Monoclonal antibody against dimethoate, detection reagent and application thereof

Abstract

The application discloses a kind of anti-methomyl monoclonal antibody, detection reagent and its application, belong to immunology and biotechnology field.The monoclonal antibody contains heavy chain variable region and light chain variable region, the heavy chain variable region and light chain variable region are all composed of complementarity determining region and framework region, the complementarity determining region is all composed of CDR1, CDR2 and CDR3.The monoclonal antibody can be used for preparing methomyl detection preparation.The methomyl colloidal gold detection test strip provided in the application has the characteristics of high sensitivity, good specificity and strong stability.

Description

Technical Field

[0001] This invention belongs to the fields of immunology and biotechnology, specifically relating to a monoclonal antibody against methomyl, a detection reagent, and their applications. Background Technology

[0002] Methomyl (MTM) is a white crystalline solid with a slight sulfurous odor. It is a highly effective, broad-spectrum carbamate insecticide, marketed as "Wanling". It kills insects by inhibiting cholinesterase, disrupting their nervous system, and is widely used in agricultural pest control.

[0003] Methomyl is a highly toxic pesticide with strong volatility and high inhalation toxicity. It has a slight irritant effect on the skin and eyes, but its dermal toxicity is relatively low, which allows it to still be used for foliar spraying under certain protective measures.

[0004] Given the above characteristics, methomyl is prone to residues in agricultural products and the environment, posing a potential hazard to the human nervous system. The National Food Safety Standard GB2763-2026, Maximum Residue Limits for Pesticides in Food, stipulates that the acceptable daily intake (ADI) for methomyl is 0.02 mg / kgbw, the maximum residue limit for cereals is 2 mg / kg, the maximum residue limit for oils and fats is 0.5 mg / kg, the maximum residue limit for vegetables, fruits, sugar crops, and beverages is 0.2 mg / kg, and the maximum residue limit for seasonings is 10 mg / kg.

[0005] GB 23200.112-2018 standard specifies that the detection method for methomyl is usually liquid chromatography-post-column derivatization. The equipment used in this method is expensive and requires highly skilled operators. In contrast, immunoassay has the advantages of low cost, high efficiency, high sensitivity, and relatively low requirements for technical personnel. It is suitable for rapid detection of large numbers of samples and on-site screening. However, it has the disadvantages of poor specificity and low affinity. In addition, it does not disclose clear VH / VL sequences or CDR information and cannot achieve genetic engineering production.

[0006] Therefore, the market urgently needs a highly specific, high-affinity, and sequence-defined monoclonal antibody against memetoxin for the development of highly sensitive and standardized immunoassay reagents. The purpose of this invention is to provide a monoclonal antibody with high specificity, high affinity, and high detection sensitivity against memetoxin, laying the foundation for establishing a colloidal gold test strip detection method and developing and promoting colloidal gold test strip products. Summary of the Invention

[0007] Therefore, the present invention provides a monoclonal antibody against methomyl, a detection reagent, and its application.

[0008] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions: In a first aspect, the present invention provides an anti-methodox monoclonal antibody, characterized in that: the monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 1; The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 2; Both the heavy chain variable region and the light chain variable region are composed of complementary determination regions and framework regions, and the complementary determination regions are composed of CDR1, CDR2 and CDR3. The amino acid sequence of CDR1 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 5; The amino acid sequence of CDR2 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 6; The amino acid sequence of CDR3 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 7; The amino acid sequence of CDR1 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 8; The amino acid sequence of CDR2 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 9; The amino acid sequence of CDR3 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 10.

[0009] Secondly, the aforementioned anti-methodox monoclonal antibody is characterized by: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 3; The nucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 4.

[0010] Thirdly, the present invention provides the application of an anti-methimox monoclonal antibody in the preparation of a detection product for detecting methomyl.

[0011] Preferably, the test product consists of methomyl test strips and microporous reagents; Preferably, the test strip is a colloidal gold test strip.

[0012] The present invention has the following advantages: The monoclonal antibody provided by this invention has high affinity and detection sensitivity for medomyl, and has the advantages of high specificity, high affinity and detection sensitivity, and convenient and rapid detection. It can be used as a raw material for colloidal gold immunoassay, laying the foundation for the research and development and promotion of colloidal gold test strips. Attached Figure Description

[0013] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0014] Figure 1 The results of SDS-PAGE identification of purified monoclonal antibodies; Figure 2 OD plotted for RIDA SOFT four-parameter method for methomyl 450nm The standard curve; Figure 3 This is a schematic diagram of the half-maximal inhibitory concentration (IC50) of the anti-methodonium monoclonal antibody provided by the present invention; Figure 4 A schematic diagram illustrating the interpretation of methomyl test strip results; Figure 5 This is a schematic diagram of the structure of the methomyl test strip. Detailed Implementation

[0015] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1: Synthesis of methomyl artificial antigen 1. The hapten was synthesized according to the hapten design method disclosed in patent CN115073337A: S1. Methomyl oxime (105 mg, 1 mmol) was dissolved in dichloromethane (5 mL), and triethylamine (111 mg, 1.1 mmol) and isocyanate (129 mg, 1 mmol) were added sequentially. The mixture was stirred at room temperature for 2 hours to complete the reaction. The mixture was washed with 10 mL of water, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain the crude product. S2. Dissolve the crude product obtained in S1 in tetrahydrofuran / water (5mL / 1mL), add lithium hydroxide monohydrate (42mg, 1.75mmol), stir at room temperature for 2 hours, evaporate to dryness, remove tetrahydrofuran from the solution, add 1mol / L dilute hydrochloric acid dropwise under ice bath to adjust the pH of the solution to weakly acidic (pH=3), extract with dichloromethane, and purify by TLC and HPLC to obtain the final product, which is methomyl hapten.

[0017] 2. Preparation of methomyl detection antigen S1. Weigh 3.7 mg of the methomyl hapten prepared above and dissolve it in 0.6 mL of N,N-dimethylformamide (DMF). Then, add 5.0 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 3.7 mg of N-hydroxysuccinimide (NHS) sequentially. Stir at room temperature and activate the reaction for 6 h to obtain the activated solution. S2. Dissolve 15 mg of bovine serum albumin (BSA) in 3 mL of carbonate buffer solution (0.05 mol / L, pH 9.6). Add the activated solution obtained in S1 dropwise to the BSA solution. Stir and react overnight at room temperature to obtain a mixed solution. S3. Dialyze and separate the mixture obtained in S2 to obtain the MTM-BSA conjugate, i.e., the immunogen (MTM-BSA), and store it in aliquots at -20℃. Similarly, the complete antigen obtained by conjugating methomyl hapten with ovalbumin (OVA) is methomyl detection antigen (MTM-OVA).

[0018] Example 2: Preparation of anti-methodox monoclonal antibody 1. Animal immunization Mice were immunized with the MTM-BSA immunogen prepared in Example 1 at a dose of 10 µg per mouse. The immunogen was diluted to the required concentration with sterile PBS. Equal volumes of adjuvant and immunogen were drawn into two 2.5 mL disposable syringes, which were then inserted into a communicating vessel and pushed 8500 times with an emulsifier. Three healthy Balb / c female mice aged 6-8 weeks and weighing 18-20 g were selected. For the first immunization, the mice were injected subcutaneously at multiple sites with the antigen emulsified with an equal volume of Freund's complete adjuvant. Every 14 days (21 days between the first and second immunizations), booster immunizations were performed with the antigen emulsified with Freund's incomplete adjuvant. After three immunizations, blood was collected from the tail tip on days 7-10. The blood was diluted 100 times with 1×PBS, centrifuged, and the supernatant was used to detect the serum titer and inhibition. If inhibition was observed and the titer reached 1:100000 or higher, a single pulse immunization could be performed, i.e., 0.5 mL of the immunogen solution was injected directly into the peritoneum. Three days later, spleen cells were collected and fused with myeloma cells, and positive wells were screened. The positive wells were cloned using the limiting dilution method to obtain and establish a hybridoma cell line that stably secretes anti-methodox monoclonal antibody.

[0019] 2. Preparation of ascites Monoclonal antibodies were prepared using an in vivo induction method. Each mouse was injected with 0.5 mL of Freund's incomplete adjuvant to purify the peritoneal cavity. After 7 days, the number of hybridoma cells in the logarithmic growth phase was adjusted to 1 × 10⁻⁶. 6Inoculate 0.5 mL of the solution into the peritoneum of each mouse at a rate of 1 / mL. Observe the ascites production of the mice daily at intervals of approximately 7 days. When the mice's abdomens are significantly distended, their mental state deteriorates, and they become immobile and near death, collect the ascites, centrifuge at 10,000 rpm for 5 minutes, remove the surface fat, and carefully aspirate the supernatant for ammonium sulfate precipitation.

[0020] 3. Antibody purification Record the name and volume of the ascites fluid. Add 3 mL of pH 4.0, 0.06 M sodium acetate buffer, mix for 5 min, add 10 µL of caprylic acid, and stir at 4°C for 15 min. Filter once through absorbent cotton using a syringe. Centrifuge at 12000 rpm for 15 min at 4°C. Collect the supernatant, add an equal volume of saturated ammonium sulfate to a final concentration of 50%, stirring continuously, and incubate at 4°C for 3 h. Centrifuge at 12000 rpm for 15 min at 4°C. Discard the supernatant, add 1.8 mL of 0.01 M PBS (pH 7.2) to the centrifuge tube until the precipitate is completely dissolved. After determining the total volume, add 1 / 2 volume of saturated ammonium sulfate to a concentration of 33%, stirring continuously, and incubate overnight at 4°C. Centrifuge at 12000 rpm for 15 min at 4°C. Discard the supernatant, add 0.45 mL of 0.01 M PBS to dissolve the precipitate. Treat the dialysis bag (boil for 5 min, rinse with pure water and check for leaks). Add the dissolved solution to a dialysis bag and dialyze overnight in 0.01M PBS. Change the dialysate, generally 2-3 times. Collect the antibody, determine its concentration, aliquot, and store at -80℃. The purified anti-methodox monoclonal antibody obtained above had a purity of approximately 95% as determined by SDS-PAGE. The results are shown below. Figure 1 .

[0021] Example 3: Sensitivity detection of anti-methodox monoclonal antibody The sensitivity of the anti-methodox monoclonal antibody was detected using an indirect ELISA method, and the results are shown in Table 1. The x-axis represents the concentrations of methomyl (0, 2.5, 5, 10, 20, 40, 80 ng / mL), and the OD values ​​corresponding to each methomyl concentration are plotted as follows: 450nm The values ​​are plotted on the ordinate using the RIDA SOFT four-parameter method to create a standard curve, and the half-maximal inhibitory concentration (IC50) is calculated. 50 ).like Figure 2 , Figure 3 The results show that curve R 2 =0.9984, IC 50 =8.4ng / mL.

[0022] Table 1. Monoclonal antibody sensitivity validation

[0023] Example 4: Specificity detection of anti-methodox monoclonal antibody The cross-reactivity of the anti-methimox monoclonal antibody with its hydrolysis product methomyl oxime and its structural analogues carbofuran, thiamethoxam, methomyl, and carbaryl was detected using an indirect ELISA method. The results showed that the cross-reactivity rate of the antibody with the above drugs was less than 2% (see Table 2), indicating that the methomyl monoclonal antibody prepared in Example 2 has high specificity.

[0024] Table 2. Validation of Monoclonal Antibody Specificity

[0025] Example 5: Cloning of the variable region gene of anti-methodox monoclonal antibody 1. Total RNA extraction Total RNA was extracted from cultured cells using a total RNA extraction kit (purchased from Tiangen Biotech (Beijing) Co., Ltd.).

[0026] 2. Synthesis of the first strand of cDNA cDNA was synthesized using the TIANScript II cDNA first-strand synthesis kit (purchased from Tiangen Biotech (Beijing) Co., Ltd.).

[0027] 3. Gene amplification Design downstream primers and upstream universal primers for Lambda, Kappa, and Heavy chains.

[0028] Primer: F (SEQ ID No. 11): AAGCGTGGTATCAACGCAGA Rκ (SEQ ID No. 12):AACATTGATGTCTTTGGGGTAGAA Rλ (SEQ ID No.13):AATCGTACACACCAGTGTGTGGG R H (SEQ ID No.14):AGGGATCCAGAGTTCCAGGT PCR amplification was performed using the first strand of cDNA as a template in a 50 μL reaction volume. The reaction volume consisted of 3 μL template, 2.5 μL each of forward and reverse primers (10 μM), 25 μL Green Taq Mix, and 17 μL ddH2O.

[0029] The PCR reaction conditions were as follows: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 1 min, for 12 cycles; 95℃ for 30 s, 56℃ for 30 s, 72℃ for 1 min, for 28 cycles; 72℃ for 7 min.

[0030] 4. Cloning and screening of PCR amplification products The PCR products were subjected to 2% agarose gel electrophoresis. The Kappa, Lambda, and Heavy chain fragments of the antibody were recovered using an agarose gel DNA recovery kit (Tiangen Biotech (Beijing) Co., Ltd.). The fragments were inserted into the pLB vector using the pLB zero-background rapid cloning kit (Beijing Tiangen). The vector was then transformed into DH5α competent cells (ampicillin-resistant). Recombinant positive clones were screened and sequenced.

[0031] 5. The variable region gene sequence and amino acid sequence of the anti-methodox monoclonal antibody in this embodiment are as follows: (1) Gene sequence of the variable region of the heavy chain (SEQ ID No. 3): CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACATTCAATAACTACTGGATAGACTGGATAAAGCAGAGGCCTGGACATGGCCTTGAATGGATTGGAGAGCATTTACCTGGAACTGGTAAAGCTCACTA CAATGAGAAGTTCAAGGACAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTTCAAGATCGGGTACTACTATGTACTACTACTACATGGACGTCTGGGGCAAAGGGACACGGTCACCG.

[0032] (2) Amino acid sequence of the variable region of the heavy chain (SEQ ID No. 1): QVQLQQSGAELMKPGASVKISCKASGYTFNNYWIDWIKQRPGHGLEWIGEHLPGTGKAHYNEKFKDKATFTADTSSNTAYMQLSSLTSEDSAVYYCSRSGTTMYYYYYMDVWGKGTRSP.

[0033] (3) Kappa chain variable region gene sequence (SEQ ID No. 4): GACATTTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAGTGTGGCCACTGCTGTAACCTGGTATCAACAGAAACCAGGGCAATCTCCTTACCCACTGATTTACTCGACATCCTATCG GAATCGTGGAGTCCCTGATCGCATCGCAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGAAATATATCAATTTTCCGCTCGCGTTCGGTGCGGGGACCAAGCTGGAACTGAAA.

[0034] (4) Amino acid sequence of the variable region of the Kappa chain (SEQ ID No. 2) DIVMTQSQKFMSTSVGDRVSVTCKASQSVATAVTWYQQKPGQSPYPLIYSTSYRNRGVPDRIAGSGSGTDFTLTISNVQSEDLAEYFCQKYINFPLAFGAGTKLELK.

[0035] 6. Variable region amino acid sequence and homology analysis Comparative analysis in the NCBI database showed that the heavy chain variable region gene sequence of the anti-methimazole monoclonal antibody had the highest homology with the mouse immunoglobulin heavy chain variable region mRNA (Igh gene) (Sequence ID: OZ390535.1), with a homology of 330 / 359, or 92%. The amino acid sequence of the heavy chain variable region had the highest homology with a partial sequence of the mouse immunoglobulin 10B7.A1 heavy chain (Sequence ID: AAB05141.1), with a homology of 95 / 116, or 82%. The light chain variable region gene sequence of the anti-methimazole monoclonal antibody had the highest homology with a partial coding region of the immunoglobulin light chain variable region mRNA in mouse hybridoma cell line F30D1 (Sequence ID: EF030738.1), with a homology of 297 / 321, or 93%. The amino acid sequence of the light chain variable region showed the highest homology with the light chain portion of mouse immunoglobulin κ (Sequence ID: CAO6191468.1), with a homology of 91 / 107, representing 85%. Homology analysis of the gene and amino acid sequences encoding the heavy and light chain variable regions of the anti-methodox monoclonal antibody showed that no sequences identical to those of this invention were found.

[0036] 7. CDR Area Analysis The CDR regions of the heavy chain variable region and the light chain variable region sequences were analyzed at https: / / www.novopro.cn / tools / cdr.html.

[0037] Antibody heavy chain CDR region: CDR-H1 (SEQ ID No.5): NYWID CDR-H2 (SEQ ID No. 6): EHLPGTGKAHYNEKFKD CDR-H3 (SEQ ID No.7):SGTTMYYYYYMDV Antibody light chain CDR region: CDR-L1 (SEQ ID No.8): KASQSVATAVT CDR-L2 (SEQ ID No.9): STSYRNR CDR-L3 (SEQ ID No.10): QKYINFPLA

[0038] Example 6: Preparation of methomyl colloidal gold test strip The test strip (such as) Figure 5 It consists of absorbent paper, nitrocellulose membrane (NC membrane), sample pad, and base plate. The specific preparation is as follows: 1. Preparation of chloroauric acid Weigh 800 mL of ultrapure water into a 1000 mL Erlenmeyer flask, add 8 mL of 1% chloroauric acid to the ultrapure water, and place the flask on a magnetic stirrer to mix and heat at speed 1. After the water boils, increase the speed to speed 6. Once the liquid is stirring, quickly add 1 mL of 0.8% reducing agent and adjust the speed to speed 5. After heating for 5 minutes and 30 seconds, stop heating and allow it to cool to room temperature. The prepared colloidal gold is pure, transparent, and free of precipitates and floating matter, with a particle size of approximately 25 nm.

[0039] 2. Preparation of methomyl monoclonal antibody-colloidal gold label (1) Add bare gold: Mix the colloidal bare gold well and take 1L of colloidal gold solution; (2) Add K2CO3: Add 0.2M K2CO3 at a ratio of 3μL / mL colloidal gold, vortex to mix, pH value is about 8.5; (3) Adding antibody: Mix 1 mL of 1 mg / mL methomyl antibody with 3 mL of 10% BSA (IgG), and then use a pipette to add the well mixed antibody dropwise to the gold, and equilibrate for 5 min; (4) Leveling: Reduce the rotation speed to stabilize the vortex at a drop of about 1 cm, and then rebalance for 1 hour; (5) Add BSA: Increase the rotation speed until the vortex drops by about 3cm, pour in 10mL of 10% BSA, balance for 3min, decrease the rotation speed to stabilize the vortex at about 1cm, and then balance for 30min. (6) Add PEG: Increase the rotation speed until the vortex drops by about 3cm, pour in 10mL of 10%PEG 20000, and balance for 5min; (7) Centrifugation: Use a benchtop high-speed centrifuge at 12,000 rpm and 4°C for 6 min; (8) Discard the supernatant: After centrifugation, gently remove the centrifuge bucket and place it on the experimental table of the negative pressure aspiration device. Do not shake or bump it to avoid disturbing the gold precipitate in the centrifuge bucket; stop aspiration when it is close to the bottom of the centrifuge bucket (about 5-6 mL remaining), and carefully remove the remaining supernatant with a 1 mL pipette to avoid aspirating the gold precipitate; (9) Resuspension: Stop aspirating when the liquid is close to the bottom of the centrifuge bucket (about 2-3 mL remaining), gently shake the centrifuge bucket to resuspend the gold precipitate, and transfer the resuspended gold precipitate to a blue cap bottle; (10) Rinsing the container: Add about 20 mL of reconstitution solution to the centrifuge container, shake gently to wash away the residual gold precipitate, transfer the liquid to the other three centrifuge containers in turn, and finally transfer it to the blue cap bottle. Repeat once. (11) Volume adjustment: Finally, adjust the volume of the gold solution to 1 / 10, i.e. 100 mL, using the blue-capped bottle, and mix well. (12) Lyophilization of microwell reagent: The microwell reagent solution was prepared by mixing methomyl monoclonal antibody-colloidal gold label and lyophilization buffer, stirring for 20 min, and dispensed into 96-well microplates at a ratio of 60 μL / well; wherein the lyophilization buffer is composed of 0.05 M phosphate buffer, 1% BSA, 2% sucrose and 2% Tween-20; the ratio of methomyl colloidal gold to the lyophilization buffer in the microwell reagent solution is 1:5; the coated 96-well microplates were placed in a vacuum freeze dryer with a preset freeze-drying program for processing. After the freeze-drying process was completed, the lyophilized gold was removed, the rubber cap was closed, and the plate was placed in an aluminum foil bag with desiccant for storage.

[0040] 3. Preparation of sample absorption pads The sample absorption pad was immersed in 0.1M phosphate buffer containing 0.5% BSA, pH=7.2 for 2 hours and then dried at 37℃ for 2 hours to obtain the sample absorption pad.

[0041] 4. Preparation of nitrocellulose membranes Methomyl artificial antigen was diluted to 1 mg / mL with phosphate buffer and coated onto a nitrocellulose membrane using a coating apparatus to form the detection line T, with a coating amount of 1.0 μg / cm. Goat anti-mouse IgG antibody was diluted to 300 μg / mL with PBS buffer (0.01 M, pH 7.2) and coated onto a nitrocellulose membrane as the control line C. The coated reaction membrane was then dried at 37°C for 5 hours to obtain the coated nitrocellulose membrane.

[0042] 5. Assembly of methomyl colloidal gold test strips The sample absorption pad, nitrocellulose membrane, and absorbent pad are sequentially attached to the base plate. The beginning of the sample absorption pad is connected to the end of the nitrocellulose membrane, and the beginning of the nitrocellulose membrane is connected to the end of the absorbent pad. The end of the sample absorption pad is aligned with the end of the base plate, and the beginning of the absorbent pad is aligned with the beginning of the base plate. This assembly forms the colloidal gold test strip. Together with the microporous reagent, it forms the methomyl colloidal gold test strip.

[0043] Example 7: Application of methomyl colloidal gold test strip 1. Test strip detection The required test strips and test samples should be brought to room temperature (20-25℃). Using a micropipette, pipette 200µL of the test sample into each well, slowly aspirating and thoroughly mixing it with the reagent in the well. After incubating at 40℃ for 3 minutes, insert the labeled test strip into the well, ensuring it is fully immersed in the solution. After incubating at 40℃ for 5 minutes, remove the test strip and follow the diagram (…). Figure 4 The judgment result is valid at other times; judgments made at other times are invalid.

[0044] 2. Interpretation of test results Negative (﹣): Both C and T lines show color, with the T line showing much stronger color than the C line, indicating that the concentration of methomyl in the sample is below the detection limit.

[0045] Positive (+): C line shows color; T line shows the same color as C line, T line shows weaker color than C line, or T line shows no color, all of which indicate that the concentration of methomyl in the sample is equal to or higher than the detection limit.

[0046] Invalid: No C line appears, indicating incorrect operation or that the test strip has deteriorated and become ineffective.

[0047] In addition to naked-eye interpretation, a colloidal gold reader can be used for result interpretation.

[0048] 3. Sensitivity testing of methomyl colloidal gold test strips Methomyl standard was diluted to 1 ng / mL and 2 ng / mL, and raw milk samples were spiked and tested according to the test strip detection method to verify the product's limit of detection. The results are shown in Table 3. The test strip provided by this invention has a detection sensitivity of 2 ng / mL for methomyl standard in raw milk.

[0049] Table 3 Sensitivity of methomyl colloidal gold test strips

[0050] 4. Specificity detection of methomyl colloidal gold test strips The test strips were tested at concentrations of 500 ng / mL for methomyl, methomyl oxime, carbofuran, aldicarb, methamidophos, and carbaryl, and the data are shown in Table 4-1. The results showed that, except for methomyl and methomyl oxime, all others were negative. This indicates that the test strips provided by this invention do not exhibit cross-reactivity with other pesticides and have good specificity.

[0051] Methomyl oxime standard was diluted to 5 ng / mL and 10 ng / mL, and raw milk samples were spiked and tested according to the test strip detection method to verify the product's limit of detection. The results are shown in Table 4-2. The test strip provided by this invention has a detection sensitivity of 10 ng / mL for methomyl oxime standard in raw milk. The results indicate that the cross-reactivity rate between this antibody and methomyl oxime is approximately 20%.

[0052] Table 4-1 Specificity of Methomyl Colloidal Gold Test Strips

[0053] Table 4-2 Sensitivity of methomyl colloidal gold test strip for methomyl oxime

[0054] 5. Stability testing of methomyl colloidal gold test strips The prepared test strips were subjected to accelerated stability tests at 4℃ and 37℃. The strips were used to detect methomyl standard (2 ng / mL) at days 0, 7, 14, and 28, with three replicates at each time point. The results are shown in Tables 5-1 and 5-2. All test results were positive. The intra-batch coefficient of variation (CV) was less than 5%, and the inter-time coefficient of variation (CV) was less than 10%, indicating that the methomyl colloidal gold test strip provided by this invention has good stability.

[0055] Table 5-1 Determination of stability index of methomyl colloidal gold test strips

[0056] Table 5-2 Stability evaluation of methomyl colloidal gold test strips

[0057] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A monoclonal antibody against metoclopramide, characterized in that: The monoclonal antibody includes a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 1; The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 2; Both the heavy chain variable region and the light chain variable region are composed of complementary determination regions and framework regions, and the complementary determination regions are composed of CDR1, CDR2 and CDR3. The amino acid sequence of CDR1 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 5; The amino acid sequence of CDR2 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 6; The amino acid sequence of CDR3 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 7; The amino acid sequence of CDR1 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 8; The amino acid sequence of CDR2 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 9; The amino acid sequence of CDR3 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No.

10.

2. The anti-methodox monoclonal antibody according to claim 1, characterized in that: The gene sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 3; The gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID No.

4.

3. The application of the anti-methomyr monoclonal antibody according to claim 1 in the preparation of methomyl detection products.

4. A methomyl testing product, characterized in that: The test product consists of a methomyl test strip and a microwell reagent. The test strip is a colloidal gold test strip, and the microwell reagent contains the anti-methomyl monoclonal antibody as described in claim 1 or 2.

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