula is shown as follows:

the specific detection method comprises the following steps:

s1, preparing an ELISA plate coated with a complete antigen of a quetiapine pesticide;

s2, adding a quinalphos pesticide standard substance or a sample to be detected into the micro-holes of the ELISA plate, and then adding the nano-antibody;

s3, adding enzyme-labeled secondary antibodies, and incubating;

s4, adding a color development liquid, and incubating;

s5, adding a stop solution and measuring;

s6, log of standard concentration of medicine ₁₀ And the value is an abscissa, the ratio of the light absorption value of each standard substance concentration to the light absorption value of the zero standard hole is taken as an ordinate, a standard curve is established, and the content of the quinalphos pesticide in the sample to be detected is calculated according to the light absorption value of the sample to be detected.

The nano antibody provided by the inventionIn the ELISA method for detecting the quetiapine pesticide, the quetiapine pesticide and IC can be detected ₅₀ The detection result of the method is accurate, the effect is good, and the stability is good, wherein the detection limit is 18.62ng/mL, the lowest detection limit is 1.93ng/mL, and the linear range is 5.14-67.45ng/mL.

The invention provides a kit for detecting quetiapine, which comprises the affinity matured nano antibody specifically recognizing quetiapine.

The invention adopts an affinity matured nano antibody obtaining method, carries out homologous modeling and molecular butt joint on the basis of an anti-quetiapine nano antibody, determines key recognition sites of the nano antibody and quetiapine, adopts a site-directed saturation mutation technology to construct an antibody mutation library, and further carries out affinity matured anti-quetiapine nano antibody through phage display and biopanning technology to obtain a mutant nano antibody M1-7E.

Preferably, the amino acid sequence of the olaquindox-resistant nano antibody 8F is EVQLQQSGGDSVQAGGSLRLSCVASGRAYCIYDVTWYRQAPGKEREFVSFIDTNDRKTYADSVEGRFTISQDKPSATVHLQMNTLKPEDTAMYYCKASVTWPCRPATYWGQGTLVTVSS.

Preferably, the key recognition sites are Gly26, arg27, ser98.

The invention has the following beneficial effects:

the invention carries out affinity maturation on the anti-quetiapine nano-antibody 8F to obtain a nano-antibody with higher affinity to the quetiapine than the wild nano-antibody (8F), the amino acid sequence of the nano-antibody is shown as SEQ ID NO.1, the nano-antibody can specifically identify the quetiapine pesticide, the sensitivity is improved by 18 percent compared with the original nano-antibody, and the minimum detection limit is reduced by 25 percent; the nano antibody provided by the invention can be used in an enzyme-linked immunoassay method for detecting the quetiapine pesticide, and can be used for detecting the quetiapine pesticide and IC ₅₀ The detection result of the method is accurate, the effect is good, the stability is good, and the method can be widely applied to detection of the quinfos pesticide residue in agricultural products.

Drawings

FIG. 1 is a three-dimensional model of nanobody 8F;

FIG. 2 is a drawing of a nanobody 8F homology modeling;

FIG. 3 is an ERRAT diagram of nanobody 8F homology model;

FIG. 4 is a Verify3D plot of nanobody 8F homology model;

FIG. 5 three-dimensional model of the interaction of nanobody 8F with quetiapine (A) and two-dimensional graph of the interaction (B);

FIG. 6 shows a nanobody 8F saturation mutagenesis protocol;

FIG. 7 is an ic-ELISA for identification of positive mutant clones;

FIG. 8 shows the standard curves of the detection of quetiapine by nanobodies M1-7E and 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 homologous modeling and molecular docking of anti-quetiapine nanobodies

1. Homologous modeling and model evaluation of anti-quetiapine nanobody

The crystal structure of the anti-parathion nanobody VHH9 obtained by laboratory researches of the inventor is used as a template, an amino acid sequence (EVQLQQSGGDSVQAGGSLRLSCVASGRAYCIYDVTWYRQAPGKEREFVSFIDTNDRKTYADSVEGRFTISQDKPSATVHLQMNTLKPEDTAMYYCKASVTWPCRPATYWGQGTLVTVSS) of the anti-quetiapine nanobody 8F is uploaded to a SWISS-MODEL online server, and a MODEL with the highest score is finally obtained through screening, wherein green represents an FR region, yellow represents a CDR1 region, blue represents a CDR2 region and red represents a CDR3 region, as shown in figure 1. To further Verify the reliability of the model, homologous models were evaluated by amino acid dihedral angles of the pull-plots and ERRAT and Verify3D indices, respectively.

The pull type diagram can be used for analyzing whether the framework structure and conformation of the model are reasonable, and the result shows that the model amino acid is partitioned into 4 areas: a core region, an additional allowed region, a substantially allowed region, and a forbidden region. The results of the pull-up plot are shown in fig. 2, wherein 95.1% of the amino acids fall in the core region and 100% of the amino acids are in the favorable positions, and the nanobody 8F homology model can be considered reliable according to the pull-up plot scoring criteria (more than 90% of the amino acids fall in the core region).

The ERRAT index shows that the number (side chain) of non-bond interactions formed between different atom type pairs within 0.35nm is theoretically more than 85 points through the three-dimensional structure of the crystallography evaluation model, which indicates that the model is reliable, and the homology model of the nanobody 8F is 88.889, as shown in fig. 3, and meets the requirements. The compatibility of the three-dimensional structure of the model with any amino acid sequence was scored by a Verify3D module, and the result is shown in fig. 4, wherein the 3D-1D score of 100% residues in the homologous model of nanobody 8F is not less than 0.2, and exceeds 80% of the gridlines, which indicates that the model is reliable. In conclusion, the resulting model is reliable and can be used for subsequent studies.

2. Molecular docking of nanobody 8F with quetiapine drug

After model evaluation, the three-dimensional model of the nanobody 8F and the structure of the quetiapine are input into a CDOCKER module in Discovery Studio software for semi-flexible butt joint, and the butt joint model with the highest score, namely the butt joint model with the lowest binding free energy is selected, as shown in figure 5.

By analyzing the docking results, we can find that the interaction between the nanobody 8F and quetiapine is mainly hydrogen bonding interaction and hydrophobic interaction. Residues involved in the interaction pocket include Val2, leu4, ala24, gly26, arg27, cys30, ile31, val34, ala97, ser98, val99, cys103, arg104, which are mainly derived from the FR1 region, CDR1 region and CDR3 region, indicating that the binding pocket of nanobody 8F is mainly composed of the FR1 region, CDR1 region and CDR3 region. Wherein Gly26 forms hydrogen bond with O atom at position 3 on the quetiapine, the bond length isSer98 and Arg27Form hydrogen bonds with H atoms at the 21-position and the 27-position on the quinalphos respectively, and the bond lengths are respectively +.>And->Gly26, ser98 and Arg27 are all located in the CDR regions of nanobody 8F, indicating that these three amino acid residues may be key sites for nanobody 8F to recognize the quetiapine drug molecule.

EXAMPLE 2 construction of Point saturation mutation library

According to the molecular docking and alanine scanning experimental results, site-directed saturation mutation is carried out on amino acids at three sites of Gly26, arg27 and Ser98, the mutation scheme is shown in figure 6, and a saturation mutation library is constructed.

The primer sequences for saturation mutagenesis are shown in Table 1 below, and first, fragment 1 was amplified using primers GLY26ARG27-F and SER98-R under the following conditions: 95 ℃ for 30s;95 ℃,15s,55 ℃,15s,72 ℃,30s,30 cycles; 72 ℃ for 10min; then, the primer GLY26ARG27-R and SER98-F are used for amplifying the fragment 2, and the amplification reaction conditions are as follows: 95 ℃ for 30s;95 ℃,15s,65 ℃,15s,72 ℃,4min,30 cycles; 72℃for 10min.

Table 1 saturation mutation primer sequence listing

Note that: italics are mutation sites, where n=a/C/G/T, k=g/T.

And after the reaction is finished, a small amount of PCR products are taken for agarose gel electrophoresis detection, if the target band is correct and single, dpnI digestion is carried out before homologous recombination, and methylated template plasmids are removed. After homologous recombination, the ligation product was transformed into E.coli TG1, the library capacity of the saturated mutation library was calculated by dilution plating, and clones were randomly picked for sequencing to verify diversity. Scraping the clone on the culture medium by using an LB culture medium, adding glycerol to adjust the concentration to 20%, subpackaging the culture medium into a 1.5mL centrifuge tube, and placing the centrifuge tube at-80 ℃ for freezing to obtain the quinfos pesticide VHH antibody saturated mutant gene bacteria library.

1mL of the mutant library was inoculated into 200mL of LB (200. Mu.g/mL of ampicillin), and cultured at 37℃and 220rpm/min to OD ₆₀₀ About 0.5. According to the infection complex ratio of 20:1 helper phage M13K07 was added and allowed to stand for infection for 30min, followed by 37℃and 220rpm/min for 1h, after which kanamycin (50. Mu.g/mL) was added and incubated overnight. The next day, centrifuge at 12000rpm/min for 20min, collect supernatant, add 1/5 of 20% PEG-NaCl solution, incubate on ice for 2h or overnight at 4 ℃. Subsequently, 12000rpm/min, centrifuging for 20min, and re-suspending the phage with PBS to obtain the olaquindox pesticide phage display nanometer antibody mutant library, sucking 10 mu L to measure the titer of the antibody library, and storing the rest at-80 ℃ for later use.

Example 3 panning and identification of anti-quetiapine nanobody mutants

The hapten H1 synthesized in the early stage of a research laboratory and the ovalbumin OVA (albumin) are coupled through an active ester method to prepare the complete antigen H1-OVA, and the structural formula of the complete antigen H1-OVA is shown as follows:

the nanobody mutant library established in example 2 was subjected to an affinity panning using H1-OVA as a coating antigen for a total of 4 rounds of panning protocols as shown in table 2 below.

TABLE 2 Point saturation mutation library panning protocol

(1) And (3) wrapping the plate: the coating antigen was diluted to 1. Mu.g/mL with PBS. Each round was coated with 3 background-removed wells (KLH, BSA and OVA,1mg/mL, 100. Mu.L/well) and coated with primary wells (100. Mu.L/well) and incubated overnight at 37 ℃. The next day, the plates were washed 2 times with PBST buffer, 120. Mu.L of blocking solution was added to each well, and incubated at 37℃for 3h. Discarding the sealing solution, 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. Centrifugation was performed at 4500rpm for 20min, and the supernatant was subjected to ic-ELISA.

(4) The specific steps of the ic-ELISA detection are as follows:

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 supernatant and 50. Mu.L of quetiapine solution at a concentration of 1. Mu.g/mL were added to each well, which was a 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 The absorbance corresponding to the potency well, B is the absorbance corresponding to the inhibition well

The results of the ic-ELISA identification of positive mutant clones are shown in FIG. 7, clones with inhibition rate exceeding 50% are sent to Rui sequencing company for gene sequencing, and the amino acid sequence is subjected to comparative analysis.

EXAMPLE 4 soluble expression and Activity characterization of mutants

Extracting the nano antibody pComb3xss-VHH plasmid by using a plasmid extraction kit, then transferring the plasmid into escherichia coli BL21 (DE 3) to be competent by a chemical conversion method, and randomly picking a monoclonal antibody for sequencing and identification. Culturing BL21 (DE 3) recombinant bacteria containing nano antibody target fragment in LB culture medium to OD ₆₀₀ The value is 0.6-0.8, 1mM inducer IPTG is added, and the induction and expression are continued for 20h at 37 ℃. The following day, cells were collected by centrifugation at 12000r/30 min. Extracting periplasmic soluble protein by a Tris-sucrose low-temperature osmotic pressure method, and purifying by Ni column affinity chromatography to obtain the soluble nano antibody.

The method takes H1-OVA as a coating source, establishes indirect competition ELISA detection, and comprises the following specific steps:

1) Coating: the coating antigen H1-OVA was diluted to 0.5. Mu.g/mL with PBS, 100. Mu.L of diluted coating antigen was added to each well, and incubated overnight at 37 ℃.

2) Closing: each well was washed 2 times with PBST buffer, patted dry, added with 120 μl of 2% nonfat dry milk (PBS dilution) and blocked in an incubator at 37 ℃ for 3 hours. Washing with PBST buffer solution for 2 times, and beating to dry at 4 ℃ for standby.

3) Incubating primary antibodies: 50 mu L of the purified nano antibody and 50 mu L of the quinalphos solution diluted into different concentrations are added into each hole, and a potency hole without adding pesticides is formed. After incubation for 30min at 37℃the plates were washed 5 times with PBST buffer and patted dry.

4) Incubating a secondary antibody: mu.L of rabbit anti-VHH-HRP secondary antibody (20000 fold dilution) was added to each well, incubated for 30min at 37℃and 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 at 37℃for 10min, 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.

And finally, detecting the sensitivity of the nano antibody obtained by screening to obtain the anti-quetiapine nano antibody M1-7E with mature affinity, wherein the amino acid sequence of the anti-quetiapine nano antibody is shown as SEQ ID NO.1, and the nucleotide sequence of the anti-quetiapine nano antibody with mature affinity is coded as SEQ ID NO.2. The quinfos pesticide is detected by adopting the nanometer antibodies M1-7E and the nanometer antibody 8F respectively, the standard curve of the quinfos pesticide is shown in figure 8, and the IC of the nanometer antibodies M1-7E ₅₀ 18.62ng/mL, 18% higher than nanobody 8F, 1.93ng/mL as the lowest detection limit, and 5.14-67.45ng/mL as the linear range.

On the basis of the anti-quetiapine nano antibody 8F, the invention carries out homologous modeling and molecular butt joint, determines key recognition sites of the nano antibody and quetiapine, adopts a site-directed saturation mutation technology to construct an antibody mutation library, and further carries out affinity maturation on the anti-quetiapine nano antibody through phage display and biopanning technology, thus obtaining a nano antibody M1-7E with higher affinity to the quetiapine than a wild type nano antibody, and the amino acid sequence of the nano antibody is shown as SEQ ID NO. 1. Compared with the original nano antibody (8F), the sensitivity of the nano antibody can be improved by 18%, and the minimum detection limit is reduced by 25%; the nano antibody provided by the invention can be used in an enzyme-linked immunoassay method for detecting the quetiapine pesticide, and can be used for detecting the quetiapine pesticide and IC ₅₀ The detection result of the method is accurate, the effect is good, the stability is good, and the method can be wider, wherein the minimum detection limit is 1.93ng/mL, the linear range is 5.14-67.45ng/mLThe method is 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.

Claims (9)

1. An affinity matured anti-quetiapine nanometer antibody with an amino acid sequence shown as SEQ ID NO. 1.

2. A gene of which the nucleotide sequence is shown as SEQ ID NO.2 and which codes for the affinity matured anti-quetiapine nanobody of claim 1.

3. A recombinant expression vector comprising the nucleotide sequence of claim 2.

4. A recombinant expression cell comprising the recombinant expression vector of claim 3.

5. Use of the nanobody of claim 1, the gene of claim 2, the recombinant expression vector of claim 3 or the recombinant expression cell of claim 4 for detecting quetiapine.

6. Use of the nanobody of claim 1, the gene of claim 2, the recombinant expression vector of claim 3 or the recombinant expression cell of claim 4 in the preparation of a kit for detecting quetiapine.

7. An enzyme-linked immunoassay method for detecting a quetiapine pesticide, which is characterized in that the nanobody of claim 1 is used, and the quetiapine artificial antigen H1-OVA is used as a coating antigen for enzyme-linked immunoassay;

the structural formula of the quinalphos artificial antigen H1-OVA is shown as follows:

8. the method of claim 7, wherein the enzyme-linked immunoassay is an indirect enzyme-linked immunoassay.

9. A kit for detecting quetiapine comprising the nanobody of claim 1.