Preparation of a novel recognition element full-length recombinant antibody of thiacloprid and application thereof in time-resolved fluorescent test strip

Abstract

The application discloses a novel thiacloprid recognition element full-length recombinant antibody preparation and application thereof in time-resolved fluorescent test paper strips, and belongs to the technical field of molecular biology and time-resolved immunological analysis rapid detection. The application provides a preparation method of thiacloprid hapten and complete antigen, and sequences of a thiacloprid full-length antibody heavy chain and light chain, wherein the amino acid sequence of the antibody heavy chain variable region is shown as SEQ ID NO. 8, and the amino acid sequence of the antibody light chain variable region is shown as SEQ ID NO. 6. The method for detecting thiacloprid by the test paper strip is simple, rapid, stable, reliable and high in sensitivity, is suitable for screening and on-site monitoring of a large number of samples, and has a good commercial application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology technology and time-resolved immunoassay rapid detection technology, specifically relating to the preparation of a full-length recombinant antibody with a novel recognition element for thiamethoxam and its application in a time-resolved fluorescent test strip. Background Technology

[0002] Neonicotinic pesticides are widely used worldwide. They bind to nicotinic acetylcholine receptors in insects, causing abnormal excitement and death through convulsive paralysis. Neonicotinic pesticides are widely present in soil, tap water, fruits, vegetables, and grains, significantly increasing the risk of human contamination. Exposure to neonicotinic pesticides can lead to adverse health effects such as cancer, chronic diseases, birth defects, and infertility. Thiacloprid (TCL), a representative neonicotinic pesticide, is a relatively polar pesticide that more easily penetrates deep into the soil, contaminates groundwater, and inevitably enters the food chain, posing a threat to public health. Currently, the maximum residue limits (MRLs) for fruits in China are 0.2-1 mg / kg, and for vegetables, they are 0.02-2 mg / kg (GB 2763-2021). Therefore, there is an urgent need to establish a rapid, sensitive, and accurate detection method.

[0003] Thiamethoxam residues in food samples have been detected using instrumental methods such as chromatography and mass spectrometry. However, these methods require expensive, large-scale equipment and complex sample preparation processes, making them unsuitable for rapid on-site detection. Immunoassay, on the other hand, does not require expensive equipment and offers lower detection costs. Antibodies are the core element of immunoassay, responsible for recognizing analytes; their specificity and sensitivity play a crucial role in immunoassay detection. Currently, the antibodies used in immunoassay are mainly monoclonal antibodies, which offer better specificity compared to polyclonal antibodies. However, due to the complexity of animal immune function, it is difficult to eliminate batch-to-batch variability in antibodies. Therefore, there is an urgent need for a recombinant antibody for detecting thiamethoxam. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing thiamethoxam hapten, complete antigen, and full-length recombinant antibody, as well as an immunochromatographic test strip for rapid and sensitive detection of thiamethoxam using a time-resolved fluorescent probe. This invention prepares full-length recombinant antibodies based on a mammalian expression system, uses fluorescent microspheres instead of traditional colloidal gold, and conjugates the fluorescent microspheres with the full-length recombinant antibody to prepare a fluorescent probe for immunochromatography. By reading the fluorescence value of the detection line on a fluorescence immunoassay analyzer, rapid quantitative analysis of thiamethoxam in fruit and vegetable samples can be performed.

[0005] This invention provides a thiamethoxam hapten, the structural formula of which is shown in Formula I:

[0006]

[0007] The present invention provides a method for preparing the above-mentioned thiamethoxam hapten, wherein the thiamethoxam technical material is subjected to a substitution reaction with mercaptopropionic acid under alkaline conditions, followed by an acidification reaction, and after separation and purification, the thiamethoxam hapten shown in Formula I is obtained.

[0008] This invention provides a complete thiamethoxam antigen, the structural formula of which is shown in Formula II:

[0009]

[0010] Among them, protein refers to the carrier protein.

[0011] The present invention provides a method for preparing the above-mentioned complete thiamethoxam antigen, wherein Formula I is coupled to a carrier protein by carbodiimide method to obtain the complete thiamethoxam antigen.

[0012] A full-length recombinant antibody against thiamethoxam, wherein the amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO. 8; the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO. 6; the amino acid sequence of the constant region of the antibody heavy chain is shown in SEQ ID NO. 9; and the amino acid sequence of the constant region of the antibody light chain is shown in SEQ ID NO. 7.

[0013] In one embodiment, the nucleotide sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO.1, and the nucleotide sequence of the variable region of the antibody light chain is shown in SEQ ID NO.2.

[0014] The present invention provides a recombinant vector containing a gene encoding the above-mentioned full-length recombinant antibody against thiamethoxam.

[0015] The present invention relates to a cell carrying the above-described recombinant vector.

[0016] This invention provides the application of the above-mentioned recombinant antibody, the above-mentioned recombinant vector, or the above-mentioned recombinant microbial cells in the preparation of a kit for detecting thiamethoxam.

[0017] In one embodiment, the kit is used for ELISA detection, Western blotting assay, immunohistochemistry, immunofluorescence, or immunochromatographic quantitative test strips.

[0018] In one embodiment, the cell is a mammalian cell line.

[0019] In one embodiment, the cells are Expi293F cells.

[0020] This invention provides a fluorescent probe, characterized in that the fluorescent probe is made by incorporating Eu... 3+ - Fluorescent microspheres are conjugated with the aforementioned full-length recombinant antibody against thiamethoxam.

[0021] This invention provides a time-resolved immunochromatographic quantitative test strip for detecting thiamethoxam. The time-resolved immunochromatographic quantitative test strip includes a sample pad, a nitrocellulose membrane, and absorbent paper. The nitrocellulose membrane has a detection line and a control line. The detection line is coated with the above-mentioned complete antigen, and the control line is coated with goat anti-mouse secondary antibody.

[0022] In one embodiment, the detection concentration of the thiamethoxam is 0.01-10 ng / mL.

[0023] In one embodiment, the amount of the thiamethoxam complete antigen is 0.2 mg / mL, and the amount of the goat anti-mouse secondary antibody complete antigen is 1 mg / mL.

[0024] In one embodiment, the detection line and the control line are 4-6 mm apart; the width of the test strip is 3-5 mm.

[0025] This invention provides a method for detecting thiamethoxam content, the method comprising the following steps:

[0026] (1) Eu 3+ - Fluorescent microspheres were conjugated with a full-length recombinant antibody against thiamethoxam as a fluorescent probe. The probe was mixed with thiamethoxam standard and incubated at 37°C for 15 min to allow for a fully competitive reaction. The mixture was then added to the sample pad of the time-resolved immunochromatographic quantitative test strip for chromatography. The fluorescence intensity values ​​of the standard at the detection line and control line were measured to obtain the T value and C value, respectively. A negative control was set up, i.e., the sample did not contain thiamethoxam, and the fluorescence intensity T0 value was measured.

[0027] (2) Use T / T0 as the abscissa and add the logarithm of the concentration of the standard as the ordinate to establish a linear standard curve.

[0028] (3) Eu 3+ - Fluorescent microspheres were coupled with a full-length recombinant antibody against thiamethoxam as a fluorescent probe. The probe was mixed with thiamethoxam standard and incubated at 37°C for 15 min to allow for a fully competitive reaction. The probe was then added to the sample pad of a time-resolved immunochromatographic quantitative test strip for chromatography. The fluorescence intensity (FI) of the standard at the detection line and control line was measured. The FI was then substituted into the standard curve obtained in step (2) to obtain the thiamethoxam content in the sample to be tested.

[0029] This invention provides an application of the above-mentioned time-resolved immunochromatographic quantitative test strip in the field of food testing.

[0030] Beneficial effects: (1) The thiamethoxam hapten and complete antigen of the present invention, by appropriately modifying the molecular structure of thiamethoxam, make it more similar to the original thiamethoxam in terms of molecular structure, stereochemistry, electronic distribution and other properties, so that the original molecular characteristic structure of thiamethoxam can be exposed on the surface of the complete antigen, which is beneficial to antibody recognition.

[0031] (2) The full-length recombinant antibody against thiamethoxam of the present invention is prepared by expression in Expi293F cells based on a mammalian expression system. The prepared recombinant antibody has a strong affinity and specificity for thiamethoxam technical. The thiamethoxam test strip prepared using this recombinant antibody has higher detection sensitivity and lower detection limit for thiamethoxam.

[0032] (3) The thiamethoxam time-resolved fluorescence test strip provided by this invention can rapidly and quantitatively detect the content of thiamethoxam in fruits and vegetables. It has high specificity and high sensitivity. When the concentration range of thiamethoxam is 0.01-10 ng / mL, the logarithm of its concentration has a linear relationship with T / T0, and the linear equation is Y=-19.113LogX+62.038, R 2 =0.9935, and the detection limit can reach 0.003 ng / mL. The detection method provided by this invention has a wide detection range, and its quantitative linear range can reach 0.01 ng / mL-10 ng / mL. The detection method is stable and reliable, with sample recovery rates of 83.28%-114.28% and coefficients of variation of less than 5.812%. The recombinant antibody against thiamethoxam is labeled with fluorescent microspheres. The fluorescent microspheres use europium, a lanthanide element, which has a higher signal value and higher sensitivity. Attached Figure Description

[0033] Figure 1 ESI-MS spectrum of thiamethoxam hapten;

[0034] Figure 2 UV scans of thiamethoxam hapten, complete antigen, and carrier protein;

[0035] Figure 3 : Target gene design scheme;

[0036] Figure 4 : SDS-PAGE results of the recombinant full-length antibody against thiamethoxam obtained using a mammalian cell expression system;

[0037] Figure 5 : ic-ELISA results of the recombinant full-length antibody against thiamethoxam obtained using a mammalian cell expression system;

[0038] Figure 6 Figure showing the results of optimizing the fluorescent probe for the thiamethoxam immunochromatographic test strip;

[0039] Figure 7 Standard curve for detecting thiamethoxam in fruits and vegetables. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0041] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0042] Example 1: Synthesis of thiamethoxam hapten

[0043] Mercaptopropionic acid (130 mg, 1.2 mmol) and sodium ethoxide (160 mg, 2.4 mmol) were added to 10 mL of dimethyl sulfoxide. After complete dissolution, thiamethoxam (290 mg, 1.2 mmol) was added while the temperature was gradually increased to 80 °C and maintained for 8 hours. The solution was adjusted to pH 3 with 1 mol / L hydrochloric acid and extracted with dichloromethane (3 × 30 mL). The organic extract was washed with water (3 × 30 mL), dried on anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation to obtain a yellow oily product, which is the compound of formula I. The obtained product was analyzed by liquid chromatography-mass spectrometry, and the ESI-MS spectrum is shown below. Figure 1 As shown in the figure. Liquid chromatography results showed a strong peak at a retention time of 2.19 min, and mass spectrometry results showed that the molecular weight of the target product in positive ion mode was 323, which was consistent with the theoretical molecular weight, indicating that the hapten shown in Formula I was successfully prepared.

[0044]

[0045] Example 2: Complete Synthesis of Thiamethoxam Antigen

[0046] 3.6 mg of thiamethoxam hapten, 6.8 mg of EDC, and 2.5 mg of NHS were dissolved in 110 μL of DMF and activated at room temperature for 4 hours to prepare solution A. 25 mg of carrier protein was weighed and dissolved in 1 mL of CBS buffer (0.05 M, pH 9.0), and stirred to prepare solution B. Solution A was added dropwise to solution B under magnetic stirring, and the reaction was carried out overnight at 4°C with magnetic stirring. The reaction solution was dialyzed against PBS at 4°C for 3 days, with the dialysate changed 4 times daily. The complete antigen was obtained and stored at 20°C for later use. The hapten prepared in Example 1 and the artificial antigen prepared in Example 2 were identified by UV wavelength scanning (220–440 nm). The maximum absorption wavelengths of the thiamethoxam hapten, OVA, and complete thiamethoxam antigen before and after conjugation were compared. The results are as follows: Figure 2As shown, OVA and hapten have UV absorption peaks at 274 nm and 253 nm, respectively. The maximum UV absorption wavelength of the antigen is 256 nm, which falls between that of OVA and hapten. These results indicate that the complete antigen (TCL-OVA) shown in Formula II was successfully prepared.

[0047]

[0048] Example 3: Construction of antibody expression plasmid and expression of full-length recombinant antibody

[0049] 1. Antibody plasmid construction

[0050] The thiamethoxam DNA sequence was optimized using the online codon optimizer GenScript based on Expi293F codon preference and obtained from a company providing gene synthesis services. A C-terminal histidine (6×HIS) tag was constructed on the thiamethoxam antibody heavy chain. Figure 3 The full-length recombinant antibody expressed can be easily purified by immobilized metal affinity chromatography (IMAC).

[0051] 2. Expression of full-length recombinant antibody

[0052] (1) Constructing plasmid DNA:

[0053] Thiamethoxam heavy chain SEQ ID NO.11:

[0054] All sequences of heavy chains

[0055] CAAGTGACCCTGAGAGAGAGCCCCGGCATGCTGCAGCCTAGCCAAACCCTGAGCCTCA

[0056] CCTGCAGCTTCAGCGGCTTCAGCATGAGCATGGGCGTGGGCTGGATCAGACAGCCTAG

[0057] CACCTTCGCCGGCAAGGGCCTGGAGTGGCTGGCTAGCATCTTCGGCTGGAACGAGAAC

[0058] AAGTACTACCCCGCCCTGATGAGCAGACTGAACACCATCAGCAGAGACACAAGCAAG

[0059] AACCTGGTGTCCTGACCGTGGCTAGCGTGGACACCGCCGACACCGGCACCTACTTCTG

[0060] CGCTAGAATCACCTACCCCTTCTTCCCCATGGACTACTGGGGCCAAGGCACAAGCGTG

[0061] ACAGTCAGCTCCGCGAAGACCACTCCTCCTTCCGTCTATCCACTTGCCCCAGGATCTGC

[0062] CGCCCAGACAAATTCAATGGTTACCCTCGGGTGTCTGGTTAAAGGATACTTTCCGGAGC

[0063] CTGTGACCGTGAGCTGGAACACAGGCTCATTGAGTAGCGGGGTTCACACTTTCCCGGC

[0064] GGTTCTGCAGAGTGACTTGTACACATTGAGCAGCAGCGTGACCGTTCCCTCCTCAACAT

[0065] GGCCATCTGAGACTGTGACGTGTAATGTGGCACACCCAGCATCCAGCACTAAAGTGGA

[0066] CAAAAAAATCGTGCCCCGAGATTGTGGCTGCAAGCCATGCATTTGCACCGTGCCCGAG

[0067] GTGAGCAGTGTGTTCATCTTTCCTCCGAAGCCTAAGGACGTCCTGACAATTACTCTGAC

[0068] CCCTAAGGTCACCTGCGTGGTCGTCGACATTTCTAAAGATGATCCTGAGGTTCAGTTCT

[0069] CATGGTTCGTCGACGACGTGGAGGTACATACGGCCCAGACCCAGCCACGGGAGGAAC

[0070] AATTTAACTCCACTTTCCGGTCTGTGTCTGAATTGCCAATCATGCATCAGGACTGGCTG

[0071] AACGGCAAGGAGTTCAAGTGTAGGGTGAACTCAGCTGCCTTCCCTGCGCCAATTGAGA

[0072] AGACCATCTCTAAGACTAAGGGAAGACCCAAAGCGCCTCAGGTGTATACTATCCCCCC

[0073] GCCCAAAGAGCAGATGGCCAAAGACAAAGTCTCTCTGACCTGCATGATCACCGACTTC

[0074] TTTCCCGAAGACATAACAGTTGAGTGGCAATGGAACGGCCAGCCCGCCGAAAACTATA

[0075] AAAACACACAGCCGATAATGAACACAAATGGAAGCTACTTCGTCTATAGCAAGCTGAA

[0076] TGTACAGAAGTCTAACTGGGAAGCTGGGAACACCTTCACCTGCTCCGTGCTGCATGAAGGCCTTCACAATCACCATACAGAGAAGAGCTTGAGTCACTCCCCCGGCAAA;

[0077] Thiacloprid light chain SEQ ID NO.12:

[0078] Full sequence of the light chain

[0079] CAAGCCGGCGTGACCCAAGAGACCCTGAGCACCACAAGCCCCGGCACCGTGACCCTGA

[0080] CCTGCAGAGAGAGCAGCACCGGCACCGTGACCACAAGCAACTACGCCAACTGGGTGC

[0081] AAGAGAAGCCCGACCACTTCACCCTGGGCCTGATCGGCGGCACCAATCATAGAGCCCC

[0082] TGGCGTCCCCGCTAGAAGCGGCTCCCTGATCGGCGACTTCAAGGCCGTGCTGACCATC

[0083] ACCGGCGCTCAGACCGAGGACGAGATGTACTTCGCCTGCGCCCTGTGGTTCGGCAACC

[0084] TGTGGGTGTTCGGCGGCGGGACCAAGCTGACCGTGCTGGGGCAGCCCAAGAGCAGCCC

[0085] TAGCGTGACCCTGTTCCCCCCTAGCAGCGAGGAGCTGGAGACCAACAAGGCCACCCTG

[0086] GTGTGCACCATCACCGACTTCTACCCCGGCGTGGTGACCGTGGACTGGAAGGTGGACG

[0087] GCACCCCCGTGACCCAAGGCATGGAGACCACACAGCCTAGCAAGCAGAGCAACAACAAGT;

[0088] pCMV vector heavy chain primers (upstream primer 5'-3', downstream primer 5'-3'):

[0089] Upstream primer: CAGCAACAGGCGTTCACTCCCAAGTGACCCTGAGAGAGAGCC (SEQ ID NO.13) → Downstream primer: GAAGGAGGAGTGGTCTTCGCGGAGCTGACTGTCACGCTTG (SEQ ID NO.14);

[0090] pCMV vector light chain primers (upstream primer 5'-3', downstream primer 5'-3'):

[0091] Upstream primer: CAGCCACCGGTGTACATTCCCAAGCCGGCGTGACCC (SEQ ID NO.15) → Downstream primer: GGGCTGCTCTTGGGCTGCCCCAGCACGGTCAGCTTGGT (SEQ ID NO.16);

[0092] 2. The connection medium is PCMV.

[0093] 3. The amplified sequences are the antibody heavy chain and light chain (because seamless cloning technology is used, the ends of the amplified heavy and light chains and the vector will have 15-25 homologous bases, that is, the amplified heavy and light chains and the vector will be longer than they are originally, in order to facilitate subsequent seamless cloning).

[0094] (2) Transfection

[0095] First, plasmid DNA was purified from cell particles using a plasmid purification kit. 75 × 10⁻⁶ ppm was added to a 125 mL flask. 6Cells were cultured and the volume was adjusted to 25.5 mL using Expi293F medium. A mixture of 30 μg plasmid (LC 18 μg and HC 12 μg) was added to 1.5 mL of serum-free Opti-MEM I medium. Then, 81 μL of cationic lipid transfection reagent was mixed with 1.5 mL of Opti-MEM I and incubated at room temperature for 5 minutes. The diluted DNA was then mixed with the diluted transfection reagent and incubated at room temperature for 20 minutes to prepare the DNA-lipid complex. The DNA-lipid complex was added to the Expi293F cells while agitating the culture flask. Cells were cultured at 37°C with 5-8% CO2 and agitated (125 rpm). 16-20 hours post-transfection, 150 μL of Enhancer I and 1.5 mL of Enhancer II solution were added. The Expi293F supernatant was harvested 6-7 days post-transfection. The supernatant was centrifuged at 12000 rpm for 20 minutes at 4°C and filtered through a 0.22 μm filter.

[0096] (3) Purification

[0097] Add 1 mL of metal affinity resin to the affinity chromatography column and equilibrate with 0.5 mL of non-denaturing lysis buffer. After equilibration 2-3 times, add the supernatant, then collect the permeate. Repeat this process 3-5 times on the column to ensure sufficient binding of the target protein. Wash the column 5 times with 0.5-1 mL of non-denaturing washing buffer to remove contaminating proteins. Finally, elute the target protein with 0.5 mL of non-denaturing elution buffer. Analyze the eluted target protein using SDS-PAGE. Figure 4 Under non-reducing conditions, a full-length antibody band (150 kDa) can be observed, while under reducing conditions, distinct heavy chain (55 kDa) and light chain bands (25 kDa) are observed. VL (light chain variable region): SEQ ID NO.6

[0098] QAGVTQETLSTTSPGTVTLTCRESSTGTVTTSNYANWVQEKPDHFTLGLIGGTNHRAPGVPARSGSLIGDFKAVLTITGAQTEDEMYFACALWFGNLWVFGGGTKLTVL;

[0099] CH (light chain constant region): SEQ ID NO.7

[0100] QAGVTQESTLTTSPGETVTLTCRSSTGTVTTSNYANWVQEKPDHLFTGLIGGTNHRAPGVPARFSGSLIGDKAVLTITGAQTEDEAMYFCALWFGNLWVFGGGTKLTVL;

[0101] VH (Heavy Chain Variable Region): SEQ ID NO.8

[0102] QVTLRESPGMLQPSQTLSLTCSFSGFSMSMGVGWIRQPSTFAGKGLEWLASIFGWNENKYYPALMSRLNTISRDTSKNLVFLTVASVDTADTGTYFCARITYPFFPMDYWGQGTSVTVSS;

[0103] CH1 (Heavy chain constant region): SEQ ID NO.9

[0104] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVSWNTGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVP;

[0105] Fc (heavy chain constant region): SEQ ID NO.10

[0106] QAGVTQESTLTTSPGETVTLTCRSSTGTVTTSNYANWVQEKPDHLFTGLIGGTNHRAP GVPARFSGSLIGDKAVLTITGAQTEDEAMYFCALWFGNLWVFGGGTKLTVL.

[0107] Example 4: Antibody ic-ELISA assay

[0108] (1) Coating: Take 0.25 μg / mL of the complete thiamethoxam antigen prepared in Example 2 for coating, 100 μL / well, and incubate at 37°C for 2 h.

[0109] (2) Washing: Pour out the solution in the plate and wash with PBST washing solution 4 to 6 times, then pat dry.

[0110] (3) Blocking: Add 250 μL / well of 5% skim milk powder blocking solution to the washed and dried 96-well plate, and react at 37°C for 2 h. Wash and dry before use.

[0111] (4) Antibody incubation: The expression antibody purified in Example 3 was added to the coated wells. 50 μL of expression antibody and 50 μL of thiamethoxam standard solution diluted with PBS were added to each well. The mixture was reacted at 37°C for 1 h. Then the mixture was washed 4 to 6 times and patted dry.

[0112] (5) Secondary antibody incubation: Add 100 μL of the enzyme-labeled secondary antibody of GOAT anti-IgG-HRP to each well, dilute the concentration appropriately, and incubate the plate at 7°C for 30 min. Then wash the plate with PBST 4 to 6 times and pat dry.

[0113] (6) Color development: Add 50 μL of TMB color development solution A and B to each well and react at 37°C in the dark for 15 min.

[0114] (7) Termination and measurement: Add 50 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using a microplate reader. 450 nm value.

[0115] The standard curve for determining the full-length recombinant antibody against thiamethoxam using ic ELISA is as follows: Figure 5 As shown, its IC 50 The value was 2.63 μg / L, indicating that the recombinant full-length antibody has good affinity for the antigen and can be used for immunoassay.

[0116] Example 5: Preparation of fluorescent probes

[0117] (1) Take 10 μL of fluorescent microspheres and add them to 800 μL of MES solution, and mix them evenly.

[0118] (2) Add 5 μL of EDC (2 mg / mL) and NHS (2 mg / mL) as activators, and activate at room temperature by shaking at 500 rpm in the dark for 30 min.

[0119] (3) After activation, add the full-length recombinant antibody of thiamethoxam prepared in Example 3 for conjugation, and shake at 500 rpm in the dark for 2 hours at room temperature.

[0120] (4) After labeling, add 10% (v / v) blocking buffer and shake at room temperature in the dark for 30 min;

[0121] (5) After sealing, centrifuge and discard the supernatant, then wash 2-3 times with 800 μL of labeled washing solution;

[0122] (6) Discard the supernatant, add 400 μL of fluorescent microsphere reconstitution solution, and obtain Eu. 3+ - The full-length recombinant antibody Eu-TCL-rAb labeled with fluorescent microspheres for thiamethoxam was stored at 4°C for later use.

[0123] Example 6: Construction of Time-Resolved Immunochromatographic Test Strips

[0124] The time-resolved immunochromatographic test strip consists of a sample pad, a nitrocellulose membrane (NC), an absorbent pad, and a PVC base plate. Using a three-dimensional spraying platform (XYZ3050, Biodot, USA), goat anti-mouse secondary antibody (1 mg / mL) and TCL-OVA (0.2 mg / mL) are sequentially sprayed onto the NC membrane as the control (C) line and the detection (T) line. Subsequently, the NC membrane is placed in a dark environment at 37°C for 2 hours. The sample pad, NC membrane, and absorbent pad are then adhered to the PVC base plate to assemble the test strip. The key to the test strip assembly is ensuring consistent transmissibility between the components. The sample pad is stacked on top of the NC membrane, with an overlap of approximately 3 mm. Similarly, the absorbent paper is stacked on top of the NC membrane, with an overlap of approximately 3 mm. The assembled plate is then cut into test strips approximately 4 mm wide using a strip cutter. These strips are assembled using a plastic base and a retainer, and then sealed and stored at 4°C for later use. A schematic diagram of the immunochromatographic structure of the test strip is shown below. Figure 6 As shown.

[0125] Example 7: Optimization of conditions for thiamethoxam immunoassay strips

[0126] (1) Optimization of EDC usage

[0127] To improve the coupling efficiency between fluorescent microspheres and antibodies, different amounts of EDC were used to activate the fluorescent microspheres. When the amount of EDC used was less than 5 μg, the binding rate of Eu-TCL to rAb decreased, and the fluorescent microspheres could not completely bind to rAb to prepare TCL-Eu-rAb. Therefore, only a small amount of TCL-Eu-rAb was captured by TCL-OVA, resulting in a decrease in the fluorescence intensity (FI) of the T-line. Furthermore, a high binding rate also reduced the fluorescence intensity of the T-line because, at the same antigen concentration, the higher the binding rate of the microspheres to the rAb, the fewer microspheres are captured by the T-line, which is detrimental to subsequent experiments. Therefore, the Eu-TCL-rAb probe exhibited optimal properties when the amount of EDC was 5 μg.

[0128] (2) Optimization of the amount of recombinant antibody used

[0129] To improve detection sensitivity, the amount of recombinant antibody used was optimized. An inappropriate amount of antibody reduced the sensitivity of TRFIA. Adding too little antibody decreased the binding of the antigen to the Eu-TCL-rAb probe, resulting in low T-line fluorescence. Excessive antibody increased the inhibition rate. The highest fluorescence and lowest inhibition rate were observed with Eu-TCL-rAb when the mass ratio of microspheres to rAb was 150:1 and the amount of antibody used was 0.67 μg.

[0130] (3) pH optimization

[0131] Hydrophobic microspheres are unstable in their natural state and tend to aggregate into clumps. Particle concentration, surface charge, and electrolyte concentration are important factors affecting the stability of microspheres. Results show that the pH of the composite solution affects the stability of Eu fluorescent microspheres, which in turn significantly impacts the sensitivity, fluorescence index (FI), and stability of TRFM-ICTS. An unsuitable pH environment neutralizes some of the surface charge of the microspheres, reduces interparticle forces, decreases the distance between particles, and leads to particle aggregation. The T-line shows the highest fluorescence at pH 6.5. Therefore, pH 6.5 is the optimal pH for preparing Eu-TCL-rAb.

[0132] (4) Optimization of dilution rate

[0133] The Eu-TCL-rAb conjugate was diluted with PBS to evaluate the effect of dilution on test strip performance. The Eu-TCL-rAb probe exhibited the highest fluorescence value at a dilution ratio of 1:10. As the dilution ratio increased, only a small amount of TCL-Eu-rAb was added to the sample pad, resulting in a decrease in the fluorescence index (FI) of the T-line. At a dilution ratio of 1:20, a significant decrease in fluorescence value and an increase in inhibition rate were observed. Therefore, the optimal dilution ratio is 1:10.

[0134] Example 8: Plotting the standard curve for thiamethoxam immunoassay strips

[0135] The method for plotting a standard curve is as follows:

[0136] Thiamethoxam standard was prepared into standard solutions of different concentrations (0.01-10 ng / mL) using PBS for detection on fluorescent test strips (the standard release solution was a 0.01 M pH 7.4 PBS solution containing 5% methanol (v:v). Eu prepared in Example 5 was then used... 3+ - Using fluorescent microsphere-labeled full-length recombinant antibody against thiamethoxam (Eu-TCL-rAb) as a fluorescent probe, 6 μL of spiked sample solution, 9 μL of fluorescent probe, and 75 mL of PBS were mixed thoroughly and slowly dropped into the sample well of the test strip. After chromatography at 37°C for 15 min, the T-line fluorescence value of the test strip was recorded using an immunoassay analyzer. Three replicates were measured for each concentration. The T-line fluorescence value of the 0 ppb standard solution was set as T0, and the T-line fluorescence value of other spiked concentrations was T. A standard curve was plotted with the logarithm of each thiamethoxam standard concentration as the x-axis and T / T0×100 (%) as the y-axis. The competitive inhibition rate was set as (1-T / T0)×100%.

[0137] like Figure 7It can be seen that as the concentration of thiamethoxam increases, the fluorescence on the T line of the test strip becomes lighter and lighter, so T / T0 decreases. When the concentration of thiamethoxam is 5 ng / mL, the fluorescence value of the T line decreases significantly. The curve showing the change in T / T0 with thiamethoxam concentration shows that when the concentration of thiamethoxam is 0.01-10 ng / mL, the logarithm of the thiamethoxam concentration has a linear relationship with T / T0, with the linear equation being Y = -19.113LgX + 62.038, R... 2 =0.9935, IC 50 =4.268 ng / mL, and the limit of detection (LOD) can reach 0.003 ng / mL.

[0138] Example 9: Performance testing of thiamethoxam immunoassay strips

[0139] 1. Specificity test

[0140] Six structural analogs (acetamiprid, thiamethoxam, imidacloprid, thiamethoxam, dinotefuran, and chlorothiazoline) with similar structures or functions to thiamethoxam were selected to evaluate the specificity of the time-resolved immunochromatographic test strip. Negative samples and positive samples of each pesticide were tested using the test strip provided by this invention, and the T-line fluorescence intensity values ​​were recorded and the IC50 was calculated. 50 Value and cross-reactivity rate (CR).

[0141] As shown in Table 1, thiamethoxam exhibits some cross-reactivity with its structural analogue acetamiprid, while showing low cross-reactivity with the other five structural analogues, indicating that the test strip has strong specificity.

[0142] Table 1. Specificity evaluation of thiamethoxam immunochromatographic test strips

[0143]

[0144]

[0145] 2. Stability test

[0146] To assess the stability of TRFM-ICTS, the test strips and Eu-TCL-rAb were placed at 4℃ and 37℃ for 15 days, respectively. Changes in T-line fluorescence were detected every two days, and stability was tested using the same and different batches of test strips. The results showed that the fluorescence intensity of the T-line did not change significantly when the test strips and Eu-TCL-rAb probe were incubated at 4℃ and 37℃ for 15 days (Table 2). The coefficients of variation were less than 5.05% and 3.01%, respectively. Therefore, TRFM-ICTS exhibits good stability at 4℃ and 37℃ and can be stored stably for a long period.

[0147] Table 2. Stability evaluation of thiamethoxam immunochromatographic test strips

[0148]

[0149] 3. Repeatability experiment

[0150] Inter-batch and intra-batch variability were used to evaluate the repeatability of the test strips. Positive samples (TCL 1 ng / mL) were added to test strips from the same and different batches. The results showed no significant difference between the T lines of the test strips, and the coefficients of variation for TCL test strips from the same batch and different batches were less than 0.40% and 5.18%, respectively (Table 3). Therefore, TRFM-ICTS has good repeatability.

[0151] Table 3 Repeatability evaluation of thiamethoxam immunochromatographic test strips

[0152]

[0153] 4. Accuracy Test

[0154] (1) Sample processing

[0155] The pear, orange, and cucumber samples were thoroughly homogenized. The homogenized sample (20 g) was placed in an Erlenmeyer flask with methanol (50 mL) and TCL standard solutions (0.5, 1, and 10 ng / mL) and shaken at 25 °C for 1 hour. After vacuum filtration, 4 g of sodium chloride was mixed with the filtrate and placed at 25 °C. Then, the supernatant (20 mL) was distilled under reduced pressure in a rotary evaporator at 45 °C. The filtrate was dissolved in acetonitrile:water (30:70, v / v) and filtered through a 0.22 μm filter membrane. Finally, the prepared sample was aliquoted and analyzed by immunoassay and HPLC-MS, respectively.

[0156] (2) Accuracy Evaluation

[0157] The accuracy of TRFM-ICTS was evaluated by preparing spiked samples by adding different concentrations of TCL standard solution (0.5, 1, and 10 ng / mL) to TCL-free samples. As shown in Table 4, the recoveries of TRFM-ICTS and HPLC-MS were compared using three different spiked samples (pear, orange, and cucumber), demonstrating strong consistency between the two methods for TCL results. The recoveries of TRFM-ICTS ranged from 79.4% to 118.6%, with relative standard deviations (RSDs) ranging from 0.68% to 11.4%, which are consistent with the results of UPLC-MS. These results demonstrate the accuracy of TRFM-ICTS. Therefore, TRFM-ICTS can serve as an effective tool for the rapid detection of TCL residues in fruit and vegetable samples, meeting market demand and possessing broad market prospects.

[0158] Table 4. Accuracy Evaluation of Thiamethoxam Immunochromatographic Test Strips

[0159]

[0160]

[0161] The variable region gene sequences of the antibody heavy and light chains are shown in SEQ ID NO.1 and SEQ ID NO.2. The full-length recombinant thiamethoxam antibody contains a heavy chain constant region and a light chain constant region. The heavy chain constant region comprises CH1 and Fc segments, wherein the nucleotide sequence of CH1 is shown in SEQ ID NO.3, the nucleotide sequence of the Fc segment is shown in SEQ ID NO.4, and the nucleotide sequence of the light chain constant region is shown in SEQ ID NO.5.

[0162] Antibody heavy chain variable region SEQ ID NO: 1:

[0163] CAAGTGACCCTGAGAGAGAGCCCCGGCATGCTGCAGCCTAGCCAAACCCTGAGCCTCACCTGCAGCTTCAGCGGCTTCAGCATGAGCATGGGCGTGGGCTGGATCAGACAGCCTAGCACCTTCGCCGGCAAGGGCCTGGAGTGGCTGGCTAGCATCTTCGGCTGGAACGAGAACAAGTAC TACCCCGCCCTGATGAGCAGACTGAACACCATCAGCAGAGACACAAGCAAGAACCTGGTGTCCTGACCGTGGCTAGCGTGGACACCGCCGACACCGGCACCTACTTCTGCGCTAGAATCACCTACCCCTTCTTCCCCATGGACTACTGGGGCCAAGGCACAAGCGTGACAGTCAGCTCC;

[0164] SEQ ID NO.2 of the variable region of the antibody light chain:

[0165] CAAGCCGGCGTGACCCAAGAGACCCTGAGCACCACAAGCCCCGGCACCGTGACCCTGACCTGCAGAGAGAGCAGCACCGGCACCGTGACCACAAGCAACTACGCCAACTGGGTGCAAGAGAAGCCCGACCACTTCACCCTGGGCCTGATCGGCGGCACCAATCATAGAGCCCCTGGCGTCCCCGCTAGAAGCGGCTCCCTGATCGGCGACTTCAAGGCCGTGCTGACCATCACCGGCGCTCAGACCGAGGACGAGATGTACTTCGCCTGCGCCCTGTGGTTCGGCAACCTGTGGGTGTTCGGCGGCGGGACCAAGCTGACCGTGCTG;

[0166] SEQ ID NO.3:

[0167] GCGAAGACCACTCCTCCTTCCGTCTATCCACTTGCCCCAGGATCTGCCGCCCAGACAAA

[0168] TTCAATGGTTACCCTCGGGTGTCTGGTTAAAGGATACTTTCCGGAGCCTGTGACCGTGAG

[0169] CTGGAACACAGGCTCATTGAGTAGCGGGGTTCACACTTTCCCGGCGGTTCTGCAGAGTG

[0170] ACTTGTACACATTGAGCAGCAGCGTGACCGTTCCCTCCTCAACATGGCCATCTGAGACT

[0171] GTGACGTGTAATGTGGCACACCCAGCATCCAGCACTAAAGTGGACAAAAAAATCGTGCCC;

[0172] SEQ ID NO.4:

[0173] CGAGATTGTGGCTGCAAGCCATGCATTTGCACCGTGCCCGAGGTGAGCAGTGTGTTCAT

[0174] CTTTCCTCCGAAGCCTAAGGACGTCCTGACAATTACTCTGACCCCTAAGGTCACCTGCGT

[0175] GGTCGTCGACATTTCTAAAGATGATCCTGAGGTTCAGTTCTCATGGTTCGTCGACGACGT

[0176] GGAGGTACATACGGCCCAGACCCAGCCACGGGAGGAACAATTTAACTCCACTTTCCGGT

[0177] CTGTGTCTGAATTGCCAATCATGCATCAGGACTGGCTGAACGGCAAGGAGTTCAAGTGT

[0178] AGGGTGAACTCAGCTGCCTTCCCTGCGCCAATTGAGAAGACCATCTCTAAGACTAAGGG

[0179] AAGACCCAAAGCGCCTCAGGTGTATACTATCCCCCCGCCCAAAGAGCAGATGGCCAAAG

[0180] ACAAAGTCTCTCTGACCTGCATGATCACCGACTTCTTTCCCGAAGACATAACAGTTGAG

[0181] TGGCAATGGAACGGCCAGCCCGCCGAAAACTATAAAAACACACAGCCGATAATGAACA

[0182] CAAATGGAAGCTACTTCGTCTATAGCAAGCTGAATGTACAGAAGTCTAACTGGGAAGCT

[0183] GGGAACACCTTCACCTGCTCCGTGCTGCATGAAGGCCTTCACAATCACCATACAGAGAAGAGCTTGAGTCACTCCCCCGGCAAA;

[0184] SEQ ID NO.5:

[0185] GGGCAGCCCAAGAGCAGCCCTAGCGTGACCCTGTTCCCCCCTAGCAGCGAGGAGCTGG

[0186] AGACCAACAAGGCCACCCTGGTGTGCACCATCACCGACTTCTACCCCGGCGTGGTGACC

[0187] GTGGACTGGAAGGTGGACGGCACCCCCGTGACCCAAGGCATGGAGACCACACAGCCTA

[0188] GCAAGCAGAGCAACAACAAGTACATGGCTAGCAGCTACCTGACCCTGACCGCTAGAGC

[0189] CTGGGAGAGACACAGCAGCTACAGCTGCCAAGTGACCCACGAGGCCACACCGTGGAGAAGAGCCTGAGCCCCGCCGAGTGCCTGTGA.

[0190] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A full-length recombinant antibody against thiamethoxam, characterized in that, The amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO. 8; the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO.

6.

2. The antibody according to claim 1, characterized in that, The nucleotide sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO.1, and the nucleotide sequence of the variable region of the antibody light chain is shown in SEQ ID NO.

2.

3. A recombinant vector containing the gene encoding the full-length recombinant antibody against thiamethoxam as described in claim 1 or 2.

4. The recombinant vector according to claim 3, characterized in that, The starting vector for the recombinant vector is the PCMV vector.

5. Recombinant microbial cells carrying the recombinant vector of claim 3.

6. The recombinant microbial cell according to claim 5, characterized in that, The recombinant microbial cells are prokaryotic or eukaryotic microbial cells.

7. The use of the recombinant antibody of claim 1 or 2, the recombinant vector of claim 3 or 4, or the recombinant microbial cell of claim 5 or 6 in the preparation of a kit for detecting thiamethoxam.

8. The application according to claim 7, characterized in that, The kit is used for ELISA detection, Western blotting, immunohistochemistry, immunofluorescence, or immunochromatographic quantitative test strips.

9. The application according to claim 7, characterized in that, Applications in the field of food testing.

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