A recombinant antibody against triazophos and application thereof

Abstract

The application discloses an anti-triazophos recombinant antibody and application, and relates to the technical field of genetic engineering and immune detection. A heavy chain variable region amino acid sequence of the anti-triazophos recombinant antibody is shown as SEQ ID NO. 1, and a light chain variable region amino acid sequence is shown as SEQ ID NO. 3, and a coding gene thereof is derived from a hybridoma cell with a preservation number of GDMCC NO: 68914. A preparation method of the anti-triazophos recombinant antibody comprises the following steps: extracting RNA from the hybridoma cell, obtaining cDNA through reverse transcription, amplifying a full-length heavy chain gene and a full-length light chain gene, and then cloning into a eukaryotic expression vector and transfecting a host cell to perform expression. The expression amount of the recombinant antibody in HEK293F cells reaches 354 mu g / mL, the IC 50 is 0.06 mu g / L, the sensitivity is high, and the anti-triazophos recombinant antibody can be used for preparing a triazophos detection product.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and immunoassay, and particularly to a recombinant antibody against triazophos and its applications. Background Technology

[0002] Triazophos (TAP) is a widely used organophosphorus insecticide, once extensively applied to the control of pests and diseases in crops such as rice, vegetables, and fruit trees. Due to its high biotoxicity and environmental risks, residues can enter the food chain through agricultural products, animal-derived foods, and environmental media, posing potential harm to the ecological environment and human health. Therefore, establishing rapid, accurate, and highly sensitive triazophos residue detection technologies has always been an important research direction in the fields of food safety and environmental monitoring. Currently, triazophos residue detection technologies mainly include two categories: instrumental analysis methods and immunoassay methods. 1. Instrumental Analysis Techniques: Existing instrumental analysis techniques mainly include gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). The basic principle is that after sample extraction and purification, the target compound is separated from impurities using a chromatographic system, and then the target compound is qualitatively and quantitatively analyzed using a detector or mass spectrometry system. These methods have advantages such as high sensitivity, good accuracy, and strong quantitative capabilities, and have become important reference methods for triazophos residue detection. However, this type of method still has the following drawbacks: (1) the instruments and equipment are expensive and the operation and maintenance costs are high; (2) the sample pretreatment process is complex; (3) the detection cycle is long; (4) the professional skills requirements for operators are high; and (5) it is difficult to meet the needs of grassroots supervision and rapid on-site screening. Therefore, it is limited in the field of rapid detection of large-scale samples. 2. Immunoassay technology based on monoclonal antibodies: In order to solve the problems of instrumental analysis methods, researchers have developed immunoassay technologies based on antigen-antibody specific recognition reactions, including enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography, fluorescence immunoassay and chemiluminescence immunoassay. The core of this type of method is to obtain high-affinity and high-specificity anti-triazole phosphorus antibodies. Currently, the publicly available anti-triazophos antibodies are mainly obtained through hybridoma technology. The process usually includes: (1) design and synthesis of triazophos hapten; (2) conjugation of hapten with carrier protein to prepare artificial antigen; (3) immunization of BALB / c mice; (4) fusion of spleen cells and myeloma cells; (5) hybridoma screening and subcloning; (6) ascites induction or in vitro culture to prepare monoclonal antibodies; (7) antibody purification and immunoassay application. This technical route has successfully obtained a variety of anti-triazophos monoclonal antibodies and established corresponding immunoassay methods. However, traditional hybridoma antibody technology still has the following shortcomings: (1) antibody production depends on long-term culture and preservation of hybridoma cell lines; (2) genetic drift, chromosome instability and decreased antibody expression may occur during continuous cell passage; (3) cell contamination, cryopreservation inactivation or cell loss may lead to the inability to recover antibody resources; (4) there are certain differences in the preparation process of different batches of ascites, which affects the consistency of antibodies; (5) animal ascites production methods have issues of animal ethics and production costs.

[0003] The reasons why the existing technology still has the above problems are: (1) Triazophos is a small molecule hapten with weak immunogenicity, making it difficult to screen high-performance antibodies; (2) Antibody affinity and specificity are significantly affected by immunogen design, individual animal differences, and screening strategies; (3) The variable region sequences of the antibody heavy chain and light chain determine its antigen recognition ability, but the currently available anti-triazophos VH / VL sequence resources that can be directly used for engineering development are still relatively limited; (4) There is a lack of anti-triazophos antibody sequence resources that have high affinity, high specificity, and good recombinant expression performance. In addition, the publicly available anti-triazophos antibody sequence resources are relatively limited, and there are few antibody sequences that can have high affinity, good specificity, and recombinant expression ability, which limits its further application in immunoassay, kit development, and industrial production.

[0004] Therefore, the present invention aims to provide a recombinant antibody against triazophos and its application, clarify and amplify the full-length heavy chain gene and light chain gene sequences of the recombinant antibody, establish a stable recombinant expression system, and provide a new core antibody resource for the detection of triazophos residues. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention proposes an anti-triazolium recombinant antibody and its application to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A recombinant antibody against triazophos, wherein the amino acid sequence of the heavy chain variable region of the recombinant antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.3.

[0007] Preferably, the amino acid sequences of the heavy chain variable region and the light chain variable region are derived from the deposit number GDMCC NO: 68914 and the taxonomic name is Murine hybridoma cell line The hybridoma cells, named in the laboratory Murine hybridoma cell line 24D; Depository address: Guangdong Provincial Center for Microbial Culture Collection; Deposit date: August 28, 2026.

[0008] This invention provides a method for preparing the anti-triazolphos recombinant antibody, comprising the following steps: (1) From the accession number GDMCC NO: 68914, the taxonomic name is Murine hybridoma cell line Total RNA was extracted from hybridoma cells and cDNA was obtained by reverse transcription. (2) Using the cDNA obtained in step (1) as a template, amplify the full-length heavy chain gene and the full-length light chain gene; the heavy chain encoded by the full-length heavy chain gene contains the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.1 and the heavy chain constant region with an amino acid sequence as shown in SEQ ID NO.5, and the light chain encoded by the full-length light chain gene contains the light chain variable region with an amino acid sequence as shown in SEQ ID NO.3 and the light chain constant region with an amino acid sequence as shown in SEQ ID NO.7; (3) The full-length heavy chain gene and the full-length light chain gene obtained in step (2) are cloned into a eukaryotic expression vector to construct a recombinant plasmid; (4) Transfect the recombinant plasmid obtained in step (3) into host cells and culture them. Collect the culture supernatant to obtain the anti-triazolium recombinant antibody.

[0009] Preferably, the eukaryotic expression vector is selected from any one of pcDNA3.4, pCAGGS, or pTT5.

[0010] Preferably, the host cell is a HEK293F cell.

[0011] Preferably, the N-terminus of the heavy chain has a first signal peptide, and the N-terminus of the light chain has a second signal peptide; the first signal peptide and the second signal peptide are each independently selected from: the mouse IgGκ signal peptide shown in SEQ ID NO.9, the tPA signal peptide shown in SEQ ID NO.10, the natural signal peptide shown in SEQ ID NO.11, the natural signal peptide shown in SEQ ID NO.12, or the engineered signal peptide shown in SEQ ID NO.13.

[0012] Preferably, the mass ratio of DNA to polyethyleneimine (PEI) in the transfection step (4) is selected from any one of 1:3, 1:4 or 1:6.

[0013] Preferably, the mass ratio of heavy to light chains in the transfection process described in step (4) is selected from any one of 1:1, 2:3, or 3:2.

[0014] This invention provides the application of the aforementioned anti-triazophos recombinant antibody in detecting triazophos residues in samples.

[0015] Preferably, the sample includes any one or more of paddy field water, chicken meat, and cowpeas.

[0016] The present invention also provides the application of the aforementioned anti-triazophos recombinant antibody in the preparation and detection of triazophos products.

[0017] Compared with the prior art, the beneficial effects of the present invention are: (1) Through combination screening of sequence form and expression vector, screening of signal peptide and stepwise optimization of transfection conditions, the optimal expression strategy of recombinant antibody was finally established: using full-length wild-type sequence, pcDNA3.4 vector and mouse IgGκ signal peptide, transient transfection was performed in HEK293F cells with C brand transfection reagent, heavy chain to light chain mass ratio of 2:3 and DNA to polyethyleneimine (PEI) mass ratio of 1:6; based on the above strategy, high affinity r-mAb 24D was successfully obtained, and the antibody expression level reached 354 μg / mL under optimized conditions, indicating that the expression system established in this study has high expression efficiency; (2) The antibody performance was evaluated using indirect competitive ELISA, and the IC50 of the recombinant antibody r-mAb 24D was found to be high. 50 The concentration was 0.06 μg / L, indicating that the antibody of the present invention has high sensitivity; (3) An icELISA method for detecting TAP residues in paddy water, cowpea, and chicken samples was established. The LOD of TAP in paddy water was 0.16 μg / L, with a recovery rate of 90.2-114.6% and a CV of 13.8%. The LOD of TAP in cowpea was 0.28 μg / kg, with a recovery rate of 85.8-114.4% and a CV of 18.6%. The LOD of TAP in chicken was 1.15 μg / kg, with a recovery rate of 80.0-111.3% and a CV of 17.9%. The results showed that the icELISA method established in this study has good sensitivity, accuracy, and precision, providing strong technical support for the detection of TAP residues in multi-matrix samples. Attached Figure Description

[0018] Figure 1 The growth morphology of the cell cluster 7 days after hybridoma cell fusion; Figure 2 Establishment of mAbs standard curves for 5 plants; Figure 3 The results of agarose gel electrophoresis of total RNA; Figure 4 Optimize 3D bar charts for antibody capture and antibody concentration detection; Figure 5 To establish the standard curve for sandwich ELISA; Figure 6 The selection of different sequence forms and vector antigen-antibody dilutions for strain 24D. Detailed Implementation

[0019] To enable those skilled in the art to better understand the technical content of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0020] Example 1: Preparation of Hybridoma Cells (1) Experimental solution system and experimental animals Carbonate buffer (CB) buffer: 0.159g Na2CO3, 0.29g NaHCO3 and 100.0mL Diswater.

[0021] Washing solution: 320.0g NaCl, 10.0g KH2PO4, 116.0g Na2HPO4·12H2O, 0.18g KCl, 1.5mL Proclin 300, 100.0mL Tween-20 and 4900.0mL Diswater.

[0022] Phosphate buffer (PBS) buffer: 0.85g NaCl, 0.02g KCl, 0.059g NaH2PO4·2H2O, 0.29g Na2HPO4·12H2O and 100.0mL Diswater.

[0023] Enzyme dilution solution: 1.3g NaH2PO4·2H2O, 8.7g Na2HPO4·12H2O, 100.0mL glycerol, 400.0mL fetal bovine serum, 2.0mL Proclin 300 and 1500.0mL Diswater.

[0024] Blocking solution: 250.0g sucrose, 250.0g casein, 12.5g Na2HPO4·12H2O, 29.0g NaH2PO4·2H2O, 2.5mL Proclin 300 and 4750.0mL Diswater.

[0025] Termination solution: 224.0 mL concentrated sulfuric acid and 1776.0 mL Diswater.

[0026] 1% agarose electrophoresis gel: 1.0 g agarose, 5.0 μL Goldview, 1×TAE to 100 mL.

[0027] 50×TAE nucleic acid electrophoresis buffer: 242.0g Tris, 18.6g EDTA-2Na, 57.1mL glacial acetic acid, and Diswater to a final volume of 500mL.

[0028] The 6-8 week old SPF-grade female BALB / c mice used in the experiment were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., and the breeding process strictly followed the standards of "Laboratory Animal Welfare and Animal Experimentation Ethics" of China Agricultural University Sanya Research Institute.

[0029] (2) Test methods The immunogen THBu-BSA and coating agent THBu-OVA used in this experiment were kindly provided by the laboratory of Associate Professor Hua Xiude, College of Plant Protection, Nanjing Agricultural University. THBu is a hapten derivative of TAP, chemically named O-ethyl, O-[1-phenyl-(1H-1,2,4-triazol-3-yl), N-(3-carboxypropyl)]thiophosphate, characterized by the introduction of butyric acid (-(CH2)3-COOH) as a linker arm. This hapten was synthesized according to the method reported by Gui in 2006 (paper title: Hapten synthesis for enzyme-linked immunoassay of the insecticide triazophos). The hapten THBu was coupled to bovine serum albumin (BSA) and ovalbumin (OVA) respectively using the active ester method to prepare the immunogen and coating agent.

[0030] (2.1) Mouse immunization (2.1.1) Immunization of BALB / c mice (2.1.1.1) Immunogen emulsification: Dilute the immunogen concentration to 1.0 mg / mL. For the first immunization, take equal volumes (1.0 mL each) of the immunogen solution and emulsify with Freund's complete adjuvant (FCA); for booster immunizations (second, third, fourth, and fifth immunizations), use the immunogen solution with Freund's incomplete adjuvant (FICA); for shock immunizations, no adjuvant is added, and the immunogen is diluted with PBS and injected directly. The specific steps of the emulsification operation are as follows: after mixing the immunogen and adjuvant 1:1, repeatedly aspirate until a uniform water-in-oil emulsion is formed. The criterion for successful emulsification is that the emulsion droplets remain intact and do not spread after being placed on the water surface.

[0031] (2.1.1.2) Immunization: Eight healthy female BALB / c mice aged 6-8 weeks and of similar weight were selected. For easy individual identification, ear tags numbered H13 to H20 were attached to their ears, followed by immunization. Each mouse received a dose of 100 μg (0.2 mL) via subcutaneous injection at multiple points on the back of the neck. The first and second immunizations were administered 4 weeks apart, and subsequent immunizations were administered 3 weeks apart. Blood was collected via the retro-orbital venous plexus on day 7 after each immunization, and serum was separated. The immune response was evaluated using icELISA.

[0032] (2.1.2) Evaluation of mouse antiserum After the third immunization, peripheral blood was collected from BALB / c mice that had been immunized for 7 days from the posterior orbital venous plexus. The blood volume was 200 μL per mouse. To avoid hemolysis, the blood was allowed to stand and then centrifuged to obtain the supernatant serum, which was then aliquoted for easy subsequent testing.

[0033] Antiserum and coating antigen were separately analyzed for each mouse. Optimal dilutions for both the coating antigen and antiserum were screened using a checkerboard method (antigen dilutions: 20K, 40K, 80K, 160K, 320K, 640K; antibody dilutions: 50K, 100K, 200K, 400K, 800K, 1600K). A series of standard concentrations were obtained by diluting the antiserum with TAP standards (purchased from Bailingwei Technology Co., Ltd.). The affinity and specificity of the mouse antiserum were then detected using icELISA. The IC50 of different mouse antiserums after the third, fourth, and fifth immunizations was also analyzed. 50 Based on the titer results, mice with the best immune response were selected for subsequent shock immunization and cell fusion experiments.

[0034] The specific experimental procedures are as follows: (a) Dilute the coating agent to an appropriate concentration using CB buffer, aspirate 100 μL / well into the microplate, incubate at 37°C for 2 h, discard the liquid in the well, wash twice with 280 μL / well washing buffer using a plate washer, and then spin dry.

[0035] (b) Add 150 μL of blocking solution to a 96-well microplate and incubate at 37°C for 1 h; discard the blocking solution, wash once with 280 μL / well washing solution and spin dry.

[0036] (c) Add 50 μL of PBS buffer or different concentration gradient standards to each well, followed by 50 μL of serially diluted antiserum. Incubate at 37°C for 30 min, then discard the reaction solution, wash twice and spin dry.

[0037] (d) Add 100 μL of diluted enzyme-labeled secondary antibody to each well, incubate at 37°C for 30 min, discard the reaction solution, wash twice and spin dry.

[0038] (e) Mix equal volumes of substrate solution, add 100 μL to each well for color development, react in the dark for 15 min, and then add 50 μL of stop solution.

[0039] (f) The absorbance was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0040] (g) Set the logarithm of the TAP standard concentration as the x-axis, OD 450 Using Origin as the ordinate, a four-parameter logistic (4PL) nonlinear curve is fitted, a standard curve is plotted, and a regression equation is established, the expression of which is shown below: Y={(A1) A2) / [1+(X / X0) P ]}+A2; Where Y represents absorbance (OD) 450 A1 represents the absorbance of the upper plateau (the asymptotic value when the standard concentration is zero), A2 represents the absorbance of the lower plateau (the asymptotic value when the maximum inhibition is reached), X represents the logarithm of the standard concentration, and X0 represents the midpoint of the curve (i.e., the X value corresponding to Y=(A1+A2) / 2, usually called IC). 50 (Half-inhibition concentration)), P represents the slope factor of the curve (Hill slope).

[0041] Data analysis process: (1) The optimal working concentrations of the coating antigen and serum were determined using the checkerboard method, and the dilutions corresponding to OD values ​​between 1.5 and 2.0 were selected as the optimal working concentrations. (2) Titer: After determining the optimal working concentration of the coating antigen, the antibody dilutions with OD values ​​in the range of 1.5 to 2.0 in the zero wells were defined as antibody titers. (3) IC 50 OD measured using standards of various concentrations 450 The values ​​were used as the ordinate, and the logarithm of the standard concentration was used as the abscissa. A nonlinear regression was performed in Origin software to establish a standard curve. A four-parameter logistic model was used to fit and analyze the data, thereby obtaining the IC50 of the antibody against the standard. 50 IC 50 The lower the value, the stronger the antibody's binding ability to the target molecule and the higher its affinity.

[0042] Experimental Results: Antiserum was collected from eight immunized mice 7-10 days after the third, fourth, and fifth immunizations, and its titer was detected by icELISA. Using 5 μg / L TAP as the inhibitory concentration, the optimal dilution of the coating antigen was determined to be 1:27000 in preliminary experiments. The results are shown in Table 1. With increasing immunization frequency, the antiserum titer of most mice showed an upward trend. Except for H15 mice, the titer of the other mice reached 1:9000 after the fifth immunization, indicating that most mice had developed high levels of specific antibodies after booster immunization. Specifically, the antiserum titers of H13, H14, H16, H18, H19, and H20 mice increased from 1:3000 after the third immunization to 1:9000 after the fifth immunization; H17 mice maintained a relatively high titer of 1:9000 after the third immunization; while the titer of H15 mice remained at 1:3000 with no significant increase. The B / B0 ratio was used to evaluate the inhibitory effect of TAP on the binding of antiserum to the coating antigen. The results showed that the B / B0 ratio of most mouse antisera gradually decreased with increasing immunization frequency, indicating a gradual increase in the antiserum's ability to recognize TAP. Among them, the B / B0 ratio of H14 mice decreased most significantly, from 50.0% after the third immunization to 20.9% after the fifth immunization; H16, H18, and H20 mice also showed a good decreasing trend. In contrast, the B / B0 ratio of H17 and H19 mice did not decrease significantly, suggesting that their antiserum's ability to recognize TAP was limited. Although H13 mice showed a high titer after the fifth immunization, they subsequently died unexpectedly, making it impossible to continue monitoring their immune response. An icELISA standard curve was established for each mouse antiserum, and the IC50 of the antiserum was calculated. 50 The results showed that the IC50 values ​​of most mouse antiserums were... 50 The concentration range was 0.29-3.11 μg / L, indicating good recognition ability for TAP. Among them, the IC50 in H14 mice... 50 The concentration was the lowest, only 0.29 μg / L, indicating the highest antiserum affinity; considering the antiserum titer, B / B0 value, and IC50... 50 As a result, H14 mice exhibited higher titers, lower B / B0 values, and the lowest IC50 values ​​after the fifth immunization. 50 The H14 mouse was selected for subsequent cell fusion and mAb screening because it had the best overall performance.

[0043] Table 1 Results of mouse antiserum detection

[0044] Note: B0: OD value without standard; B: OD value with standard; Binding rate calculation formula: (B / B0)×100%.

[0045] (2.2) Preparation of triazophos monoclonal antibody (2.2.1) Shock immunization in mice Four days prior to cell fusion, mice exhibiting high titers and affinity in serum tests were given a pulse immunization. The immunogen dose was typically controlled at 0.3-0.5 mg / mouse, with adjustments made based on the mice's weight, health status, and mental state. The required amount of immunogen was drawn using a sterile syringe, diluted to 0.5 mL with PBS buffer, and administered via intraperitoneal injection.

[0046] (2.2.2) Resuscitation, culture and preparation of SP2 / 0 myeloma cells Five days prior to the cell fusion experiment, SP2 / 0 myeloma cells were resuscitated and expanded. On the day of cell fusion, SP2 / 0 myeloma cells in the logarithmic growth phase with 80%-90% confluence were selected. Semi-adherent cells were gently detached using a pipette and collected into centrifuge tubes. Generally, the amount of myeloma cells required per immunized mouse corresponds to the cell volume in 9-well culture tubes. The collected cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the outer wall of the centrifuge tubes was sterilized with 75% ethanol before being transferred to a laminar flow hood. The cell pellet was gently tapped at the bottom of the tube to loosen it, and 30 mL of DMEM medium was added for resuspending, ready for subsequent cell fusion experiments.

[0047] (2.2.3) Preparation of feeder cells Healthy mice that had not received immunization were euthanized by cervical dislocation and immediately immersed in 75% ethanol for 5 minutes for surface disinfection. Aseptic procedures were performed in a laminar flow hood, with the operator's hands and the work surface disinfected with alcohol. Sterile syringes, forceps, scissors, and HAT culture medium were prepared. The mice were fixed in a supine position, the abdominal skin was lifted, and a small incision was made with sterile scissors. The skin was gently dissected laterally along the incision to fully expose the peritoneum. Then, sterile instruments were changed, the peritoneum was gently lifted, and culture medium was slowly injected into the peritoneal cavity using a syringe. The abdomen was gently massaged for 1 minute to promote the release of cells into the fluid. The peritoneal fluid was then slowly collected. This process was repeated to improve cell recovery rate. Peritoneal cells from four mice were collected and added to HAT culture medium (hypoxanthine-aminopterin-thymidine medium) to a final volume of 200 mL. After thorough mixing, the cells were aliquoted into 96-well cell culture plates, with 100 μL of cell suspension in each well.

[0048] (2.2.4) Preparation of immune spleen lymphocytes Prior to cell fusion, all relevant reagents, including HAT medium, DMEM medium, and polyethylene glycol (PEG), were preheated in a 37°C water bath. Mice immunized 4 days prior were euthanized by cervical dislocation and sterilized in 75% ethanol. Under aseptic conditions in a laminar flow hood, the spleen was carefully removed from the abdominal cavity, its attached fat and connective tissue removed, and placed on a sterile filter membrane. An appropriate amount of erythrocyte lysis buffer was added, and the spleen tissue was gently ground with a sterile grinding rod to fully release lymphocytes. DMEM medium was then quickly added to terminate the lysis reaction. The resulting cell suspension was filtered through a filter membrane into centrifuge tubes, and medium was added to the appropriate volume. The cells were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were then resuspended in a small amount of DMEM, tissue debris was removed, and the medium was replenished to the final volume for use as a source of splenic lymphocytes for subsequent fusion.

[0049] (2.2.5) Cell fusion The prepared spleen lymphocytes and myeloma cells were centrifuged separately at 1000 rpm for 5 min, and the supernatant was discarded and the cells were gently resuspended. The two cell types were mixed at a 5:1 ratio, and DMEM medium was added and mixed thoroughly before centrifugation to remove the supernatant as completely as possible. The cell pellet was gently dispersed and pre-warmed at 37°C. Then, pre-warmed PEG was slowly added dropwise over 30 seconds while continuously and gently mixing to promote cell fusion. After the fusion reaction was complete, pre-warmed DMEM medium was gradually added to terminate the PEG effect, and the total volume was replenished before centrifugation. After discarding the supernatant, the cells were resuspended in HAT selective medium and diluted to an appropriate volume. The cells were then aliquoted into 96-well plates containing feeder cells and incubated at 37°C in a 5% CO2 incubator. Continuous observation of cell growth after fusion revealed that most cells gradually adhered to the wells and formed well-defined, relatively regular cell colonies, exhibiting typical hybridoma cell clonal growth characteristics. Figure 1 ).

[0050] (2.2.6) Screening of hybridoma cells Cell growth was observed daily after fusion, and initial screening was performed when the cell clusters reached a suitable size. First, the culture supernatant was analyzed using icELISA to determine antibody secretion, and the detection conditions were optimized based on different coating antigen concentrations. Based on this, positive wells were further analyzed using icELISA to comprehensively evaluate antibody titer and binding rate, screening out cell wells with high affinity and good specificity. In subsequent screening, the screening range was gradually narrowed down (OD value between 1.68 and 2.53, B / B0 value between 9.7% and 28.5%) based on cell growth status and antibody performance indicators, with positive wells having cell clusters of 2 or less preferentially selected for further subcloning. The results are shown in Table 2.

[0051] (2.2.7) Subcloning of hybridoma cells Sufficient feeder cells are needed to support single-cell growth before subcloning. First, the distribution of cell clusters in the positive wells is observed under a microscope. Under aseptic conditions, single cell clusters are aspirated and seeded into 96-well plates containing feeder cells. Single-cell isolation and culture are achieved through serial dilution. For wells containing multiple cell clusters, the above operation is repeated to obtain monoclonal source cells. After confirming the source of a single cell cluster, three consecutive rounds of subcloning are required to consistently obtain a single cell cluster, confirming the acquisition of a stable hybridoma cell line. Simultaneously, some cells are cryopreserved for subsequent experiments. The results are shown in Table 2. After multiple rounds of subcloning screening, five hybridoma cell lines capable of stably secreting anti-TAP antibodies were finally obtained.

[0052] Table 2. Screening and subcloning of hybridoma cells

[0053] (2.2.8) Preparation of monoclonal antibodies by ascites induction method Eight healthy BALB / c mice were selected, and each mouse was injected intraperitoneally with 0.5 mL of liquid paraffin to induce ascites formation. One week later, hybridoma cells that had grown to 80%-90% confluence and were in the logarithmic growth phase were collected from culture plates. Cells cultured in half-well 6-well plates or 2-well 24-well plates could be selected for collection. After centrifugation at 1000 rpm for 5 min, the supernatant was discarded, the bottom of the tube was gently tapped, and 4 mL of DMEM culture medium was added to resuspend the cells. The cell suspension was then aspirated into a 5 mL syringe for later use. The cell suspension was injected intraperitoneally into the mice at a dose of 0.5 mL per mouse, and the experimental animals were numbered and marked. Ascites was aspirated daily when obvious abdominal distension was observed in the mice until the mice died. After each collection of ascites, the cells were centrifuged at 4000 rpm for 10 min, and the supernatant was collected and stored at -20℃ for later use.

[0054] (2.2.9) Purification of monoclonal antibodies and detection of antibody affinity (a) Affinity column pretreatment: The Protein A affinity chromatography column stored at 4°C was taken out and allowed to stand at room temperature for 30 min to return to room temperature.

[0055] (b) Column equilibration: Use a syringe to add 10 mL of Binding Buffer to equilibrate the affinity column, controlling the flow rate at 1-2 mL / min (20-40 drops / min) to ensure that the packing material in the column is in full contact with the buffer.

[0056] (c) Sample processing: Take 2 mL of ascites fluid sample, add 7 mL of Binding Buffer and mix well. Filter through a 0.22 μm filter membrane to remove particulate impurities. The resulting filtrate is used as the sample to be purified.

[0057] (d) Sample loading onto the column: The treated sample is slowly loaded into the affinity column at a flow rate of 1-2 mL / min to ensure sufficient binding of the antibody to the Protein A ligand. The overflow solution is collected and labeled for later use.

[0058] (e) Column washing: Add 10 mL Binding Buffer to wash to remove unbound or weakly bound contaminating proteins. Discard the washing buffer.

[0059] (f) Preparation of eluent collection: Prepare 12 0.6 mL centrifuge tubes and number them. Add 100 μL of neutralization buffer to each tube beforehand for subsequent collection of eluent and pH neutralization.

[0060] (g) Antibody elution: Add 10 mL of Elution Buffer to the affinity column to elute the IgG bound to the column packing material. Gently mix immediately after each collection to ensure complete neutralization of the solution.

[0061] (h) Column equilibration recovery: Rinse the affinity column again with 10 mL Binding Buffer to restore it to its initial equilibrium state.

[0062] (i) Column packing regeneration: Add 5 mL of 0.1 M NaOH to regenerate the column packing and let it stand at room temperature for 15 min.

[0063] (j) Re-equilibrate: Rinse the affinity column with 10 mL Binding Buffer to remove residual alkali and restore a suitable buffer environment.

[0064] (k) Column preservation treatment: Add 5 mL of 20% ethanol solution to protect the column packing, then tighten the seals at both ends of the column and store it at 4°C.

[0065] (l) Antibody post-processing: The absorbance of the eluent in each collection tube at 280 nm was measured using Nanodrop. Based on the measurement results, fractions with antibody concentrations higher than 0.2 mg / mL were combined. The combined antibody solution was then transferred to a dialysis bag with a molecular weight cutoff of 8 kDa, and dialyzed for 3 days with buffer changes after 8 hours. The purified antibody was stored at -20°C.

[0066] The performance of these five candidate antibodies was evaluated using icELISA. Based on the optimized concentrations of the antibodies and coating antigens, standard curves for the five antibodies were established. Figure 2 Test results showed that the IC50 values ​​of the five mAbs (5A11, 25C7, 24C3, 11A8, 24D) were... 50The concentrations were 0.25 μg / L, 0.20 μg / L, 0.25 μg / L, 0.24 μg / L, and 0.18 μg / L, respectively, and mAb 24D showed the highest affinity. The 24D hybridoma cells were ultimately preserved for research. The hybridoma cells (… Murine hybridoma cell line The taxonomic name of 24D is Murine hybridoma cell line It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, deposited on August 28, 2026, with accession number GDMCC NO: 68914.

[0067] (2.2.10) Identification of monoclonal antibody subtypes The specific operating steps are as follows: (a) Dilute the antigen that specifically binds to mAb with CB and add 100 μL to each well of the ELISA plate. Incubate at 37°C for 2 h or at 4°C for 12 h.

[0068] (b) Wash the microplate once with PBS-T buffer.

[0069] (c) Add 200 μL of ELISA blocking solution to each well and incubate at 37°C for 2 h, or at 4°C for 16 h.

[0070] (d) After discarding the blocking solution, wash three times with PBS-T and spin dry using a spin bar.

[0071] (e) Collect the culture supernatant of the hybridoma cells to be tested, or dilute the ascites fluid appropriately with PBS-T (usually 1000-fold). Set up 16 wells for each sample to be tested, and add 100 μL to each well. Incubate at 37°C for 30 min.

[0072] (f) After removing the sample solution, wash the microplate 5 times with PBS-T buffer and spin dry.

[0073] (g) Add HRP-labeled antibody subtype detection reagent to each well, with 100 μL of each labeled antibody added to 2 wells, for a total of 16 wells; if no duplicate wells are set, add 1 well of each type, for a total of 8 wells. Then incubate at 37°C for 30 min.

[0074] (h) Discard the enzyme-labeled secondary antibody solution, wash 5 times with PBS-T buffer, and spin dry with a spin bar.

[0075] (i) Pipette 100 μL of the colorimetric solution into the well and react at 37 °C in the dark for 20 min. Then add 50 μL of the stop solution. Read the absorbance at a wavelength of 450 nm and determine the result accordingly.

[0076] The results showed that the 24D heavy chain subtype of hybridoma cells was IgG2a, and the light chain was Lambda(λ) type.

[0077] (2.3) Gene sequencing of triazophos monoclonal antibody (2.3.1) Extraction of total RNA from hybridoma cells Sample pretreatment: After discarding the cell culture supernatant, cells can be directly lysed in culture dishes. For standard 6-well plates or 3.5cm diameter petri dishes, add 500μL of Buffer CRL, ensuring complete coverage of the cell surface, and then repeatedly pipette to detach the cells. Total RNA was extracted from hybridoma cells using the silica gel membrane column method. The results showed an RNA concentration of 478.2 ng / μL and an OD... 260 nm / OD 280 The nm ratio was 1.99, indicating high sample purity. Further verification of RNA integrity using agarose gel electrophoresis showed that clear and intact 28S and 18S rRNA bands were observed in lanes 1-5, with no obvious degradation, meeting the requirements for subsequent molecular biology experiments. Figure 3 ).

[0078] (2.3.2) cDNA synthesis and full-length gene amplification To obtain the antibody-encoding gene sequence, the extracted total RNA was first subjected to genomic DNA removal. Under RNase-free conditions, the RNA sample was mixed with the gDNA removal system and incubated briefly at 42°C to eliminate genomic contamination. Subsequently, it was directly added to the reverse transcription reaction system (Hiscript III qRT SuperMix), and cDNA synthesis was completed by reacting at 37°C for 15 min. The reaction was then terminated by a brief treatment at 85°C. The synthesized cDNA can be used for subsequent amplification or cryopreservation (short-term at -20°C, long-term at -70°C) to avoid repeated freeze-thaw cycles.

[0079] Using the obtained cDNA as a template, specific primers were designed to amplify the full-length heavy and light chain sequences of IgG1 and IgG2a antibodies by PCR. The total volume of the PCR reaction system was 50 μL, including high-fidelity amplification enzyme system, template cDNA, and upstream and downstream primers. The amplification program was set as follows: 98℃ pre-denaturation for 1 min; followed by 30 cycles (98℃ denaturation, 55℃ annealing, 72℃ extension), with the extension time adjusted according to the fragment length; the final extension reaction was performed at 72℃. The amplification products were detected by 1% agarose gel electrophoresis, and the target fragments were screened according to the band size. The DNA was purified using a gel extraction kit. The purified PCR products were stored at -20℃ after concentration determination for subsequent cloning experiments.

[0080] The results showed that both the heavy chain and light chain amplification products exhibited a single, clear band, indicating that the target fragment was successfully amplified with good specificity.

[0081] (2.3.3) Gene cloning and sequencing based on zero-background vectors Blunt-end ligation: PCR products are efficiently cloned using the pLB zero-background rapid cloning kit, and the insert fragment is ligated into a positively selected vector. The ligation product can be directly transformed into commonly used E. coli strains. First, the PCR product undergoes end repair treatment. Under aseptic conditions, the DNA fragment is mixed with the blunt-end ligation reaction system, and the ends are smoothed after a brief low-temperature treatment and high-temperature inactivation. Then, a linearized vector and T4 DNA ligase are added, and the ligation reaction is completed at an appropriate temperature to obtain the recombinant plasmid.

[0082] Transformation: The ligation product was transformed into competent *E. coli*. The specific procedure was as follows: the ligation product was added to competent cells and incubated on ice to allow DNA to adhere to the cell surface; subsequently, a 42°C heat shock was performed to promote DNA entry into the cells, followed by rapid recovery on ice. Preheated antibiotic-free LB medium was added, and the cells were cultured at 37°C with shaking for 45 min to induce antibiotic resistance gene expression and complete cell resuscitation. After resuscitation, the bacterial culture was evenly spread onto LB agar plates containing ampicillin and incubated upside down at 37°C for 12-16 h. Single colonies were screened and subsequent sequencing analysis confirmed the accuracy of the antibody heavy and light chain gene sequences, providing a reliable template for recombinant antibody expression.

[0083] Gene sequencing analysis: Five colonies were randomly selected from each plate, cultured in a small amount of LB liquid medium, and sent to Qingke Biotechnology Co., Ltd. for sequencing. SnapGene software was used to assemble, organize, and translate the antibody nucleotide sequences, which were then further translated into amino acid sequences for sequence alignment. The nucleotide sequence lengths of mAb 24D heavy chain and light chain were 1338 bp and 645 bp, respectively.

[0084] The light chain gene sequence of mAb 24D antibody is shown below: Heavy chain variable region (VH) amino acid sequence (SEQ ID NO.1): EVKLVESGGGLLKPGGSLTLSCAASGFTFSNYVMSWVRQTPEKRLEWVASITSSGTTYSPDSVKGRFTFSRDNVRNVLYLQMSSLRSEDTAIYYCVRVTHYYGTDYWGQGTTVTVSS.

[0085] Heavy chain variable region (VH) nucleotide sequence (SEQ ID NO.2): GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTACTGAAGCCTGGAGGGTCCCTGACACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACTATGTCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATCCATTACTAGCAGCGGTACCACCTATTCTCCAGACAGTGTGAAGGGCCGATTTACCTTCTCAAGGGATAATGTAAGGAACGTCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATTTATTACTGTGTAAGAGTCACCCATTATTACGGGACCGACTACTGGGGCCAAGGCACCACTGTCACAGTCTCCTCA。

[0086] Light chain variable region (VL) amino acid sequence (SEQ ID NO.3): QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGAKLTVL。

[0087] Light chain variable region (VL) nucleotide sequence (SEQ ID NO.4): CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCACTGGGTGTTCGGTGGAGGAGCCAAACTGACTGTCCTA。

[0088] Heavy chain constant region (CH) amino acid sequence (SEQ ID NO.5): AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG.

[0089] Heavy chain constant region (CH) nucleotide sequence (SEQ ID NO.6): GCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGG。

[0090] Light chain constant region (CL) amino acid sequence (SEQ ID NO.7): GQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTPPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS.

[0091] Light chain constant region (CL) nucleotide sequence (SEQ ID NO.8): GGCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCCACCTTCCTCTGAAGAGCTCGAGACTAACAAGGCCACACTGGTGTGTACGATCACTGATTTCTACCCAGGTGTGGTGACAGTGGACTGGAAGGTAGATGGTACCCCTGTCACTCAGGGTATG GAGACAACCCCGCCTTCCAAACAGAGCAACAACAAGTACATGGCTAGCAGCTACCTGACCCTGACAGCAAGAGCATGGGAAAGGCATAGCAGTTACAGCTGCCAGGTCACTCATGAAGGTCACACTGTGGAGAAGAGTTTGTCCCGTGCTGACTGTTCC.

[0092] Example 2: Construction and Optimization of Recombinant Triazophos Antibody Based on HEK293F Cells (1) Monoclonal antibody heavy and light chain gene sequence analysis The antibody gene sequences obtained from sequencing were imported into NCBI BLAST for homology alignment. Simultaneously, bioinformatics tools such as ClustalW, VBASE2, and DNAMAN were used to perform multi-faceted analysis of the sequences. Furthermore, IMGT / V-QUEST was used to functionally annotate the antibody variable region sequences, clarifying the origins of the V, D, and J gene fragments, and dividing the antibody structural regions, including the framework region (FR) and complementarity-determining region (CDR). These analyses provide a theoretical basis for antibody structural characterization and subsequent engineering modifications.

[0093] (2) Construction and screening of recombinant full-length antibody sequences and expression vectors (2.1) Construction of expression carrier (2.1.1) To systematically evaluate the impact of codon optimization strategies on antibody expression levels, three different forms of expression sequences were designed and constructed based on the full-length heavy and light chain sequences of mAb24D obtained through sequencing. Specifically, these included: ① the natural full-length sequence without any optimization (wild-type, WT); ② a construct with overall codon optimization of the full-length sequence (full-length optimized type); and ③ a construct with codon optimization only for the variable region (V region) while the constant region (C region) retained its original sequence (V-region optimized type).

[0094] (2.1.2) After obtaining the antibody expression sequences in three forms, the corresponding full-length heavy chain gene and full-length light chain gene were cloned into three commonly used eukaryotic expression vectors pcDNA 3.4, pTT5, and pCAGGS, respectively, to construct different expression combination systems. The effects of vector background on antibody expression levels were systematically compared. The heavy chain encoded by the full-length heavy chain gene contains the heavy chain variable region as shown in SEQ ID NO.1 and the heavy chain constant region as shown in SEQ ID NO.5. The light chain encoded by the full-length light chain gene contains the light chain variable region as shown in SEQ ID NO.3 and the light chain constant region as shown in SEQ ID NO.7. (2.1.3) After the above three types of target genes are synthesized, they are cloned into the corresponding eukaryotic expression vectors according to the vector multiple cloning site design. Both the heavy chain and light chain expression frameworks adopt conventional secretory expression structures, including signal peptide sequences to guide antibody secretion.

[0095] (2.1.4) The ligation product was digested with restriction endonucleases and sequenced to confirm the correctness and integrity of the inserted sequence. The synthesized bacterial culture containing the plasmid was streaked, and single colonies were picked and amplified. Single colonies from the plate were then inoculated into 20 mL of LB medium containing Amp and cultured at 37°C and 220 rpm for 12-16 h with shaking. Subsequently, plasmid DNA was extracted using an endotoxin-free miniprep kit, and the DNA concentration was detected by UV spectrophotometry. After the constructed heavy and light chain expression plasmids were verified by sequencing, they were co-transfected into HEK293F cells at a 1:1 mass ratio for transient expression. Polyethyleneimine (PEI)-mediated transfection was used; PEI is a cationic polymer transfection reagent. After transfection, cells were cultured at 37°C, 5% CO2, and on a shaker. Cell culture supernatant was collected after a certain time for subsequent antibody detection and quantitative analysis.

[0096] (2.2) Transient transfection Taking a 6 mL transfection system as an example: Incubate the plasmid DNA at room temperature for at least 5 minutes beforehand, and prepare 5.4 mL of cell culture in a 25 mL shaker. Complex A consists of 0.3 mL ExpiCD HEK293 Plus medium, 3 μg H plasmid, and 3 μg L plasmid; Complex B consists of 0.3 mL ExpiCD HEK293 Plus medium and 24 μL PEI 40000 (1 mg / mL).

[0097] The specific steps are as follows: (2.2.1) Add complex B to complex A and mix by vortexing or gently blowing 2-3 times; (2.2.2) Incubate the mixture at room temperature for 10-20 min, mixing once every 5 min during this period; (2.2.3) Slowly add the mixture to the prepared cells while gently shaking the cells to mix them. (2.2.4) Incubate the cells in a shaker: 37℃, 5% CO2, 120 rpm (axis distance 50 mm). (2.2.5) 24 hours after transfection (cell concentration 5-7×10⁻⁶) 6 Add 1% EmCD supplement 1 to the cells and add 3% EmCD HEK293 feed medium (purchased from Jiangsu Aimeiyi Biotechnology Co., Ltd.). (2.2.6) Five days after transfection, centrifuge at 6000 rpm for 10 min and collect the supernatant for testing.

[0098] (2.3) Establishment of monoclonal antibody sandwich ELISA method mAb sandwich ELISA is a commonly used method for quantitative detection of antibody concentration. In this experiment, the capture antibody was goat anti-mouse IgG antibody, and the detection antibody was goat anti-mouse IgG enzyme-labeled secondary antibody. A standard curve was established using IgG antibody of known concentration as the standard antibody. First, the working concentrations of the coating antibody and the detection antibody were optimized using the checkerboard method, and then the standard curve was established under optimal conditions.

[0099] The specific operating steps are as follows: (2.3.1) Goat anti-mouse IgG at a concentration of 1.84 mg / mL was diluted to 10,000, 20,000, 40,000, 80,000, and 160,000, and 100 μL / well was added to a 96-well plate. PBS was used as the negative control. The plate was coated at 37°C for 2 h and washed three times with PBST. (2.3.2) Add 200 μL of blocking solution to each well, incubate at 37°C for 1 h, and then wash the plate three times with PBST; (2.3.3) Dilute the standard antibody solution to 400 μg / L, incubate at 37°C for 30 min, and then wash the plate three times with PBST; (2.3.4) Goat anti-mouse IgG enzyme-labeled secondary antibody was diluted to 5000, 7500, 10000, and 20000, and 100 μL / well was added to a 96-well plate. After incubation at 37°C for 30 min, the plate was washed three times with PBST. (2.3.5) Prepare the colorimetric solution fresh and use immediately. Mix equal volumes of the substrate solution and add 100 μL to each well. React at room temperature in the dark for 15 min. (2.3.6) Add 50 μL / well of stop solution; (2.3.7) Read the OD value at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) instrument (dual wavelength: 620 nm is the reference filter wavelength).

[0100] Experimental results are as follows Figure 4 As shown, within the range of capture antibody dilutions of 1:160000-1:10000 and detection antibody dilutions of 1:20000-1:5000, the OD value increased from 0.56 to 3.88, showing an overall increasing trend. This indicates that antibody concentration has a significant impact on the detection signal, and the signal gradually approaches saturation at higher concentrations. The positive / negative ratio (P / N) was used as an evaluation index to screen different antibody dilution combinations. It was found that when the capture antibody dilution was 1:10000 and the detection antibody dilution was 1:20000 (40 μg / L), the detection signal tended to plateau, and the P / N value reached its maximum of 35.31, indicating that the system had the best signal-to-noise ratio and detection performance under these conditions. Considering both signal intensity and detection sensitivity, the above conditions were determined as the optimal working concentration for the sandwich ELISA and used for the subsequent establishment of the standard curve.

[0101] (2.4) Establishment of the sandwich ELISA standard curve Under the above optimized conditions, a standard curve for mouse IgG sandwich ELISA was established.

[0102] (2.4.1) Dilute goat anti-mouse IgG to the optimal concentration, add 100 μL / well to a 96-well plate, incubate at 37°C for 2 h, and wash the plate three times with PBST. (2.4.2) Add 200 μL of blocking solution per well, incubate at 37 °C for 1 h, and then wash the plate three times with PBST; (2.4.3) Add 100 μL of TAP standard solution to each well, dilute the IgG standard solution with PBS at different dilution gradients, incubate at 37°C for 30 min, and wash 3 times with PBST; (2.4.4) Dilute goat anti-mouse IgG enzyme-labeled secondary antibody to the optimal concentration, add 100 μL / well to a 96-well plate, incubate at 37°C for 30 min, and wash the plate three times with PBST. (2.4.5) Color development: The color development solution should be prepared fresh and used immediately. Mix equal volumes of the substrate solution, add 100 μL to each well, and react at room temperature in the dark for 15 min. (2.4.6) Termination: Add 50 μL of termination solution per well; (2.4.7) Measurement: Read the OD value at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) instrument (dual wavelength: 620 nm is the reference filter wavelength).

[0103] Plotting the standard antibody concentration on the x-axis, the corresponding OD 450 The values ​​are plotted on the ordinate, with three replicates for each concentration. A nonlinear standard curve was fitted using Origin software. The x-axis represents the concentration of the standard antibody detected, and the corresponding OD values ​​are plotted on the slant axis. 450 The values ​​are plotted on the ordinate, and a standard curve for this detection method is generated using Origin software. The LOD and linear range are obtained by substituting the blank value plus three times the standard deviation into the nonlinear fitting formula. The standard antibody IgG concentration and OD are then compared. 450 The linear equation of the value converts the calculated OD value into a quantitative value.

[0104] Experimental results are as follows Figure 5 As shown, the detection signal exhibits a good linear relationship with the target analyte concentration within the range of 20-200 μg / L, and the correlation coefficient (R0) of the linear regression equation is high. 2 The values ​​were all greater than 0.99, and the linear equation was Y = 0.01026X + 0.01361. Further calculations showed that the LOD of this method was 0.96 μg / L, and the linear detection range was 20-400 μg / L, indicating that the established sandwich ELISA method has good quantitative analysis capabilities.

[0105] (2.5) Detection of affinity and titer of recombinant antibody The affinity assay for the transfected recombinant antibody was performed as shown in Example 1 (2.1.2). First, the optimal antigen-antibody dilution was determined using the checkerboard method, and then a standard curve was established to detect its IC50. 50 .

[0106] The specific steps for detecting the titer of recombinant antibodies are as follows: (2.5.1) Coating: Dilute the coating agent to 0.1 μg / L with coating buffer, coat 100 μL / well of the microplate, incubate at 37°C for 2 h, pour out the liquid in the well, wash the plate three times with PBS-T, and spin dry. (2.5.2) Sealing: Add 150 μL of sealing solution to each well, incubate at 37°C for 1 h, pour out the liquid in the well, and spin dry with a spin bar. (2.5.3) Binding: Add 50 μL of PBS and 50 μL of serially diluted antibody to each well in sequence, incubate at 37°C for 30 min, pour out the liquid in the well, wash the plate three times with PBST, and spin dry with a spin plate. (2.5.4) Add enzyme label: Add 100 μL of HRP-labeled goat anti-mouse enzyme label antibody diluted 5000 times to each well, incubate at 37℃ for 30 min, pour out the liquid in the well, wash the plate three times with PBST, and spin dry with a spin plate. (2.5.5) Color development: The color development solution should be prepared fresh and used immediately. Mix equal volumes of the substrate solution, add 100 μL to each well, and react at room temperature in the dark for 15 min. (2.5.6) Termination: Add 50 μL of stop solution to each well; (2.5.7) Measurement: Read the OD value of each well at 450nm using an ELISA reader (dual wavelength: 620nm is the reference filter wavelength).

[0107] Plot the antibody dilution factor on the x-axis and the average OD value on the y-axis. 450 The values ​​are plotted on the ordinate using Origin software to create an antibody titer curve. In this study, antibody titer was defined as the supernatant dilution factor corresponding to a final OD value of 1.5 when the concentration of the coating antigen was fixed at 0.1 μg / L.

[0108] (2.7) Screening of signal peptides Since different signal peptides exhibit varying transport efficiencies in the protein secretion pathway, significantly impacting antibody expression levels, a comparative screening of signal peptide sequences was conducted to further improve the secretory expression efficiency of recombinant antibodies. Based on the identified optimal sequence-vector, three signal peptide sequences from different sources were introduced into the N-terminus of both the heavy and light chains: a signal peptide derived from the mouse immunoglobulin κ chain (mouse-IgGκ), a classic secretory signal peptide; a tissue plasminogen activator signal peptide (tPA), a commonly used high-secretion efficiency signal peptide; and the antibody's own natural signal peptide and an engineered signal peptide designed by the original company. Corresponding expression plasmids were constructed, and the signal peptides and their amino acid sequences are shown in Table 3. High-purity plasmids were obtained using a small-scale plasmid extraction method. Subsequently, the heavy and light chain plasmids were mixed in an equal mass ratio and transiently transfected into HEK293F cells using a polyethyleneimine (PEI)-mediated method. Five days after transfection, cell culture supernatant was collected, and antibody production was detected and compared using the sandwich ELISA method described in step (2.4) above, thereby evaluating the effect of different signal peptides on antibody expression levels. The affinity of recombinant antibodies secreted by different signal peptides was also measured to ensure that good affinity was maintained while maintaining high antibody production.

[0109] Table 3. Signal peptides and their amino acid sequences

[0110] (2.8) System optimization of transfection conditions In polyethyleneimine (PEI)-mediated transient transfection systems, improving transfection efficiency typically relies on the systematic optimization of key process parameters. The PEI-to-DNA mass ratio directly affects the complex particle size, charge properties, and cellular uptake efficiency; the amount of DNA affects intracellular transcriptional load and toxicity balance; and the antibody heavy-to-light chain mass ratio determines the assembly efficiency of secretory intact IgG. Therefore, these factors are considered core variables influencing antibody expression. In recent years, studies have proposed that multi-factor optimization (such as response surface methodology or orthogonal factorial experiments) can more comprehensively assess the combined impact of different parameters on expression levels. In this experiment, three main parameters were selected: PEI reagent brand (A, B, C), DNA-to-PEI mass ratio (1:3, 1:4, 1:6), and heavy-to-light chain mass ratio (1:1, 2:3, 3:2) for orthogonal factorial experiments. Other parameters, such as DNA volume, cell density at transfection, and transfection shaker conditions, were kept consistent. PEI reagent A was purchased from APExBIO, PEI reagent B from Shanghai Beyotime Biotechnology Co., Ltd., and PEI reagent C from Beijing Sizhengbai Biotechnology Co., Ltd. Specific optimized parameters are shown in Table 4. Five days after transfection, the supernatant was collected, and the transfected antibody yield and r-mAb affinity were detected using a sandwich ELISA method. A more stable and higher-yielding general condition combination was selected.

[0111] Table 4 Optimization parameters for transfection conditions

[0112] (3) Experimental results (3.1) Results of codon optimization and carrier selection To systematically evaluate the combined impact of codon optimization and expression vector on r-mAb performance, this study investigated antibody yield, IC50, and other parameters. 50 A comprehensive evaluation of efficacy was conducted to select the advantageous combination of high expression and high affinity.

[0113] (3.1.1) Triazophos recombinant antibody yield analysis Table 5 shows the r-mAb yields, with r-mAb 24D yields ranging from 19.71 to 132.47 μg / mL. Among different codon optimization formats and vector combinations, the combinations with the highest r-mAb 24D yields were: full-length wild-type sequence / vector pcDNA3.4 (132.47 μg / mL), full-length optimized sequence / vector pTT5 (85.45 μg / mL), and full-length optimized sequence / vector pcDNA3.4 (82.5 μg / mL). The first combination was significantly superior to the latter two. In summary, both the full-length wild-type sequence / vector pcDNA3.4 and the full-length optimized sequence / vector pTT5 combinations exhibited high yields in antibodies and have the potential to be used as universal plasmid combinations.

[0114] Table 5. r-mAb yield and transfection antibody affinity for different sequence formats and vectors

[0115] (3.1.2) Analysis of recombinant antibody titer Under uniform antibody concentration (2 μg / mL) and coating precursor concentration (0.1 μg / L), the titers of r-mAbs expressed in different combinations were determined (expressed as dilution factors at OD = 1.5-2.0). The results showed that the 24D titers of r-mAbs ranged from 49.8 to 103.2, with the full-length wild-type sequence / pcDNA3.4 combination exhibiting the highest titer (103.2), and the full-length optimized sequence / pTT5 combination exhibiting the lowest titer (49.8).

[0116] (3.1.3) Recombinant antibody affinity assay (3.1.3.1) Optimization of coating agent and antibody dilution The dilution of the coating antigen and antibody was optimized using a checkerboard titration method. The results are as follows: Figure 6 As shown, under the condition of TAP standard concentration of 0.2 μg / L, when the original coating dilution factor is 1:40000, the OD value of the zero standard well is in the range of 1.5-2.0 and the binding rate is the lowest. This condition is determined to be the optimal original coating concentration. Based on this, the suitable dilution range for r-mAb 24D is 1:600.

[0117] (3.1.3.2) Determination of r-mAb affinity The IC50 of r-mAb 24D was determined by icELISA under different sequence and vector combinations. 50 The concentration range was 0.03–0.29 μg / L (Table 5). The lowest IC50 values ​​were observed in both the full-length wild-type sequence / pcDNA3.4 combination. 50 The value, i.e., the highest affinity, is r-mAb24D, which is 0.03 μg / L.

[0118] (3.2) Results of signal peptide screening Based on the advantageous sequence-vector combinations obtained from the aforementioned screening, this study introduced different signal peptides for comparative screening to further improve the secretory expression efficiency of r-mAb. The same transient expression system was used, and antibody yield, titer, and IC50 were considered. 50 The indicators were comprehensively evaluated to screen for advantageous signal peptides suitable for anti-TAP r-mAb expression.

[0119] (3.2.1) Analysis of recombinant antibody yield Different signal peptides had varying effects on the expression yield of r-mAb 24D, indicating that the type of signal peptide significantly influenced antibody secretion levels. Specifically, the mouse IgGκ signal peptide showed the highest yield in r-mAb 24D at 146.56 μg / mL, demonstrating good secretion-promoting ability; followed by the antibody's native signal peptide and engineered signal peptide, with yields of 99.20 μg / mL and 79.83 μg / mL, respectively. tPA signal peptides did not show any effective secretion-promoting effect.

[0120] (3.2.2) Affinity of recombinant antibody (3.2.2.1) Optimization of coating agent and antibody dilution Test results showed that, with a TAP standard concentration of 0.2 μg / L, and a coating dilution factor of 1:40000, the OD value of the zero-scale well was in the range of 1.5-2.0, and the binding rate was lowest. This condition was determined to be the optimal coating working concentration. Based on this, the suitable dilution range for r-mAb 24D is 1:600.

[0121] (3.2.2.2) Affinity determination of r-mAb The affinity of r-mAb 24D after transfection with different signal peptides was tested, and the results showed that the IC50 of r-mAb 24D was [missing information]. 50 The concentrations ranged from 0.04 to 0.11 μg / L, with mouse IgGκ signal peptide at 0.04 μg / L, native antibody signal peptide at 0.04 μg / L, engineered signal peptide at 0.11 μg / L, and tPA signal peptide at 0 μg / L. Overall, the IC50 concentrations under each signal peptide condition were [not specified in the original text]. 50 The fluctuation range was small, and no clear effect of different signal peptides on affinity was observed. In summary, the type of signal peptide has no significant effect on antibody affinity; its main function is to regulate the secretion and expression efficiency of antibodies, while its effect on antigen-antibody binding ability is relatively limited.

[0122] (3.3) Results of Optimization of Transfection Conditions After completing the expression construction and signal peptide optimization, in order to further improve the stability and yield of the r-mAb transient expression system, this study systematically optimized the transfection conditions to determine an efficient transfection protocol suitable for anti-TAP r-mAb expression.

[0123] (3.3.1) Optimization of PEI transfection reagent The transfection yield of r-mAb24D was compared using three transfection reagents (A, B, and C) at different heavy / light chain mass ratios and DNA / PEI mass ratios. The results showed that different transfection reagents had a significant impact on antibody expression efficiency.

[0124] In r-mAb 24D, reagent A yielded 56-297 μg / mL with an average yield of 138.1 μg / mL; reagent B yielded 6.17-174.8 μg / mL with an average yield of 64.2 μg / mL; and reagent C yielded 134-354 μg / mL with an average yield of 247.1 μg / mL. Reagent C consistently achieved the highest expression levels. Based on average yield, the expression levels of reagent C in the 24D system were 1.79 times and 3.85 times higher than those of reagents A and B, respectively. These results indicate that reagent C exhibits superior transfection efficiency and expression capacity in various antibody systems.

[0125] (3.3.2) Optimization of the heavy-light chain mass ratio For r-mAb 24D antibody, the yield ranged from 7.98 to 354 μg / mL with an average yield of 161.53 μg / mL at a mass ratio of 1:1; the yield ranged from 15.6 to 297 μg / mL with an average yield of 176.18 μg / mL at a mass ratio of 2:3; and the yield ranged from 6.17 to 225 μg / mL with an average yield of 110.95 μg / mL at a mass ratio of 3:2.

[0126] In summary, the highest average yield was obtained when the heavy chain to light chain mass ratio was 2:3. Calculated on average, the expression level of 24D under this condition was 1.1 times and 1.6 times that under the 1:1 and 3:2 conditions, respectively; the results indicate that appropriately increasing the light chain ratio (2:3) is beneficial to improving antibody expression efficiency.

[0127] (3.3.3) Optimization of DNA to polyethyleneimine (PEI) mass ratio For r-mAb 24D antibody, the yield ranged from 6.17 to 238 μg / mL with an average yield of 106.41 μg / mL at a mass ratio of 1:3; the yield ranged from 20.4 to 290 μg / mL with an average yield of 132.07 μg / mL at a mass ratio of 1:4; and the yield ranged from 110 to 354 μg / mL with an average yield of 210.18 μg / mL at a mass ratio of 1:6.

[0128] In summary, the r-mAb 24D antibody achieved the highest average yield at a PEI:DNA mass ratio of 1:6. On average, the expression level of r-mAb 24D antibody at the 1:6 ratio was 1.59 times and 1.98 times higher than that at the 1:4 and 1:3 ratios, respectively. These results indicate that increasing the amount of polyethyleneimine (PEI) within a certain range can enhance transfection efficiency, thereby increasing antibody expression levels.

[0129] (3.3.4) High-yield combined antibody affinity Based on the optimization of the above transfection conditions, the four r-mAbs with the highest yields from r-mAb 24D were selected for subsequent performance evaluation. The working concentration combination of antigen and antibody was further optimized using the icELISA method. With a TAP standard concentration of 0.1 μg / L, and ensuring the OD value of the zero-label well was within the range of 1.5-2.0, the lowest binding rate was used as the screening index to optimize the original coating concentration. The results showed that the system had the lowest binding rate at a coating concentration of 1:40000, indicating that the antigen-antibody binding was in a relatively optimal equilibrium under this condition, thus determining it as the optimal working concentration of the coating. Under this condition, the optimal dilution range for the two antibodies was further determined: 1:800 for r-mAb 24D, showing that the r-mAb 24D antibody could obtain an ideal detection signal at a lower working concentration, indicating its strong effective binding ability. IC50 50 The concentration ranged from 0.04 to 0.08 μg / L, with minimal overall fluctuations. No clear effect of different transfection conditions on antibody affinity was observed.

[0130] Example 3: Establishment of an immunoassay method for triazophos residues in food (3.1) Solution system 2% skim milk powder: 10g skim milk powder and 0.49L Diswater.

[0131] 5% skim milk powder: 10g skim milk powder and 0.19L Diswater.

[0132] (3.2) Optimization of the icELISA method (3.2.1) Optimization of coating agent and antibody dilution The optimal concentrations of the antigen and antibody were determined using a checkerboard method. The competitive standard concentration in the optimization procedure was 0.1 μg / L. The antigen was diluted in two-fold increments from the lowest dilution of 1:2500 to the highest dilution of 1:320000, with eight dilutions used for coating. Simultaneously, mAb 24D (monoclonal antibody) and r-mAb 24D (recombinant monoclonal antibody) were diluted in two-fold increments from the lowest dilution of 1:2500 to the highest dilution of 1:320000, with the eight antibody dilutions and eight antigen dilutions forming a checkerboard pattern. The optimal antigen-to-antigen and antibody concentration combination was determined when B0 was between 1.5 and 2.0, and the B / B0 ratio was minimized.

[0133] The test results showed that the optimal dilution for the coating antigen (1.81 mg / mL) was 1:40000. The optimal dilution for mAb24D (2.40 mg / mL) was 1:40000; and the optimal dilution for r-mAb 24D (260 μg / mL) was 1:4000.

[0134] As shown in Table 6, under the above conditions, the B0 value remained stable between 1.5 and 2.0, and the B / B0 value was 38.6-45.7%. Considering both signal intensity and binding rate, the pairing concentrations of the coating antigen and each antibody were determined to be the optimal working concentrations for the TAP icELISA method.

[0135] Table 6 Selection of Optimal Antigen-Antibody Concentrations

[0136] Table Notes: The antigen concentration was 1.81 mg / mL, which was diluted 40,000 times to a concentration of 45.25 μg / L. The mAb 24D concentration was 2.40 mg / mL, which was diluted 40,000 times to a concentration of 60 μg / L. The r-mAb 24D concentration was 260 μg / mL, which was diluted 4,000 times to a concentration of 65 μg / L. The mAb standard concentration was 0.2 μg / L, and the r-mAb standard concentration was 0.1 μg / L.

[0137] (3.2.2) Optimization under multiple conditions (3.2.2.1) Coating solution screening: CB buffer and PBS buffer were compared, and the results were obtained by analyzing B0 and IC50. 50 The changes determine the optimal encapsulation system.

[0138] (3.2.2.2) Coating conditions optimization: The reaction effects under the three conditions were compared: incubation at 4℃ for 16h, 25℃ for 2h, and 37℃ for 2h.

[0139] (3.2.2.3) Screening of blocking liquid types: Three blocking liquids were selected, of which blocking liquid #1 was a BSA system, and blocking liquids II and III were 2% skim milk powder and 5% skim milk powder, respectively. Their effects on background signal and affinity were compared for screening.

[0140] (3.2.2.4) Optimization of sealing time: Compare the effects of sealing at 37℃ for 1 hour, sealing at 37℃ for 2 hours, and sealing at 4℃ for 16 hours on detection performance.

[0141] (3.2.2.5) Optimization of competitive reaction time: Three competitive reaction times were set at 37℃ for 20 min, 30 min, and 40 min. The B0 and IC values ​​were analyzed. 50 The change determines the optimal reaction time.

[0142] (3.2.2.6) Optimization of secondary antibody dilution buffer: Two different antibody dilution buffer systems were selected (1# enzyme dilution buffer formula: Na2HPO4·12H2O 5g, NaH2PO4·2H2O 0.9g, glycerol 50mL, Proclin 1mL, fetal bovine serum 1mL and Diswater 750mL; 6# enzyme dilution buffer formula: Na2HPO4·12H2O 5g, NaH2PO4·2H2O 0.9g, NaCl 8.5g, glycerol 50mL, BSA 15g and Diswater 750mL) and their effects on the detection signal were compared.

[0143] (3.2.2.7) Screening of working concentration of secondary antibody: The enzyme-labeled secondary antibody was diluted to 1:1000, 1:3000 and 1:5000 respectively, and the affinity and background signal were compared by comprehensive comparison.

[0144] The above optimizations were all achieved through comprehensive analysis of B0 and IC. 50 and B0 / IC 50 The optimal encapsulation system is determined by the changes.

[0145] The optimization results are shown in Table 7. Table 7 Summary of Antibody Condition Optimization Results

[0146] (3.2.3) Establishment of the standard curve The detection was performed under the above-mentioned optimal conditions, and the method was the same as (2.1.2) in Example 1.

[0147] (3.3) Comparison between traditional monoclonal antibodies and recombinant antibodies (3.3.1) Determination of affinity The detection method is as described in Example 1 (2.1.2).

[0148] Test Results: Standard curves for mAb 24D and r-mAb 24D were established at the optimal antigen-antibody dilutions and based on the optimal conditions summarized in Table 7. The IC50 values ​​of the antibodies were... 50 The concentrations were 0.16 μg / L and 0.06 μg / L, respectively. 2 All values ​​are greater than 0.99, indicating good curve fitting correlation. Overall, the IC50 of r-mAb is... 50 It has a higher affinity than mAb.

[0149] (3.3.2) Specificity determination Specificity is one of the main indicators for evaluating monoclonal antibodies. Antibody specificity is determined by measuring cross-reactivity. Under the optimal conditions determined above, the specificity of the antibody using the icELISA method was investigated. TAP and some structural analogs of TAP, such as chlorpyrifos, methyl parathion, fenthion, fenitrothion, and methamidophos, were selected for assay. Standard curves were plotted and the IC50 values ​​of each antibody in different standards were calculated. 50 Using the TAP standard test results as a reference, the cross-reactivity rate was calculated using the following formula: Cross-reactivity rate (%) = IC 50 (TAP) / IC 50 (Competitors) × 100%; The test results are shown in Table 8. The IC of mAb 24D... 50 IC50 of 0.06 μg / L r-mAb 24D 50 The concentration was 0.06 μg / L, and the CR values ​​against chlorpyrifos, methyl parathion, fenthion, fenitrothion, and methamidophos were all below 0.02, indicating negligible cross-reactivity. This demonstrates that both mAb 24D and r-mAb 24D exhibit good specificity.

[0150] Table 8 Cross-reactivity results

[0151] (3.3.3) Determination of potency The detection method is as described in Example 1 (2.1.2).

[0152] Test results: When the coating agent was diluted to 0.1 mg / mL and the antibody was uniformly diluted to 2 μg / L, the titers of the two antibodies were monitored, and there was no significant difference in the titers of mAb and the corresponding r-mAb.

[0153] (3.3.4) Stability determination Antibody stability was systematically evaluated from three aspects: pH tolerance, ionic strength tolerance, and organic solvent tolerance.

[0154] (3.3.4.1) Determination of salt ion tolerance concentration range: Antibody dilution solutions with different NaCl concentrations were prepared to achieve final concentrations of 0 mM, 72.5 mM, 145 mM, 290 mM, 580 mM, 1160 mM, 2320 mM, and 5000 mM. Under each condition, the antibody was adjusted to the same working concentration, and detection was performed according to the standard icELISA procedure. The antibody's tolerance to changes in ionic strength was assessed by signal changes.

[0155] Test results show that mAb 24D maintains good performance in the range of 72.5-1160mM, while r-mAb 24D shows large fluctuations in detection signal under different salt ion concentrations and does not exhibit a stable tolerance range.

[0156] (3.3.4.2) pH tolerance range determination: The antibody was diluted to its optimal working concentration by adjusting the pH of the antibody dilution buffer (3.0-11.0), including 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0, before icELISA detection. The changes in OD values ​​under each condition were recorded, and inhibition curves were plotted to assess the stability of the antibody in different acidic and alkaline environments.

[0157] Test results show that mAb 24D is relatively stable in the pH range of 5-10, and its BO / IC ratio is [not specified]. 50 The ratio was highest at pH 8; r-mAb 24D tolerated pH 5-11, reaching its optimum at pH 9. Both showed relatively weak tolerance to acidic environments, while exhibiting better stability under alkaline conditions, indicating strong adaptability to alkaline environments.

[0158] (3.3.4.3) Methanol tolerance range determination: The methanol content in the antibody diluent was adjusted to 0%, 1.25%, 2.5%, 5%, 10%, 20%, 40%, and 60% to detect the antibody under the same antibody concentration conditions. The changes in OD value were recorded and inhibition curves were constructed to analyze the stability of the antibody under different organic solvent concentration conditions.

[0159] Test results show that mAb 24D and r-mAb 24D have similar OD values ​​and B0 / IC ratios within the methanol concentration range of ≤10%. 50 The ratio change was small, demonstrating good stability. When the methanol concentration exceeded 10%, the antibody's IC50 value... 50 Both values ​​were significantly elevated, indicating a marked decrease in antibody recognition ability; under 20% methanol conditions, the antibodies essentially lost their effective ability to recognize the antigen. Comprehensive analysis showed that the two antibodies had essentially the same tolerance range to methanol, both able to tolerate 10% methanol solution.

[0160] (3.3.4.4) Acetonitrile tolerance concentration range determination: The final concentration gradient of acetonitrile was set using a similar method: 0%, 0.625%, 1.25%, 2.5%, 5%, 10%, 20%, 40%. After diluting the antibody to the working concentration, icELISA detection was performed. The antibody's tolerance to acetonitrile was evaluated by comparing the changes in detection signals under different conditions.

[0161] Test results show that mAb 24D has a stable range of 0%-1.25%. In contrast, r-mAb 24D exhibits greater signal fluctuations under acetonitrile conditions, with lower OD values ​​and lower B0 / IC values. 50 The ratio changes all exceeded 2-fold, failing to demonstrate a clear stable tolerance range. Comprehensive analysis indicates that mAb 24D exhibits better overall adaptability to acetonitrile environments than its corresponding r-mAb 24D, suggesting differences in the response of different antibody expression forms to organic solvents.

[0162] (3.4) Addition and recovery test of actual samples (3.4.1) Sample pretreatment Water samples (paddy field water) were collected from paddy fields in Yazhou Bay Town, Sanya City, Hainan Province. After collection, the water samples were filtered through a membrane and stored. Cowpea and chicken samples were purchased from a supermarket in Yazhou Bay, Sanya City, Hainan Province. After washing and drying, the samples were thoroughly pulverized using a high-speed blender, aliquoted, and stored at -20℃. 10g of blank sample was weighed into a 50mL centrifuge tube, 30mL of PBS buffer containing 2% methanol was added, vortexed for 5 minutes, sonicated for 5 minutes, and centrifuged at 4000rpm for 5 minutes. The supernatant was diluted at different ratios (water sample directly diluted, paddy field water diluted 10 times, cowpea sample diluted 50 times, and chicken sample diluted 100 times) to prepare standard solutions of different concentrations of the TAP to be tested. By establishing a standard curve of the matrix at different dilution ratios, the minimum dilution ratio with the least impact on the detection system was selected to eliminate the influence of the matrix on the detection method.

[0163] (3.4.2) Establishment of curves in blank matrix Following the method described in Example 3 (3.4.1), various samples were pretreated, and standard curves were established in the matrices of chicken, cowpea, and paddy field water samples. The fit between the standard curves at different dilution factors and the standard curves in PBS solution was compared to determine the optimal dilution factor. All parameters of the standard curves in the actual sample matrices needed to be converted to true values ​​according to the dilution factor (true value = detected value × dilution factor). The test results showed that the blank matrix curves of each sample could fit the standard curves in PBS.

[0164] (3.4.3) Determination of detection limit After pretreatment of various samples according to the method in Example 3 (3.4.1), they were detected by icELISA. The LOD value was obtained by calculating the average value + 3SD. Considering the affinity index, the experimental antibody for the samples was r-mAb 24D.

[0165] The test results are shown in Table 9. The LOD values ​​of TAP in the samples of paddy field water, cowpea, and chicken were 0.16 μg / L, 0.28 μg / kg, and 1.15 μg / kg, respectively.

[0166] (3.4.4) Determination of accuracy and precision To evaluate the accuracy and stability of the method, recovery rate and coefficient of variation were added as the main indicators. Three blank samples were selected, and the results were analyzed from the IC50 data. 20 -IC 80 Three spiking levels (low, medium, and high) were set for the value range. After icELISA detection, the results were statistically analyzed, and the recovery rate was calculated (spiking recovery rate = [(actual detected concentration / spiking concentration) × 100%]).

[0167] The test results are shown in Table 9. The linear ranges were 0.32-3.62 μg / L, 0.66-13.25 μg / kg, and 2.40-29.28 μg / kg, respectively. The recoveries in paddy field water were 90.2-114.6%, with a CV of less than 13.8%; the recoveries in cowpeas were 85.8-114.4%, with a CV of less than 18.6%; and the recoveries in chicken were 80.0-111.3%, with a CV of less than 17.9%. These results indicate that the icELISA method established in this study for detecting TAP in paddy field water, cowpeas, and chicken samples meets the requirements of residue detection methods and has high accuracy and precision.

[0168] Table 9. Results of Triazophos Addition and Recovery

[0169] Note: Rice paddy water is measured in μg / L, while cowpea and chicken meat are measured in μg / kg.

[0170] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant antibody against triazophos, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-triazolium recombinant antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

3.

2. The anti-triazolphos recombinant antibody according to claim 1, characterized in that, The amino acid sequences of the heavy chain variable region and the light chain variable region are derived from accession number GDMCC NO: 68914, and the taxonomic name is [missing information]. Murine hybridoma cell line Hybridoma cells; deposited at Guangdong Provincial Center for Microbial Culture Collection, deposit date: August 28, 2026.

3. A method for preparing the anti-triazolphos recombinant antibody according to claim 1, characterized in that, Includes the following steps: (1) From the accession number GDMCC NO: 68914, the taxonomic name is Murine hybridoma cell line Total RNA was extracted from hybridoma cells and cDNA was obtained by reverse transcription. (2) Using the cDNA obtained in step (1) as a template, amplify the full-length heavy chain gene and the full-length light chain gene; the heavy chain encoded by the full-length heavy chain gene contains the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.1 and the heavy chain constant region with an amino acid sequence as shown in SEQ ID NO.5, and the light chain encoded by the full-length light chain gene contains the light chain variable region with an amino acid sequence as shown in SEQ ID NO.3 and the light chain constant region with an amino acid sequence as shown in SEQ ID NO.7; (3) The full-length heavy chain gene and the full-length light chain gene obtained in step (2) are cloned into a eukaryotic expression vector to construct a recombinant plasmid; (4) Transfect the recombinant plasmid obtained in step (3) into host cells and culture them. Collect the culture supernatant to obtain the anti-triazolium recombinant antibody.

4. The method according to claim 3, characterized in that, The eukaryotic expression vector is selected from any one of pcDNA3.4, pCAGGS, or pTT5.

5. The method according to claim 3, characterized in that, The host cell was HEK293F cell.

6. The method according to claim 3, characterized in that, The heavy chain has a first signal peptide at its N-terminus, and the light chain has a second signal peptide at its N-terminus; the first signal peptide and the second signal peptide are each independently selected from: the mouse IgGκ signal peptide shown in SEQ ID NO.9, the tPA signal peptide shown in SEQ ID NO.10, the natural signal peptide shown in SEQ ID NO.11, the natural signal peptide shown in SEQ ID NO.12, or the engineered signal peptide shown in SEQ ID NO.

13.

7. The method according to claim 3, characterized in that, In step (4), the mass ratio of DNA to polyethyleneimine in the transfection is selected from any one of 1:3, 1:4 or 1:

6.

8. The method according to claim 3, characterized in that, In step (4), the mass ratio of heavy to light chains in the transfection is selected from any one of 1:1, 2:3 or 3:

2.

9. The use of the anti-triazophos recombinant antibody according to claim 1 in detecting triazophos residues in samples.

10. The application according to claim 9, characterized in that, The samples were selected from any one or more of paddy field water, chicken meat, and cowpeas.

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