Monoclonal antibody hybridoma cell strain of forchlorfenuron, monoclonal antibody, product, colloidal gold test strip and application

Abstract

The application belongs to the technical field of animal genetic engineering, and particularly relates to a forchlorfenuron monoclonal antibody hybridoma cell strain, a monoclonal antibody, a product, an enzyme-linked immunoassay kit, a colloidal gold test strip and application 。 The application provides a forchlorfenuron monoclonal antibody hybridoma cell strain; the preservation number of the hybridoma cell strain LBN-9G9 is CGMCC NO. 45170. The forchlorfenuron monoclonal antibody is prepared by using a hybridoma cell strain capable of secreting the forchlorfenuron monoclonal antibody; the forchlorfenuron monoclonal antibody can realize high-sensitivity detection of forchlorfenuron; the obtained forchlorfenuron monoclonal antibody cell strain can be used for preparing an enzyme-linked immunoassay kit, a colloidal gold immunochromatography test strip and detecting forchlorfenuron residues in food, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of animal genetic engineering technology, specifically relating to chlorpyrifos monoclonal antibody hybridoma cell lines, monoclonal antibodies, products, colloidal gold test strips and their applications. Background Technology

[0002] Forchlorfenuron (CPPU), also known as 1-(2-chloro-4-pyridyl)-3-phenylurea, is a highly active cytokinin-like plant growth regulator. Pre-harvest, it induces callus growth, accelerates cell mitosis, promotes cell enlargement and differentiation, promotes bud development, prevents flower and fruit drop, promotes fruit enlargement, and increases yield. Post-harvest, it significantly controls leaf yellowing and post-harvest diseases in various fruits and vegetables, and aids in post-harvest preservation. Therefore, it is widely used in the agricultural production of navel oranges, citrus fruits, kiwifruit, grapes, watermelons, cucumbers, and other fruits and vegetables.

[0003] CPPU is registered for use in crops such as kiwifruit, melons, grapes, watermelons, and cucumbers. In practice, the pursuit of large fruit size and high yield often leads to high-dose, high-concentration spraying, resulting in shortened shelf life and reduced nutrient and flavor compounds in these fruits. Furthermore, CPPU residues in fruits and other foods may have potential effects on humans and other organisms in the environment. For example, the U.S. Environmental Protection Agency has pointed out that long-term exposure to CPPU in humans can lead to protein metabolism disorders and emaciation, and high doses of CPPU may induce delayed puberty initiation in female rats.

[0004] Currently, the main methods for detecting chlorpyrifos are instrumental methods, including gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), Raman spectroscopy, and ion mobility spectrometry (IMP). Instrumental methods offer high sensitivity and accuracy, but require expensive equipment and complex pretreatment processes, making them unsuitable for rapid on-site detection. Furthermore, due to their simple pretreatment and high throughput, antigen-antibody binding-based immunoassays are widely used as a supplement to instrumental methods for chlorpyrifos detection. Immunoassays are rapid, simple, sensitive, and low-cost, making them suitable for large-volume on-site sample testing. Colloidal gold immunochromatography, in particular, offers significant advantages in on-site detection. The basis of using colloidal gold immunochromatography for chlorpyrifos detection is the specific recognition of chlorpyrifos antigen and antibody, resulting in monoclonal antibodies with high specificity and sensitivity to chlorpyrifos. However, the detection sensitivity of existing chlorpyrifos monoclonal antibodies is currently low. Summary of the Invention

[0005] This invention provides hybridoma cell lines, monoclonal antibodies and their preparation methods, colloidal gold test strips and kits, and the chlorpyrifos monoclonal antibody prepared using the chlorpyrifos hapten of this invention has high detection sensitivity.

[0006] To address the above problems, the present invention provides the following technical solution:

[0007] This invention provides a chlorpyrifos monoclonal antibody hybridoma cell line, the hybridoma cell line LBN-9G9, with the accession number CGMCC NO.45170.

[0008] This invention provides a chlorpyrifos monoclonal antibody, which is secreted by the hybridoma cell line or its passaged cell line described in the above technical solution.

[0009] Preferably, the IC50 of the chlorpyrifos monoclonal antibody is... 50 The values ​​ranged from 0.19 to 0.22 ng / mL.

[0010] This invention provides a product for detecting chlorpyrifos, the product comprising the chlorpyrifos monoclonal antibody described in the above technical solution; the product includes reagents, test strips, or kits.

[0011] This invention provides a colloidal gold immunochromatographic test strip for detecting chlorpyrifos, comprising a gold-labeled pad, a nitrocellulose membrane, and absorbent paper; the gold-labeled pad is coated with a colloidal gold-labeled chlorpyrifos monoclonal antibody as described in claim 2; the nitrocellulose membrane comprises a C-line and a T-line in sequence; the C-line is coated with goat anti-mouse IgG; and the T-line is coated with chlorpyrifos-OVA coating agent.

[0012] Preferably, the amount of the colloidal gold-labeled antichlorpyrifos monoclonal antibody used is 30–60 μg.

[0013] Preferably, the concentration of the chlorpyrifos-OVA coating agent on the T-line is 3.5–4.5 mg / mL.

[0014] Preferably, the concentration of goat anti-mouse IgG coated on the C line is 0.18–0.22 mg / mL.

[0015] This invention provides the application of the chlorpyrifos monoclonal antibody hybridoma cell line, the chlorpyrifos monoclonal antibody, the product, the colloidal gold test strip, or the kit described in the above-mentioned technical solutions in the detection of chlorpyrifos.

[0016] The beneficial effects of this invention are as follows: This invention provides a chlorpyrifos monoclonal antibody hybridoma cell line, LBN-9G9, with the accession number CGMCC NO.45170. The chlorpyrifos monoclonal antibody secreted by this hybridoma cell line exhibits high specificity. Using the chlorpyrifos monoclonal antibody of this invention, highly sensitive detection of chlorpyrifos can be achieved, with a detection IC50 value of [missing information]. 50The value was 0.19 ng / mL. The obtained chlorpyrifos monoclonal antibody cell line can be used to prepare colloidal gold immunochromatographic test strips for the detection of chlorpyrifos residues in food, which has good application prospects.

[0017] Biological Preservation Instructions

[0018] The hybridoma cell line LBN-9G9 was deposited on August 1, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNo.45170. The address of the depository is No.3, No.1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1 The MALDI-TOF-MS spectrum of the BSA standard in Example 1 is shown below.

[0021] Figure 2 The MALDI-TOF-MS spectrum of the complete antigen obtained by conjugating chlorpyrifos hapten and BSA in Example 1 is shown.

[0022] Figure 3 The image shows the MADLI-TOF-MS spectrum of the OVA standard from Example 1.

[0023] Figure 4 The MALDI-TOF-MS spectrum of the complete antigen obtained by conjugating chlorpyrifos hapten and OVA in Example 1 is shown.

[0024] Figure 5 These are monoclonal antibody cells secreted by the hybridoma cell line LBN-9G9 of Example 3;

[0025] Figure 6 These are monoclonal antibody cells secreted by the hybridoma cell line LBN-9A10 of Example 3;

[0026] Figure 7 These are monoclonal antibody cells secreted by the hybridoma cell line LBN-1B6 of Example 3;

[0027] Figure 8 The standard curve for the monoclonal antibody secreted by the hybridoma cell line LBN-9G9 in Example 3;

[0028] Figure 9 This is the standard curve for detecting chlorpyrifos in cucumber samples using the chlorpyrifos enzyme-linked immunosorbent assay kit in Example 4;

[0029] Figure 10The results of pH optimization for antibody-labeled colloidal gold in Example 4;

[0030] Figure 11 This is a chromatogram of the optimized combination of different concentrations of coating antigen and secondary antibody in Example 4;

[0031] Figure 12 The standard curve (solvent standard) for detecting chlorpyrifos using the chlorpyrifos test strip in Example 4 is shown.

[0032] Figure 13 The standard curve for detecting chlorpyrifos in cucumber samples using colloidal gold immunochromatographic test strips in Example 4 is shown.

[0033] Figure 14 The image shows the colorimetric pattern of the colloidal gold immunochromatographic test strip used in Example 4 to detect the standard of chlorpyrifos containing cucumber matrix;

[0034] Figure 15 This is a colorimetric image of the spiked recovery of cucumber detected by the colloidal gold test strip in Example 4. Detailed Implementation

[0035] This invention provides a chlorpyrifos monoclonal antibody hybridoma cell line, including one or more of hybridoma cell lines LBN-9G9, 1B6, and 9A10; the hybridoma cell line LBN-9G9 has the accession number CGMCC NO.45170; the hybridoma cell lines 1B6 and 9A10 are stored in the laboratory.

[0036] In this invention, the method for preparing the chlorpyrifos monoclonal antibody hybridoma cell line that produces chlorpyrifos monoclonal antibodies preferably includes the following steps:

[0037] After obtaining the complete chlorpyrifos antigen, mice were sequentially immunized with the first, second, third, and fourth immunizations, and then subjected to a sprint immunization to obtain immunized mice. Spleen cells from the immunized mice were extracted and fused with myeloma cells to obtain fused cells. The fused cells were then screened for hybridomas.

[0038] This invention first prepares the chlorpyrifos complete antigen. In this invention, the chlorpyrifos complete antigen is obtained by coupling the chlorpyrifos hapten with a carrier protein. The structure of the chlorpyrifos complete antigen when the carrier protein is BSA is shown in Formula I-1; the structure of the chlorpyrifos complete antigen when the carrier protein is OVA is shown in Formula I-2.

[0039]

[0040]

[0041] The structure of the chlorpyrifos hapten described in this invention is shown in Formula II. The preparation method of the chlorpyrifos hapten described in this invention is described in (Xiao Shimei, Yan Aiping, Xiao Fang, Wan Yiqun, Guo Lan. Synthesis and identification of chlorpyrifos artificial antigen [J]. Food Science, 2016, 37(21):183-188. Xiao Shimei. Construction of gold nanoparticle colorimetric and immunoassay methods for detecting pesticide residues in the environment and food [D]. Nanchang University, 2017.).

[0042]

[0043] After obtaining the chlorpyrifos hapten, the present invention preferably uses the active ester method to activate the carboxyl group of the chlorpyrifos hapten with the structure shown in Formula II. The present invention preferably involves mixing the chlorpyrifos hapten with the structure shown in Formula II, NHS, EDC, and DMF for activation to obtain an activated chlorpyrifos hapten solution. The preferred mass of the chlorpyrifos hapten is 27.6 mg, the preferred mass of NHS is 18.2 mg, the preferred mass of EDC is 30.3 mg, and the preferred volume of DMF is 1.5 mL. The preferred activation temperature is 4°C, the preferred activation time is 12 h, and the activation is preferably carried out under magnetic stirring.

[0044] After obtaining the activated chlorpyrifos hapten solution, the present invention preferably centrifuges the activated chlorpyrifos hapten solution to obtain the chlorpyrifos hapten supernatant, which is used for coupling with the carrier protein to obtain the complete chlorpyrifos antigen. The centrifugation speed is preferably 6000 rpm, and the centrifugation time is preferably 5 min.

[0045] In this invention, the buffer solution for the carrier protein is preferably a phosphate-buffered saline (PBS) buffer for the carrier protein. In this invention, the concentration of the carrier protein in the buffer solution is preferably 0.005–0.015 M, more preferably 0.01 M. In this invention, the pH value of the PBS buffer is preferably 7.2–7.8, more preferably 7.4.

[0046] In this invention, the conjugation of chlorpyrifos hapten with a carrier protein yields the complete chlorpyrifos antigen. The preferred molar ratio of chlorpyrifos hapten to carrier protein during conjugation is (30-60):1, more preferably (50-60):1. When the carrier protein is BSA, the actual conjugation ratio of chlorpyrifos hapten to BSA is preferably between 10:1 and 25:1, more preferably 19.70:1, and the preferred molar ratio is 50:1. When the carrier protein is OVA, the preferred conjugation ratio of chlorpyrifos hapten to OVA is preferably between 1:1 and 10:1, more preferably 3.45:1, and the preferred molar ratio is 60:1. The preferred reaction temperature for the conjugation reaction is 2-6°C, more preferably 4°C; the preferred reaction time is 4-8 hours, more preferably 4 hours; and the preferred conjugation reaction is carried out under stirring conditions. The coupling ratio mentioned in this invention refers to the ratio of the hapten to the carrier protein. The calculation formula is coupling ratio = (Mp - Mstd) / Mh, where "Mp" represents the molecular weight of the chlorpyrifos antigen conjugate, "Mstd" represents the molecular weight of the BSA / OVA standard, and "Mh" represents the molecular weight of the chlorpyrifos hapten.

[0047] After coupling, the present invention preferably further includes dialysis purification of the reaction solution of the obtained chlorpyrifos hapten and carrier protein to obtain the complete chlorpyrifos antigen. In the present invention, the dialysis buffer used for dialysis purification is preferably PBS buffer; the pH value of the PBS buffer is preferably 7.2-7.6, more preferably 7.4. In the present invention, the molecular weight cutoff of the dialysis membrane used for dialysis purification is preferably 3000-4000, more preferably 3500. In the present invention, the number of dialysis purification cycles is preferably 2-6 times, more preferably 6 times.

[0048] In this invention, the chlorpyrifos complete antigen is preferably dispensed and flash-frozen in liquid nitrogen at -30 to -15°C, more preferably at -20°C.

[0049] This invention provides a method for preparing the chlorpyrifos monoclonal antibody hybridoma cell line described in the above technical solution, comprising the following steps: sequentially immunizing mice with chlorpyrifos complete antigen as an immunogen, resuscitating and expanding myeloma cells, preparing feeder cells, preparing spleen cells, cell fusion and screening, to obtain the chlorpyrifos monoclonal antibody hybridoma cell line.

[0050] After obtaining the complete chlorpyrifos antigen, the present invention uses the complete chlorpyrifos antigen as an immunogen to immunize mice, more preferably by sequentially performing a first immunization, a second immunization, a third immunization, a fourth immunization, and a sprint immunization on the mice to obtain immunized mice.

[0051] The female mice used in this invention are preferably 7-week-old purebred BALB / c healthy female mice. The immunization process preferably includes a first immunization, a second immunization, a third immunization, a fourth immunization, and a sprint immunization. The first immunization is preferably an emulsion of equal volumes of chlorpyrifos-BSA complete antigen and Freund's complete adjuvant. The second, third, and fourth immunizations are preferably an emulsion of equal volumes of chlorpyrifos-BSA complete antigen and Freund's incomplete adjuvant, injected intraperitoneally and dorsally into the mice. The preferred immunization dose is 100 μg / mouse of chlorpyrifos-BSA complete antigen. The preferred temperature for the first emulsification is 2–8°C, more preferably 4°C. The preferred emulsification time is 4–8 hours, more preferably 4 hours. The sprint immunization is preferably performed three to five days before the fusion of mouse spleen cells and mouse myeloma cells. During the sprint immunization, chlorpyrifos-BSA complete antigen is injected directly without adjuvant. The preferred immunization dose of chlorpyrifos-BSA complete antigen during the sprint immunization is 50 μg / mouse.

[0052] In this invention, the time interval between the first immunization, the second immunization, the third immunization, and the fourth immunization is preferably 14 days, and the time interval between the sprint immunization and the fusion is 3 to 5 days.

[0053] After obtaining immunized mice, the present invention isolates spleen cells from the immunized mice. The spleen cells from the immunized mice are extracted and fused with myeloma cells to obtain fused cells. Preferably, the myeloma cells are resuscitated and expanded in culture one day before cell fusion, and the myeloma cells are preferably SP2 / 0 myeloma cells of the same strain as the immunized mice. The myeloma cell resuscitation and expansion culture of the present invention preferably utilizes DMEM culture medium and 20% complete culture medium. The present invention does not have specific limitations on the source of the DMEM culture medium and 20% complete culture medium; conventional commercially available products are acceptable.

[0054] The hybridoma cell line LBN-9G9 of this invention is obtained by fusing mouse spleen cells and mouse myeloma cells immunized with the invention; the ratio of mouse myeloma cells to mouse spleen cells is 5–10:1. In this invention, the extraction of spleen cells from immunized mice is preferably performed 3–5 days after the last immunization. After obtaining spleen cells and SP2 / 0 myeloma cells, this invention preferably mixes SP2 / 0 myeloma cells and mouse spleen cells at a cell ratio of 5–10:1 and then centrifuges to perform cell fusion. The preferred cell ratio of mouse myeloma cells to mouse spleen cells during fusion, 5–10:1, improves cell fusion efficiency.

[0055] This invention does not impose any particular limitation on the fusion process; any fusion method well-known to those skilled in the art can be used. Preferably, the cell fusion method of this invention involves adding 1 mL of PEG at a constant rate to a mixed pellet of SP2 / 0 myeloma cells and mouse spleen cells over 1 minute, rotating the centrifuge tube in the same direction during the addition, mixing the liquid for 30 seconds, aspirating it back into a 1 mL pipette, allowing the liquid to stand for 30 seconds in the pipette, and then dripping it back into the centrifuge tube at a constant rate over 30 seconds. After the cell fusion is completed, it is preferable to add DMEM culture medium to the cell mixture to terminate the reaction.

[0056] After obtaining the fused cells, the present invention cultured the fused cells. Preferably, after fusing SP2 / 0 myeloma cells with mouse spleen cells, the cells were cultured in 96-well plates with 2% HAT resuspended cells containing feeder cells. After 7-10 days, hybridoma cells secreting specific antibodies (clonal antibodies against chlorpyrifos) were screened based on growth. After obtaining the hybridoma cells, the present invention preferably used an indirect competitive enzyme-linked immunosorbent assay (ELISA) to detect the inhibition rate of the chlorpyrifos monoclonal antibody. Hybridoma cells with an inhibition rate greater than 80% were preferably subjected to subclonal culture and cryopreservation.

[0057] This invention provides a chlorpyrifos monoclonal antibody, which includes the chlorpyrifos monoclonal antibody secreted by the hybridoma cell line described in the above technical solution.

[0058] In this invention, mice are preferably injected intraperitoneally with the hybridoma cell line LBN-9G9, hybridoma cell line 1B6, or hybridoma cell line 9A10 described in the above technical solution. After 7-10 days, the ascites fluid of the mice is collected, centrifuged, purified, and then the chlorpyrifos monoclonal antibody is obtained.

[0059] The purification described in this invention involves collecting ascites fluid with good titer, allowing it to stand, centrifuging it, pretreating it with saturated ammonium sulfate, and then dialysis to obtain purified chlorpyrifos monoclonal antibody. The saturated ammonium sulfate pretreatment is performed under ice bath conditions and includes a first saturated ammonium sulfate pretreatment and a second saturated ammonium sulfate pretreatment. During the first saturated ammonium sulfate pretreatment, the volume ratio of ascites fluid, PBS solution, and saturated ammonium sulfate solution is (1-2):(1-2):(2-3), more preferably 1:1:2. During the second saturated ammonium sulfate pretreatment, the volume ratio of PBS solution to saturated ammonium sulfate solution is preferably (1-2):(1-2), more preferably 2:1. The centrifuged product after the first saturated ammonium sulfate pretreatment undergoes a second saturated ammonium sulfate pretreatment.

[0060] Unless otherwise specified, the present invention does not have any particular limitation on the source of the mouse myeloma cells, BALB / c female mice, Freund's incomplete adjuvant, Freund's complete adjuvant, BSA, OVA, ammonium sulfate, and PBS solution, and conventional commercially available products may be used.

[0061] This invention provides a product for detecting chlorpyrifos, the product comprising the chlorpyrifos monoclonal antibody described in the above technical solution; the product includes reagents, test strips or kits.

[0062] This invention provides a colloidal gold immunochromatographic test strip for detecting chlorpyrifos, comprising a gold-labeled pad, a nitrocellulose membrane, and absorbent paper; the gold-labeled pad is coated with a colloidal gold-labeled chlorpyrifos monoclonal antibody; the nitrocellulose membrane comprises a C-line and a T-line; the C-line is coated with goat anti-mouse IgG; and the T-line is coated with chlorpyrifos-OVA coating antigen.

[0063] The preferred amount of the colloidal gold-labeled chlorpyrifos monoclonal antibody used in this invention is (30-60) μg, more preferably (30-50) μg, even more preferably (30-40) μg, and more preferably 30 μg; the preferred concentration of the chlorpyrifos OVA coating agent on the T line used in this invention is 1.5-4 mg / mL, more preferably 2-4 mg / mL, even more preferably 3-4 mg / mL, and more preferably 4 mg / mL.

[0064] The concentration of the goat anti-mouse IgG coated on the C-line of the present invention is preferably 0.2-1 mg / mL, more preferably 0.2-0.8 mg / mL, even more preferably 0.2-0.4 mg / mL, and more preferably 0.2 mg / mL.

[0065] The preferred combination of concentrations of the chlorpyrifos-OVA-coated antigen on the T-line and the goat anti-mouse IgG-coated antigen on the C-line in this invention is as follows: the concentration of the chlorpyrifos-OVA-coated antigen is preferably 2 mg / mL and the concentration of the goat anti-mouse IgG is preferably 1 mg / mL; or the concentration of the chlorpyrifos-OVA-coated antigen is preferably 1.5 mg / mL and the concentration of the goat anti-mouse IgG is preferably 0.8 mg / mL; or the concentration of the chlorpyrifos-OVA-coated antigen is preferably 3 mg / mL and the concentration of the goat anti-mouse IgG is preferably 0.4 mg / mL; or the concentration of the chlorpyrifos-OVA-coated antigen is preferably 4 mg / mL and the concentration of the goat anti-mouse IgG is preferably 0.2 mg / mL.

[0066] This invention provides the application of the chlorpyrifos monoclonal antibody hybridoma cell line, the chlorpyrifos monoclonal antibody, the product, or the colloidal gold test strip described in the above-mentioned technical solutions in the detection of chlorpyrifos.

[0067] The chlorpyrifos monoclonal antibody secreted by the hybridoma cell line of this invention has high specificity. Using the chlorpyrifos monoclonal antibody of this invention, high-sensitivity detection of chlorpyrifos can be achieved, with a detection IC50 value of [missing information]. 50 The value was 0.19 ng / mL. The range of chlorpyrifos test strips is 4.6–76.9 ng / mL.

[0068] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0069] The reagents used in the examples are as follows:

[0070] Freund's complete and incomplete adjuvants, bovine serum albumin (BSA), ovalbumin (OVA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), polyethylene glycol 2000 (PEG-2000), hypoxanthine and thymidine (HT) medium supplements, hypoxanthine, aminopterin and thymidine (HAT), dimethyl sulfoxide (DMSO), TMB (3,30,5,50-tetramethylbenzidine) substrate solution, and 50% (w / v) polyethylene glycol solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Gibco BRL (Paisley, Scotland). DMEM basal medium, glutamine solution, and penicillin-streptomycin solution were all selected from Thermo Fisher Scientific (Waltham, MA, USA). N,N-Dimethylformamide (DMF) was provided by Tianjin Shengsi Biochemical Technology Co., Ltd. (Tianjin, China). Yamanaka peroxidase-labeled goat anti-mouse IgG (IgG-HRP) was purchased from Jackson Immunological Research Laboratories Co., Ltd. Other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. Colloidal gold (0.01%, w / v) 40nm and colloidal gold reconstitution solution were purchased from Jieyi Biotechnology Co., Ltd. (Shanghai, China).

[0071] 0.01M PBS buffer (pH=7.4): Dissolve 8g sodium chloride, 2.92g dodecahydrate and disodium hydrogen phosphate, and 0.2g potassium dihydrogen phosphate in water and bring the volume to 1L.

[0072] 0.02M PB buffer: 2.92g dodecahydrate and disodium hydrogen phosphate, 0.2g potassium dihydrogen phosphate dissolved in water and brought to a final volume of 500mL.

[0073] Saturated ammonium sulfate: Weigh an excess of ammonium sulfate (>450g) and dissolve it in 500mL of distilled water. Heat and stir until completely dissolved. When the temperature drops to room temperature, ammonium sulfate crystals will precipitate.

[0074] 20% DMEM complete culture medium: 1% penicillin and streptomycin, 1% L-glutamine, 20% fetal bovine serum, 78% DMEM culture medium.

[0075] 2% HAT medium: 1% penicillin and streptomycin, 1% L-glutamine, 20% fetal bovine serum, 69% DMEM medium, 2% HAT.

[0076] Coating buffer: 1.5g sodium carbonate, 2.94g sodium bicarbonate, add water to a final volume of 1000mL.

[0077] Sample diluent (PBSTG): 8g sodium chloride, 2.92g dodecahydrate and disodium hydrogen phosphate, 0.2g potassium dihydrogen phosphate, 1g gelatin dissolved in an appropriate amount of water, then 1mL Tween-20 was added to bring the volume to 1L.

[0078] Substrate buffer: Dissolve 0.1 g potassium sorbate and 46.04 g potassium dihydrogen citrate in water and bring the volume to 1000 mL.

[0079] TMB chromogenic solution: Substrate buffer with TMB and hydrogen peroxide, prepared fresh before use.

[0080] Plate washing solution: Dissolve 8g sodium chloride, 2.92g dodecahydrate and disodium hydrogen phosphate, and 0.2g potassium dihydrogen phosphate in an appropriate amount of water, then add 1mL Tween-20 and bring the volume to 1L.

[0081] Termination solution (1 mol / L) hydrochloric acid: Measure 87.72 mL of concentrated hydrochloric acid and add it along the wall of a beaker to 800 mL of water while stirring. Transfer the solution to a 1 L volumetric flask, add water to the mark, and mix well.

[0082] Example 1: Preparation of chlorpyrifos complete antigen

[0083] To obtain the immunogenic complete antigen, the carboxyl group of the chlorpyrifos hapten was activated using the active ester method. Chlorpyrifos was then conjugated to a carrier protein. The immunogen was obtained by conjugating the hapten with BSA (bovine serum albumin), and the coating agent was obtained by conjugating the hapten with OVA (ovalbumin). The specific method for preparing the chlorpyrifos complete antigen is as follows: 27.6 mg of chlorpyrifos hapten, 18.2 mg of NHS (N-hydroxysuccinimide), and 30.3 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were weighed and fully dissolved in 1.5 mL of DMF (N,N-dimethylimide). The mixture was then magnetically stirred at 25°C for 4 h. After the reaction was complete, if turbidity was observed, the mixture was centrifuged at 6000 rpm for 5 min. The supernatant of the activated chlorpyrifos hapten was used for subsequent conjugation to the carrier protein. Weigh 20 mg of BSA and dissolve it in 2 mL of 0.01 M phosphate-buffered saline (PBS). Weigh 10 mg of OVA and dissolve it in 1 mL of 0.01 M PBS. Take the activated chlorpyrifos hapten supernatant and slowly add it dropwise to the pre-dissolved BSA and OVA PBS buffers, respectively. Stir overnight at 4°C. The molar ratios of chlorpyrifos hapten supernatant to BSA carrier protein are 40:1, 50:1, and 60:1, respectively, and the molar ratios of chlorpyrifos hapten supernatant to OVA carrier protein are 40:1, 50:1, and 60:1, respectively. Dialyze the reaction solutions of chlorpyrifos hapten supernatant with BSA carrier protein and chlorpyrifos hapten supernatant with OVA carrier protein six times with 4 L of 0.01 mol / L PBS (pH 7.4) for 4 h each time to remove unreacted hapten or other small molecules. After complete dialysis, the chlorpyrifos complete antigen was aliquoted into 1 mg / mL portions, flash-frozen in liquid nitrogen, and stored at -20°C for later use.

[0084] The obtained clopidogrel complete antigen was identified: The clopidogrel complete antigen was identified using MALDI-TOF-MS, and the conjugation ratio of BSA, OVA, and clopidogrel hapten was calculated. Based on the change in the mass-to-charge ratio of the molecular ion peaks before and after conjugation, the conjugation ratio of clopidogrel hapten to protein was calculated. The BSA standard detection results are shown below. Figure 1 , Figure 1 The peak value was 67217.314. Figure 1 The x-axis represents the mass-to-charge ratio (m / z) of the ions in the BSA standard, and the y-axis represents the intensity of the ion current; the complete antigen obtained by conjugating chlorpyrifos hapten and BSA is shown in [reference needed]. Figure 2 , Figure 2 The peak value was 74068.026; Figure 2 The x-axis represents the mass-to-charge ratio (m / z) of the ions of the complete antigen obtained from the coupling diagram of chlorpyrifos hapten and BSA, and the y-axis represents the intensity of the ion current; the OVA standard test results are shown in [reference needed]. Figure 3 , Figure 3 The peak value was 44630.281. Figure 3 The x-axis represents the mass-to-charge ratio (m / z) of the OVA standard ions, and the y-axis represents the intensity of the ion current; the complete antigen diagram obtained by conjugating chlorpyrifos hapten and OVA is shown in the figure. Figure 4 , Figure 4 The peak value was 45829.570. Figure 4 The x-axis represents the mass-to-charge ratio (m / z) of the complete antigen obtained by conjugating chlorpyrifos hapten and OVA, and the y-axis represents the intensity of the ion current. The conjugation ratio described in this invention refers to the ratio of hapten to carrier protein binding, calculated as: Conjugation Ratio = (Mp - Mstd) / Mh, where "Mp" represents the molecular weight of the chlorpyrifos antigen conjugate, "Mstd" represents the molecular weight of the BSA / OVA standard, and "Mh" represents the molecular weight of the chlorpyrifos hapten. Calculations show that the conjugation ratio of chlorpyrifos hapten to BSA is 19.70:1. The conjugation ratio of chlorpyrifos hapten to OVA is 3.45:1. Based on the obtained actual conjugation ratios, the conjugation effect is good and it can be used for immunization and coating.

[0085] Example 2: Preparation of chlorpyrifos monoclonal antibody hybridoma cell line

[0086] 1. Mouse immunization

[0087] Seven-week-old purebred BALB / c healthy female mice (Beijing Spaford Biotechnology Co., Ltd.) were selected. Six mice were immunized with chlorpyrifos-BSA complete antigen (conjugation ratio 19.70:1) prepared in Example 1, and named mice 1, 2, 3, 4, 5, and 6, respectively. The chlorpyrifos-BSA complete antigen was emulsified with an equal volume of adjuvant, and 0.1 mL was injected intraperitoneally and 0.05 mL subcutaneously on the back of each mouse. The immunization dose of chlorpyrifos-BSA complete antigen was 100 μg per mouse. Freund's complete adjuvant was used for the initial immunization, and Freund's incomplete adjuvant was used for subsequent immunizations.

[0088] 2. Myeloma cell resuscitation and expanded culture

[0089] One week prior to fusion, myeloma cells were resuscitated and expanded. The selected myeloma cells were SP2 / 0 myeloma cells of the same strain as the immunized mice in step 1. Cells were removed from a liquid nitrogen container, thawed in a 37°C water bath, resuspended in pre-warmed DMEM medium (pre-warmed in a 37°C water bath), centrifuged to remove the supernatant, and then transferred to 20% DMEM complete culture medium. The cells were cultured in a 37°C incubator containing 5% CO2. When the cells reached 80% of the bottom area of ​​the 6-well plate (logarithmic growth phase), they were passaged 2-3 times in 20% DMEM complete culture medium. SP2 / 0 myeloma cells with good growth, uniform size, and good light-blocking properties were selected for the fusion experiment.

[0090] 3. Preparation of feeder cells

[0091] On the day of fusion, 6-8 week old blank mice of the same strain as the mice fused in step 1 were selected, and feeder cells were collected from the peritoneal cavity of the mice for subsequent hybridoma cell culture. The specific steps were as follows: the blank mice were excised by removing their eyeballs and bleeding, then soaked in alcohol. Using sterilized forceps and scissors, the abdomen of the mice was cut open, and preheated DMEM (37°C) was injected into the abdomen using a syringe. After mixing, the cells were centrifuged, the supernatant was discarded, and the cells were resuspended in 2% HAT medium. 100 μL of the solution was then seeded into 96-well cell culture plates and cultured at 37°C in a 5% CO2 incubator for use on the same day.

[0092] 4. Preparation of spleen cells

[0093] Select the immunized mice from step 1, remove blood by gouging out the eyeballs, soak them in alcohol, and use sterilized forceps and scissors to cut open the mouse abdomen and remove the spleen. Place the spleen in a culture dish containing 20 mL of preheated (37°C) DMEM. Use a syringe needle to poke a dozen small holes on both sides of the spleen, and repeatedly blow with DMEM until it turns white, ensuring that all spleen cells are blown out. Centrifuge, discard the supernatant, and resuspend in DMEM for later use. At the same time, discard the supernatant from the SP2 / 0 myeloma cells in 20% DMEM complete culture medium from step 2, resuspend in DMEM, centrifuge, discard the supernatant, and then resuspend in DMEM for later use.

[0094] 5. Cell fusion:

[0095] The SP2 / 0 myeloma cells from step 4 were mixed with mouse spleen cells at a ratio of 5–10:1, centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. The cell fusion operation was then performed. The specific steps for cell fusion are as follows:

[0096] Add 1 mL of PEG at a constant rate over 1 minute, rotating the centrifuge tube in the same direction during the addition process.

[0097] Mix the liquid for 30 seconds, then aspirate it back into a 1 mL pipette.

[0098] The liquid was allowed to stand for 30 seconds in a pipette.

[0099] Drop the solution back into the centrifuge tube at a constant rate within 30 seconds.

[0100] After fusion is complete, DMEM culture medium is added to the cell mixture to terminate the reaction, as follows:

[0101] First minute: Add 1 mL of DMEM culture medium at a uniform rate;

[0102] Second and third minutes: Add 4 ml of LDM medium at a uniform rate;

[0103] Fourth and fifth minutes: Add 20 mL of DMEM culture medium at a uniform rate;

[0104] Preheated 2% HAT resuspended cells were added to prepared 96-well culture plates containing feeder cells, 100 μL per well, and cultured in a constant temperature incubator at 37°C containing 5% CO2.

[0105] After fusion, observe the status for 3 days, and then select hybridoma cells that secrete specific antibodies from chlorpyrifos monoclonal antibodies 7-10 days later based on their growth.

[0106] 6. Screening of specific hybridoma cells

[0107] After the hybridoma cells from step 5 grew to a certain size, they were detected using an indirect competitive enzyme-linked immunosorbent assay (ELISA). For the first detection, positive wells with an OD value greater than 3.0 were added with 20% complete culture medium. The next day, the inhibition rate of the selected positive wells was measured using clopyralid at an inhibition concentration of 1000 ng / mL. Hybridoma cell lines with an inhibition rate greater than 80% were screened using the above method. These cell lines were then expanded and cloned using limiting dilution. Indirect competitive ELISA was then used to screen for hybridoma cell lines with an OD value greater than 3.0 and an inhibition rate greater than 80%, which were then cultured and cryopreserved. Three clopyralid monoclonal antibody hybridoma cell lines were obtained and named LBN-9G9, LBN-1B6, and LBN-9A10, respectively. The monoclonal antibody secreted by the LBN-9G9 hybridoma cell line is shown in the figure. Figure 5 Monoclonal antibody cells secreted by the hybridoma cell line LBN-1B6 were observed. Figure 7 Monoclonal antibody cells secreted by hybridoma cell line LBN-9A10 were observed. Figure 6 .according to Figure 5 , Figure 6 , Figure 7 It can be seen that the monoclonal cells are growing well and can be positively detected for inhibition by chlorpyrifos. The cell supernatant was detected by indirect competitive enzyme-linked immunosorbent assay and the inhibition rate was greater than 80%.

[0108] The serum titer and inhibition rate of chlorpyrifos in mice after the third and fourth immunizations in step 1 were determined by indirect competitive enzyme-linked immunosorbent assay (ELISA).

[0109] The indirect competitive enzyme-linked immunosorbent assay (ELISA) procedure is as follows: The chlorpyrifos OVA-coated antigen was diluted with coating buffer and added to each well of a 96-well plate (100 μL). The plate was incubated at 37°C for 3 hours, washed three times with wash buffer, and chlorpyrifos standard and antibody were diluted with PBSTG (50 μL per well). The plate was incubated at 37°C for 30 minutes, washed three times with wash buffer, and 100 μL of horseradish peroxidase-labeled goat anti-mouse IgG diluted with PBSTG was added to each well. The plate was incubated at 37°C for 30 minutes, washed three times, and 100 μL of TMB chromogenic buffer was added. The reaction was stopped by adding 50 μL of stop solution after reacting at 25°C for 15 minutes. The OD value was measured and calculated at 450 nm.

[0110] The results of mouse blood tests used for fusion are shown in Tables 1 and 2. In Tables 1 and 2, C represents the negative control well, I represents the inhibition well (positive well), and IR represents the inhibition rate (%).

[0111] The inhibition rate is calculated as follows: Inhibition rate = (OD value of negative control well - OD value of positive well) / OD value of negative control well * 100%.

[0112] Table 1. Serum titer and inhibition rate of chlorpyrifos in mice 6 after the third immunization.

[0113]

[0114] Table 2. Serum titer and chlorpyrifos inhibition rate of mice 6 after the fourth immunization.

[0115]

[0116] According to Tables 1 and 2, a mouse serum inhibition rate of over 80% indicates good efficacy and the fusion procedure can be performed.

[0117] Example 3: Preparation and purification of chlorpyrifos monoclonal antibody

[0118] Six- to eight-week-old female BALB / c mice were selected and injected with paraffin one week prior to treatment to disrupt their immune system. The chlorpyrifos monoclonal antibody hybridoma cell lines LBN-9G9, LBN-1B6, and LBN-9A10 obtained in Example 2 were cultured, the supernatant was discarded, and the cells were resuspended in DMEM and injected into the mice. Approximately 0.3 mL was injected into each mouse, resulting in approximately 10 cells. 6After the mice's abdomens swelled (7-10 days), ascites fluid was collected and placed at room temperature for 0.5 hours, then incubated overnight at 4°C. After centrifugation, the supernatant was used to determine the ascites fluid titer using an indirect competitive enzyme-linked immunosorbent assay (ELISA). The ascites fluid supernatant with good titers was further purified. The ascites fluid was incubated overnight and centrifuged at 4°C. One volume (mL) of ascites fluid was added to one volume (mL) of PBS, and two volumes of saturated ammonium sulfate solution were added with stirring in an ice bath. The mixture was incubated at 4°C for 4 hours. The precipitate obtained after centrifugation was dissolved in two volumes (mL) of PBS, and one volume (mL) of saturated ammonium sulfate solution was added with stirring in an ice bath. The mixture was incubated at 4°C for 4 hours. The precipitate obtained after centrifugation was dissolved in one volume (mL) of PBS and placed in a dialysis bag. Dialyzed four times with PBS and twice with PB, each time for 4 hours. The dialyzed antibodies were aliquoted and lyophilized in a vacuum freeze dryer at -80°C. The powder was stored at -20°C for later use. The lyophilized powders of chlorpyrifos monoclonal antibody secreted by hybridoma cell line LBN-9G9, LBN-9A10, and LBN-1B6 were respectively prepared to a concentration of 1 mg / mL for antibody titer determination. The antibody titer was determined by indirect competitive enzyme-linked immunosorbent assay (ELISA). The determination method and calculation formula were the same as those for serum titer detection in Example 2. The determination results are shown in Tables 3, 4, and 5 (C represents control well, I represents inhibition well, IR represents inhibition rate (%), and - represents data that could not be measured).

[0119] Table 3. Titer of chlorpyrifos monoclonal antibody secreted by hybridoma cell line LBN-9G9

[0120]

[0121]

[0122] Table 4. Titer of chlorpyrifos monoclonal antibody secreted by hybridoma cell line LBN-9A10

[0123]

[0124]

[0125] Table 5. Titer of chlorpyrifos monoclonal antibody secreted by hybridoma cell line LBN-1B6

[0126]

[0127] As shown in Tables 3-5, the monoclonal antibody secreted by the hybridoma cell line LBN-9G9, when the coating agent was diluted 64,000 times and the monoclonal antibody was diluted 16,000 times, had an OD value of 1.0596 in the control well and an inhibition rate of 89.5%, which was the best compared to the other two cell lines. It can be used to establish subsequent immunoassay methods.

[0128] Identification and characterization of chlorpyrifos monoclonal antibodies:

[0129] a. Antibody sensitivity: The IC50 of the monoclonal antibody secreted by the hybridoma cell line LBN-9G9 was determined using an indirect competitive enzyme-linked immunosorbent assay (ELISA). 50 The value was 0.19 ng / mL, IC50 20 ~IC 80 The concentration ranged from 0.04 to 0.87 ng / mL (see standard curve). Figure 8 This indicates that the antibody has good sensitivity to clopidogrel and can be used to establish clopidogrel immunoassay. The procedure for the indirect competitive enzyme-linked immunosorbent assay is the same as above. Figure 8 B / B0 represents the absorbance value of the positive well / the absorbance value of the negative control well.

[0130] b. Antibody Specificity Identification: Antibody specificity should be considered an important factor affecting the detection of analytes in samples. To assess the specificity of monoclonal antibodies to CPPU, the cross-reactivity (CR) of the following six structurally similar antibodies was evaluated using an indirect competitive enzyme-linked immunosorbent assay (procedure steps as above). The CR value was calculated using the formula: CR (%) = IC50 of chlorpyrifos. 50 ICs with structural analogues 50 ×100%. The results are shown in Table 6 below (in the table, "-" indicates that IC could not be measured). 50 The monoclonal antibody secreted by the hybridoma cell line LBN-9G9 showed cross-reactivity with thiamethoxam (CR value: 20.2%), but no cross-reactivity with the other five structural analogs (CR < 0.01%). These results indicate that the monoclonal antibody secreted by the hybridoma cell line LBN-9G9 has good specificity.

[0131] Table 6. Specificity of chlorpyrifos monoclonal antibodies secreted by hybridoma cell line LBN-9G9

[0132]

[0133]

[0134] Example 4: Application of chlorpyrifos monoclonal antibody

[0135] The principle of the indirect competitive enzyme-linked immunosorbent assay kit is to fix a certain concentration of antigen or antibody onto the surface of a polystyrene microplate by physical adsorption, add the sample to be tested, and indirectly reflect the presence or quantity of the antigen or antibody being tested by the color intensity of the enzyme label.

[0136] The principle of colloidal gold immunochromatography is based on a nitrocellulose membrane as a carrier, with the analyte and secondary antibody serving as the test line and control line, respectively. During sample loading, the analyte competes with the analyte for binding to the gold-labeled antibody. As the analyte concentration increases, its ability to compete with the analyte for binding to the gold-labeled antibody increases, leading to a weakening of the visual signal of the test line. When the analyte concentration is sufficiently high, the visual signal of the test line disappears completely, and the excess gold-labeled antibody binds to the secondary antibody in the control line. If the sample does not contain the analyte, both the test line and control line will have a large number of colloidal gold particles, resulting in similar color intensities for the two lines.

[0137] The clonal antibody secreted by the hybridoma cell line LBN-9G9 prepared in Example 3 was used to prepare an enzyme-linked immunosorbent assay (ELISA) kit and a colloidal gold immunochromatographic test strip, and an immunoassay method was established for the detection of chlorpyrifos in actual samples. The cucumber samples used in the experiment were purchased from a supermarket and were blank samples confirmed to be chlorpyrifos-free by LC-MS / MS. Spiked samples were blank samples with different concentrations of chlorpyrifos standard solution added: 0 ng / g, 100 ng / g, 200 ng / g, 500 ng / g, and 1000 ng / g. The recovery rate was calculated as: Recovery rate = (Measured amount / Added amount) * 100%. The specific steps are as follows:

[0138] 1. Preparation of chlorpyrifos enzyme-linked immunosorbent assay kit

[0139] The chlorpyrifos OVA coating agent (1 mg / mL) was diluted with coating buffer and added to 100 μL per well of a 96-well plate. The plate was incubated at 37°C for 3 h and then washed 3 times with washing buffer.

[0140] Weigh 1g of cucumber sample, add chlorpyrifos standard solutions of different concentrations, let stand for 30min, add 20mL of PBSTG solution containing 10% acetonitrile to extract chlorpyrifos, centrifuge at 8000rpm for 10min, take the supernatant and dilute it twice with PBSTG solution containing 10% acetonitrile for later use.

[0141] The chlorpyrifos standard and the anti-chlorpyrifos monoclonal antibody secreted by the hybridoma cell line LBN-9G9 were diluted with PBSTG solution containing 10% acetonitrile. 50 μL of chlorpyrifos standard and 50 μL of antibody diluent were added to each well of the standard curve. 50 μL of sample diluent and 50 μL of antibody diluent were added to each well of the sample curve. The plates were incubated at 37°C for 30 min, washed three times with washing buffer, and 100 μL of HRP (horseradish peroxidase)-IgG diluted in PBSTG was added to each well. After incubation at 37°C for 30 min, the plates were washed three times, 100 μL of TMB chromogenic solution was added, and the reaction was stopped by adding 50 μL of stop solution after reacting at 25°C for 15 min. The OD value was detected at 450 nm, and the data were analyzed using Origin software. The standard curve is shown below. Figure 9As shown, Figure 9 In the diagram, B / B0 represents the absorbance value of the positive well / the absorbance value of the negative control well. The recovery rate was calculated by adding the recovered absorbance values ​​to the curve, as shown in Table 7 below.

[0142] 2. Preparation of chlorpyrifos test strips:

[0143] 2.1 Preparation of Gold-Labeled Antibody: The pH, detection line, and coating line concentrations were optimized to select the optimal conditions. Nine 1.5 mL centrifuge tubes were used, each containing 1 mL of colloidal gold solution. 2 μL of 10 μL of K₂CO₃ (0.1 M) was added to each tube and mixed thoroughly. 15 μL of chlorpyrifos monoclonal antibody (2 mg / mL) was added to each tube, and the mixture was incubated at room temperature for 3 min. The color change of the solution was observed, and the lowest pH value at which the solution remained red was recorded as the optimal pH of 8.0 (with the addition of 7 μL of potassium carbonate). The pH optimization results for antibody-labeled colloidal gold are shown in [the table below]. Figure 10 .

[0144] This experiment used high-speed centrifugation to label the antibody. 1 mL of colloidal gold solution was placed in a 1.5 mL centrifuge tube, and 7 μL of K₂CO₃ (0.1 M) was added to adjust the pH. After reacting at room temperature for 10 min, 30 μL of 10% BSA solution was added for blocking for 10 min. The mixture was then centrifuged at 8000 r / min for 10 min (4℃). The resulting precipitate was resuspended in 600 μL of colloidal gold reconstitution solution to obtain the colloidal gold-labeled chlorpyrifos monoclonal antibody 9G9.

[0145] The amount of chlorpyrifos monoclonal antibody added was 15 μL, and the concentration of chlorpyrifos monoclonal antibody was 2 mg / mL. Different combinations of coating antigen and secondary antibody (goat anti-mouse IgG) were used: ① coating antigen 2 mg / mL and secondary antibody 1 mg / mL, ② coating antigen 1.5 mg / mL and secondary antibody 0.8 mg / mL, ③ coating antigen 3 mg / mL and secondary antibody 0.4 mg / mL, ④ coating antigen 4 mg / mL and secondary antibody 0.2 mg / mL. The optimization results of different combinations of coating antigen and secondary antibody concentrations are shown in [the table below]. Figure 11 The test strip prepared with PB buffer should produce a negative result, i.e., a T / C value greater than 1. Based on this requirement and the color development results, the concentration of chlorpyrifos OVA-coated antigen was finally selected as 4 mg / mL; the concentration of goat anti-mouse IgG coated on the C line was 0.2 mg / mL.

[0146] 2.2 Preparation of gold-labeled pads: Cut the gold-labeled pads into thin strips of 1cm*30cm, evenly spread the optimized colloidal gold-labeled chlorpyrifos monoclonal antibody 9G9 resuspension on the gold-labeled pads, place them on iron mesh, and dry at 37℃ for 1h for later use.

[0147] 2.3 Test strip assembly: Assemble the test strips by overlapping the gold-labeled pad with the NC membrane by 1-2 mm, the sample pad with the gold-labeled pad by 1-2 mm, and the absorbent pad with the NC membrane by 1-2 mm. Cut the strips into 4 mm x 60 mm strips using an automatic cutter. The gold-labeled pad is coated with colloidal gold-labeled chlorpyrifos monoclonal antibody; the nitrocellulose membrane includes a C-line and a T-line; the C-line is coated with goat anti-mouse IgG; and the T-line is coated with chlorpyrifos-OVA-coated antigen.

[0148] 2.4 Performance Evaluation of Test Strips

[0149] a. Organic solvent stability of the test strip

[0150] To investigate the effects of methanol and acetonitrile, two organic solvents, on the test strips, 0%, 1%, 10%, 20%, 50%, and 100% of the organic solvents were added to the test strips, and the changes in the C and T lines were observed. Finally, 10% methanol was selected as the extractant.

[0151] b. Detection range of chlorpyrifos

[0152] A standard curve for chlorpyrifos was prepared using 10% methanol-PB. The T / C value read by the card reader was used to plot the curve using Origin software. Figure 12 As shown, Figure 12 In the diagram, B / B0 represents the (T / C) value measured in the positive sample / the (T / C) value measured in the negative control sample; the IC is calculated by software. 50 The value was 21.2 ng / mL, and the detection range was 4.6–76.9 ng / mL.

[0153] c. Detection stability of the test strip

[0154] To evaluate the stability of the test strips during application, a 50 ng / mL chlorpyrifos standard solution was prepared. Ten test strips were randomly selected and tested against the blank control and the 50 ng / mL standard solution, respectively. The test results were interpreted using a card reader. The RSDs for the negative control (CK) and the positive control (50 ng / mL) were 7.9% and 2.0%, respectively, demonstrating that the test strips have good stability and can be used for practical testing.

[0155] 2.5 Application of test strips

[0156] a. Matrix extraction and recovery experiment

[0157] Weigh 1g of blank sample, add chlorpyrifos standard solutions of different concentrations, let stand for 30min, add 10mL of 10% methanol-PB extraction solution, shake and extract for 30s, centrifuge at 8000r / min for 5min, take the supernatant and drop it into the sample well of the chlorpyrifos test strip, read the result with the card reader after 10min and record the T / C value. The colorimetric diagram is shown below. Figure 15 As shown.

[0158] b. Establishment of standard curves

[0159] Weigh 1g of blank sample, add 10mL of 10% methanol-PB extraction solution, shake and centrifuge at 8000r / min for 5min, collect the supernatant to prepare and plot a standard curve, and read the result using a card reader after 10min to record the T / C value. The colorimetric graph is shown below. Figure 14 As shown below, the obtained T / C values ​​were fitted using Origin software to obtain a standard curve. Figure 13 The spiked sample values ​​were substituted into the curve, and the results were calculated using software. The recovery rate was calculated using the above recovery rate calculation formula, as shown in Table 7. Figure 13 , Figure 14 , Figure 15 As shown in Table 7, the test strip detection recovery rate is high, and the chlorpyrifos monoclonal antibody 9G9 can be used to establish the colloidal gold test strip method in cucumber samples.

[0160] Figure 13 In the figure, B / B0 represents the (T / C) value measured in the positive sample / the (T / C) value measured in the negative control sample. Figure 14 In the table, 0, 1, 2, 5, 10, 20, 50, 100, 200, 500, and 1000 represent chlorpyrifos concentrations of 0 ng / g, 1 ng / g, 2 ng / g, 5 ng / g, 10 ng / g, 20 ng / g, 50 ng / g, 100 ng / g, 200 ng / g, 500 ng / g, and 1000 ng / g, respectively (each concentration is repeated 3 times). Figure 15 In the table, ① represents a spiked amount of 100 ng / g, ② represents a spiked amount of 200 ng / g, ③ represents a spiked amount of 500 ng / g, and ④ represents a spiked amount of 1000 ng / g.

[0161] 3. Instrumental analysis verification: The detection method refers to GB 23200.110-2018.

[0162] Weigh 10g (accurate to 0.01g) of the sample into a 50mL centrifuge tube, add chlorpyrifos standard solutions of different concentrations, let stand for 30min, add 10mL of acetonitrile, and vortex for 3min; add 4g of anhydrous magnesium sulfate and 1g of sodium chloride to the centrifuge tube, then centrifuge at 4000r / min for 5min, take 1.5mL of the supernatant and put it into a 2mL centrifuge tube containing 50mg PSA and 150mg of anhydrous magnesium sulfate; vortex for 30s, centrifuge at 5000r / min for 5min, take the supernatant and filter it through an organic filter membrane for HPLC-MS / MS detection.

[0163] Weigh 10g of blank sample, add extraction buffer and process as above, obtain supernatant, filter through an organic filter membrane, and prepare a standard curve for HPLC-MS / MS detection. The LC-MS / MS recovery rates are shown in Table 7 below. The instrument conditions are as follows:

[0164] Liquid chromatography conditions: column A C18 column (100 mm × 2.1 mm, 1.7 μm) was used at a column temperature of 40 °C. The injection volume was 5 μL, and the mobile phase flow rate was 0.3 mL / min. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.01 M ammonium formate-methanol. The elution gradient was as follows: [0, 1.5] min, 10% A, 90% B; [1.5, 2.5] min, 90% A, 10% B; [2.6, 4] min, 10% A, 90% B.

[0165] Mass spectrometry conditions: Ion pairs and collision voltages: 248 / 129 (quantitative ion pairs), collision voltage 30 eV; 248 / 155 (qualitative ion pairs), collision voltage 17 eV; Scan mode: multiple reaction monitoring (MRM); Residence time: 100 ms; Scan mode: ESI+; Collision gas: N2; Collision gas flow rate: 3 L / min; Heating gas: N2; Heating gas flow rate: 10 L / min; DL temperature: 250 °C; Heating block temperature: 400 °C; Drying gas flow rate: 10 L / min.

[0166] Table 7. Results of enzyme-linked immunosorbent assay (ELISA), colloidal gold test strip (LIFA), and liquid chromatography-mass spectrometry (LC-MS / MS) addition and recovery of cucumber samples.

[0167]

[0168] Note: ND means not detected, and Negative means negative.

[0169] As shown in Table 7, the enzyme-linked immunosorbent assay (ELISA) kit and colloidal gold test strip (LIFA) prepared using chlorpyrifos monoclonal antibody 9G9 exhibit high detection accuracy and good repeatability, with no significant difference from HPLC-MS / MS analysis results. The immunoassay method established using chlorpyrifos monoclonal antibody 9G9 has high sensitivity and can be used for the detection of chlorpyrifos in food.

[0170] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A chlorpyrifos monoclonal antibody hybridoma cell line, characterized in that, The accession number for the hybridoma cell line LBN-9G9 is CGMCC NO.45170.

2. A chlorpyrifos monoclonal antibody, characterized in that, The chlorpyrifos monoclonal antibody is secreted by the hybridoma cell line or its passaged cell line as described in claim 1.

3. The chlorpyrifos monoclonal antibody according to claim 2, characterized in that, The IC50 of the chlorpyrifos monoclonal antibody 50 The value was 0.19 ng / mL.

4. A product for detecting chlorpyrifos, characterized in that, The product includes the chlorpyrifos monoclonal antibody as described in claim 2; the product includes reagents, test strips, or kits.

5. A colloidal gold immunochromatographic test strip for detecting chlorpyrifos, characterized in that, Includes gold-labeled pads, nitrocellulose membranes, and absorbent paper; The gold-labeled pad is coated with the chlorpyrifos monoclonal antibody of claim 2, which is labeled with colloidal gold; the nitrocellulose membrane includes C-lines and T-lines in sequence; The C-line is coated with goat anti-mouse IgG; the T-line is coated with chlorpyrifos-OVA and the original coating. The amount of the colloidal gold-labeled anti-chlorpyrifos monoclonal antibody used was 30 μg; The concentration of the chlorpyrifos-OVA coating agent on the T line was 4 mg / mL; The concentration of goat anti-mouse IgG coated on the C line was 0.2 mg / mL.

6. The use of the chlorpyrifos monoclonal antibody hybridoma cell line of claim 1, or the chlorpyrifos monoclonal antibody of claim 2 or 3, or the product of claim 4, or the colloidal gold immunochromatographic test strip of claim 5 in the detection of chlorpyrifos.

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