Triazophos hapten, antigen, antibody and triazophos test strip and application

Abstract

The application belongs to the technical field of immunology, and particularly relates to a triazophos hapten, an antigen, an antibody, a triazophos detection test strip and application. The triazophos antigen is obtained by coupling a triazophos hapten and a carrier protein, and can be used for preparing a triazophos antibody and a triazophos detection reagent. A monoclonal cell strain OXY secreting a triazophos monoclonal antibody is provided, and can be used for preparing a triazophos monoclonal antibody. The monoclonal antibody and the hybridoma cell strain are used to prepare a triazophos detection test strip. The test strip has strong specificity, high sensitivity, and a minimum detection limit of 3.152 ng / mL. The test strip is simple, rapid, and has strong timeliness. The test strip is a colloidal gold chromatographic detection test strip, and can be operated on site without any other large instruments. After a sample liquid is added to the test strip, the detection result can be determined within 10 min, and the test strip has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of immunology technology, specifically relating to a triazophos hapten, antigen, antibody, triazophos test strip, and its application. Background Technology

[0002] Triazophos, a moderately toxic, non-systemic organophosphorus broad-spectrum insecticide, acaricide, and nematicide, possesses advantages such as high selectivity, high efficiency, broad spectrum, low toxicity, easy decomposition, and low residue, and has been widely used in agriculture, forestry, and animal husbandry. Currently, these two types of pesticides are widely used in the cultivation of traditional Chinese medicinal herbs. Although the use of these pesticides has brought significant benefits to people, excessive use can pose potential harm to humans. For example, triazophos induces neurotoxicity in animals by inhibiting acetylcholinesterase activity; simultaneously, it has estrogenic effects, which may affect animal reproductive performance.

[0003] Currently, the detection of triazophos residues mainly relies on instrumental analysis and enzyme-linked immunosorbent assay (ELISA). Instrumental analysis methods, including gas chromatography, liquid chromatography, and gas chromatography-mass spectrometry, while possessing high sensitivity and specificity, depend on expensive equipment, specialized operators, cumbersome and time-consuming sample pretreatment processes, and the consumption of large amounts of organic solvents, making them difficult to meet the needs of rapid on-site screening. ELISA, a labeled immunological technique, combines the specificity of antigen-antibody reactions with the specificity and sensitivity of highly efficient enzyme catalysis, simplifying the detection process to some extent. However, the ELISA method still has the following disadvantages: (1) ELISA detection generally takes 3-4 hours to produce results, which is a long detection time; (2) Due to inconsistent sample loading, sample loading time, washing conditions, and operators, ELISA results in large repeatability differences and poor repeatability; (3) ELISA detection requires the use of various instruments or reagents, and the operation steps are relatively complex, requiring personnel trained in relevant experimental instruments to operate; (4) ELISA has high production costs, with relatively many and expensive consumables, sample volumes, reagent volumes, and reagent types; (5) ELISA requires a storage and transportation temperature of 2-8 ℃, which is relatively high; (6) ELISA sample loading is relatively complex and prone to interwell contamination, which can lead to false positive results and affect the accuracy of the test.

[0004] Based on the shortcomings of the aforementioned methods, colloidal gold immunochromatography has been widely used due to its advantages such as short testing time, high accuracy, good repeatability, simple operation, and low cost. Colloidal gold immunoassay (CGIA) is a novel labeled immunoassay technique that uses colloidal gold as a tracer marker in antigen-antibody reactions. Colloidal gold has a large specific surface area and good biocompatibility, allowing it to non-covalently bind to the labeled substance through physical adsorption. Its labeling technology is simple, rapid, and pollution-free, and the results do not require expensive laser detection instruments; only ordinary optical instruments are needed, and identification can even be achieved with the naked eye, making it particularly suitable for rapid on-site screening. However, existing colloidal gold immunochromatographic methods still have room for improvement in terms of detection accuracy and sensitivity (detection limit), and their stability and reproducibility need further optimization. Therefore, developing a rapid detection method for triazole phosphate colloidal gold immunochromatography that combines high sensitivity, high accuracy, and good stability to meet the increasingly stringent requirements for residue monitoring and rapid food safety detection has become a key technical problem urgently needing to be solved by those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings and defects of existing technologies for detecting triazophos by providing a method for preparing a gold standard chromatography test strip for rapid detection of triazophos in Angelica sinensis, enabling faster, more sensitive, and simpler detection of triazophos residues in traditional Chinese medicinal materials. The specific method is as follows: In a first aspect, the present invention provides a triazole phosphate hapten, the structural formula of which is shown in formula (I): .

[0006] In a second aspect, the present invention provides a triazophos complete antigen, obtained by coupling the aforementioned triazophos hapten with a carrier protein.

[0007] Preferably, the carrier protein is one or more of bovine serum albumin, ovalbumin, thyroprotein, or human serum albumin.

[0008] A third aspect of the present invention provides the use of the aforementioned triazole phosphate hapten in the preparation of hybridoma cell lines that secrete triazole phosphate monoclonal antibodies.

[0009] In a fourth aspect, the present invention provides a monoclonal cell line OXY that secretes a triazophos monoclonal antibody, said monoclonal cell line OXY being deposited on November 12, 2025 at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 46733.

[0010] In a fifth aspect, the present invention provides a triazophos monoclonal antibody, wherein the monoclonal antibody is prepared by secretion from the monoclonal cell line OXY.

[0011] In a sixth aspect, the invention provides the use of the monoclonal antibody in the detection of triazophos.

[0012] In a seventh aspect, the present invention provides a triazophos test strip, wherein the test strip comprises the monoclonal antibody.

[0013] An eighth aspect of the present invention provides the application of the triazophos test strip in any of the following aspects: (1) Preparation of triazophos detection kit; (2) Preparation of a triazophos detection device; (3) Detection of triazophos.

[0014] The beneficial effects of this invention are: (1) The present invention provides a triazophos hapten, which is the one with the best effect obtained by the inventor after screening multiple haptens. The hapten can be used to prepare triazophos antigen, triazophos antibody and detection reagent. (2) The present invention also provides a complete triazophos antigen, which is obtained by coupling the triazophos hapten with a carrier protein. The complete triazophos antigen can be used to prepare triazophos antibodies and triazophos detection reagents. (3) The present invention also provides a monoclonal cell line OXY that secretes triazophos monoclonal antibody. The cell line is obtained by immunizing animals with triazophos complete antigen and can be used to prepare triazophos antibody. (4) The present invention also provides a triazophos monoclonal antibody, wherein the triazophos monoclonal antibody is obtained by secretion from the monoclonal cell line OXY.

[0015] (5) This invention also provides a triazophos test strip, which has high specificity and sensitivity, with a detection limit of 3.152 ng / mL; it is also simple, rapid, and timely. Using colloidal gold chromatography test strips, no other large instruments are required, and the test results can be determined within 10 minutes after the test strip is added to the sample solution. It can detect triazophos residues in Chinese medicinal materials more quickly, sensitively, and conveniently; the result determination is vivid, intuitive, accurate, simple, and clear; it is not prone to false positives and false negatives; it saves costs, has a wide range of applications, and is easy to promote; it has broad market prospects and significant economic and social benefits. Attached Figure Description

[0016] Figure 1 A schematic diagram of antigen synthesis via UV light involving hapten and carrier protein coupling.

[0017] Figure 2 Schematic diagram of the cross-sectional structure of the triazole phosphorus colloidal gold chromatography test strip;

[0018] Note: 1. Sample pad, 2. Coating film, 3. Absorbent pad, 4. Detection line (T line), 5. Control line (C line), 6. Backing plate.

[0019] Figure 3 Results of determining the colloidal gold chromatography test strip curve of triazole phosphide in PBS; Note: From left to right, the values ​​are: 0, 20, 50, 100, and 200 ng / mL.

[0020] Figure 4 Quantitative calibration curve of triazole phosphorus colloidal gold chromatography test strip in PBS.

[0021] Figure 5 Results of the determination of the colloidal gold chromatographic test strip curve of triazole phosphorus in Angelica sinensis extract; Note: From left to right, the values ​​are: 0, 20, 50, 100, 200, and 500 μg / kg.

[0022] Figure 6 Quantitative calibration curve of triazole phosphorus colloidal gold chromatography test strip in Angelica sinensis extract. Detailed Implementation

[0023] The embodiments described below are merely illustrative of the invention and should not be construed as limiting the scope or content of the invention. The invention will be further illustrated below through these embodiments.

[0024] Unless otherwise specified, the preparation process conditions described below are all standard operations in the prior art.

[0025] Unless otherwise specified, all reagents used in the following preparation processes are commercially available.

[0026] The reagents involved in the following examples are as follows: Resuspension: 10% (w / v) sucrose, 1% (w / v) BSA, 0.1% (v / v) Triton X-100, pH 7.4; First buffer: 10mM Na2HPO4·12H2O, 3% (w / v) BSA, 0.1% (v / v) Triton X-100, 0.01% (v / v) Proclin 300; Second buffer: 20mM Na2B4O7·10H2O, 1% (w / v) BSA, 0.8% (w / v) NaCl, 0.05% (v / v) SDS-L, 0.01% (v / v) Proclin 300, surfactant S9: 0.5% (v / v), Tween-20: 0.1% (v / v); Coating solution: 20mM Na2HPO4·12H2O, surfactant S9: 0.5% (v / v).

[0027] Example 1: Preparation of triazophos hapten and monoclonal antibody 1. Preparation of triazophos antigen Purchase the CAS: 57451-70-4 structure, and hydrolyze the cyano group in the structure to obtain the structure (Ⅰ) with a carboxyl group. The specific derivatization steps are as follows: dissolve the purchased compound in 100 μL of pure methanol solution, add it dropwise to 5 mL of 0.1M sodium hydroxide solution, stir at 80℃ for 4 h to obtain compound (Ⅰ).

[0028]

[0029] 2. Preparation of immunoantigens 0.1 mmol of the synthesized compound (Ⅰ), 0.3 mmol of N-hydroxysuccinimide, and 0.3 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were dissolved in anhydrous N,N-dimethylformamide and stirred to obtain an activated hapten solution, i.e., solution A. 6 mg of bovine serum albumin was diluted with carbonate buffer to obtain solution B. Solution A was slowly added to solution B to carry out the reaction to obtain the reaction solution. After the reaction was completed, the reaction solution was dialyzed with phosphate buffer to obtain immunoantigen (Ⅱ).

[0030]

[0031] 3. Preparation of coating antigen The synthesized compound (Ⅰ), 0.3 mmol of N-hydroxysuccinimide, and 0.3 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were dissolved in anhydrous N,N-dimethylformamide and stirred to obtain an activated hapten solution, i.e., solution A. 6 mg of ovalbumin was diluted with carbonate buffer solution to obtain solution B. Solution A was slowly added to solution B to carry out the reaction to obtain the reaction solution. After the reaction was completed, the reaction solution was dialyzed with phosphate buffer to obtain the coated antigen.

[0032] 4. Preparation of triazophos monoclonal antibody and purification of ascites fluid (1) Acquisition of immunity in animals The prepared immunoantigen was emulsified with an equal amount of Freund's adjuvant and then administered to BALB / c mice via subcutaneous injection at multiple sites on the back of the neck (except for sprint immunization). The first immunization used complete Freund's adjuvant at a dose of 100 μg / mouse. For multiple booster immunizations, incomplete Freund's adjuvant was used at a dose halved to 50 μg / mouse. For sprint immunization, no adjuvant was used; the adjuvant was diluted directly with physiological saline and injected intraperitoneally at a dose halved to 25 μg / mouse. The interval between the first and second booster immunizations was one month, the interval between multiple booster immunizations was 21 days, and the interval between sprint immunization and the last booster immunization was 18-21 days. The titer and inhibition of the designed antigen in mouse serum were observed using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), and mice with the best serum assay results were selected for fusion.

[0033] The results are shown in Table 1, which shows the titer and inhibition measured by blood collected from the tail of mice after the fifth immunization. The inhibition competition rate was optimal when defined within the working point titer range.

[0034] Table 1. Results of mouse serum immunization by indirect competitive enzyme-linked immunosorbent assay (ELISA).

[0035] (2) Cell fusion and screening Three days after the sprint immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 4000) method. Cell screening was conducted on day 7, in two steps: first, positive cell wells were selected using intracellular ELISA; second, triazophos was used as a standard, and its inhibitory effect on positive cells was determined using intracellular ELISA. Cell wells showing good inhibition of the triazophos standard were selected for subcloning using limiting dilution, and the same method was used for detection seven days later.

[0036] At least three subclonings were performed using the method described above, ultimately obtaining the triazophos monoclonal cell line OXY. This monoclonal cell line OXY was named OXYBXMD and was deposited on November 12, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46733. The depository address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Telephone: 010-64807596, Fax: 010-64807288, Email: cgmcc@im.ac.cn.

[0037] (3) Preparation of monoclonal antibodies 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1×10 6Ascites fluid was collected from the triazophos monoclonal cell line OXY starting from day 7, and the ascites fluid was purified for antibody using the caprylic acid-saturated ammonium sulfate method.

[0038] Under slightly acidic conditions, octanoic acid can precipitate other proteins in the ascites fluid besides IgG immunoglobulin. After centrifugation, the precipitate is discarded, and then an equal volume of saturated ammonium sulfate solution is used to precipitate the IgG type monoclonal antibody. After centrifugation, the supernatant is discarded, and the antibody is dissolved in 0.01M phosphate buffer solution (pH 7.4), then dialyzed to desalt. The purified triazole phosphate monoclonal antibody is then stored at -20 °C.

[0039] Example 2: Preparation of Triazole Phosphate Colloidal Gold Chromatography Test Strip It should be noted that in this embodiment, the inventors only provided the optimal test strip preparation method. However, in the actual scientific research process, the inventors optimized multiple parameters to obtain the following method.

[0040] 1. Preparation of colloidal gold-monoclonal antibody complex The pH of a colloidal gold solution with a particle size of 15 nm was adjusted to 8.2 with 0.1 M K2CO3. 10 μg of triazole phosphate monoclonal antibody was diluted with resuspension and added to the colloidal gold solution. After standing for 45 min, 10% BSA solution was added, mixed well, and allowed to stand for 2 h. Then, it was centrifuged at 4 ℃ and 9500 rpm for 45 min, the supernatant was discarded, and the resulting precipitate was resuspended with resuspension.

[0041] 2. Preparation of micropores for gold nanoparticles The colloidal gold-monoclonal antibody complex was diluted 5-fold with the first buffer and added to a 96-well microplate at a density of 50 μL per well. The plate was then lyophilized and sealed in aluminum foil for later use.

[0042] 3. Preparation of sample pads The glass fiber membrane was immersed in the second buffer solution for 5 hours, then removed and dried overnight at 37 °C to obtain the sample pad. It was then sealed in an aluminum foil bag, dried, and stored for later use.

[0043] 4. Preparation of coating membrane The triazophos-coated antigen was diluted to 0.3 mg / mL and 0.1 mg / mL using coating solution. Then, two lines were drawn on the nitrocellulose membrane using a spraying device at a spray volume of 0.8 pL / cm. The membrane was then dried in a drying oven at 37 ℃ for 12 h to obtain two detection lines. The goat anti-mouse polyclonal antibody was diluted to 0.1 mg / mL using coating buffer and then streaked onto a nitrocellulose membrane at a speed of 0.8 mL / cm using a spray membrane applicator. This streak was parallel to the two detection lines. The membrane was then dried in a drying oven at 37 °C for 12 h to obtain the control line.

[0044] 5. Assembly of test strips The absorbent pad, sample pad, and nitrocellulose membrane are adhered to a PVC base plate, with the upper end of the nitrocellulose membrane partially overlapping the absorbent pad and the lower end partially overlapping the sample pad. Then, the strips are cut and assembled. The assembled test strip structure is as follows: Figure 2 As shown.

[0045] 6. Detection Principle The detection principle of the colloidal gold chromatography test strip is that the coating antigen on the T line competes with the target analyte in the sample for binding to the colloidal gold-labeled monoclonal antibody. When there is no target analyte in the sample, the colloidal gold-labeled monoclonal antibody binds only to the coating antigen on the T line, resulting in a darker T line. When the sample contains the target analyte, the colloidal gold-labeled monoclonal antibody binds to the target analyte first, reducing binding to the coating antigen on the T line, resulting in a lighter T line. As the amount of target analyte increases, the T line gradually becomes lighter. The color intensity of the T and C lines can be obtained using a colloidal gold analyzer. A standard inhibition curve of the colloidal gold chromatography test strip can be obtained by plotting (T1+T2) / C on the ordinate and the target analyte concentration on the abscissa using OriginPro 8.5.

[0046] Example 3: Performance Testing and Practical Application of Triazole Phosphate Colloidal Gold Chromatography Test Strips 1. Detection limit experiment Triazophos standards were added to PBS buffer to final concentrations of (0, 20, 50, 100, and 200 ng / mL), and the test strips prepared in Example 2 were used for testing. Each sample was tested three times.

[0047] Add 150 μL of sample solution to the microwell of the gold standard and incubate at 37 °C for 3 min. Then, take 100 μL of reaction solution and drop it vertically onto the sample pad and react for 8 min.

[0048] Test strip results as follows Figure 3 As shown, with the increase of triazophos concentration, the color of the test line for triazophos detection on the test strip gradually becomes lighter. The standard curve obtained by detecting different concentration gradients is as follows: Figure 4 As shown in the figure, the results corresponding to this curve indicate that the limit of detection for triazophos pesticide residues by this method is 3.152 ng / mL, which meets the detection requirements.

[0049] 2. Stability test The triazole phosphate colloidal gold chromatography test strips prepared in Example 2 were placed in a sealed bag with a desiccant and stored in an oven at 45 °C. They were taken out after 3, 7, 14, 21, 28 and 30 days, respectively. The triazole phosphate standard solution was diluted with PBS and then the stability of the test strips was tested.

[0050] The results showed no significant changes in the test results after 30 days. Storage at 45℃ for 30 days is equivalent to storage at room temperature for 12 months.

[0051] Example 4: Detection and Analysis of Triazophos Pesticide Residues in the Traditional Chinese Medicine Angelica sinensis Weigh 10 g (±0.1 g) of homogenized sample into a 50 mL plastic centrifuge tube. Each sample is divided into two groups: a negative sample and a positive sample. The negative sample is left untreated, while the positive sample is treated with triazophos standard at concentrations of 0, 20, 50, 100, 200, and 500 μg / kg. Then, add 3 mL of methanol to both the negative and positive samples, vortex for 3 min, and centrifuge at 3500 rpm for 5 min. Transfer 1 mL of the supernatant to a 5 mL centrifuge tube, incubate under nitrogen at 40 °C, then reconstitute with 100 μL of methanol solution, and finally dilute 10-fold with PBS solution containing 1% 870.

[0052] The test strips prepared in Example 2 were used for testing. 150 μL of sample solution was added to the microwells of the gold standard and incubated at 37°C for 3 min. 100 μL of reaction solution was then vertically added to the sample pad, and the reaction was allowed to proceed for 8 min. Each sample was tested three times. Accurate data could be obtained by reading the colloidal gold analyzer. A standard inhibition curve of the triazole phosphate colloidal gold chromatography test strip in the Angelica sinensis sample could be obtained using OriginPro 8.5.

[0053] like Figure 5 , 6 As shown, the detection results using the standard inhibition curve of the colloidal gold chromatography test strip indicate that the limit of detection for triazophos pesticide residues using this method is 10.525 μg / kg. Furthermore, this method can quantitatively analyze the residue limits of triazophos in actual Angelica sinensis samples. This demonstrates that the triazophos detection method of this invention can meet the requirements for detecting actual samples.

[0054] In summary, this invention provides a triazophos hapten, which is the most effective one obtained by the inventors after screening multiple haptens. This hapten can be used to prepare triazophos antigens, triazophos antibodies, and detection reagents. This invention also provides a complete triazophos antigen, which is obtained by conjugating the triazophos hapten with a carrier protein. This complete triazophos antigen can be used to prepare triazophos antibodies and triazophos detection reagents. This invention also provides a monoclonal cell line OXY that secretes triazophos monoclonal antibodies. This cell line is obtained by immunizing animals with the complete triazophos antigen and can be used to prepare triazophos antibodies. This invention further provides a triazophos monoclonal antibody, which is obtained by secretion from the monoclonal cell line OXY. This invention also provides a triazophos test strip, which is highly specific and sensitive, with a detection limit of 3.152 ng / mL. It is also simple, rapid, and timely, using a colloidal gold chromatography test strip, requiring no other large instruments and can be operated on-site. After adding the sample solution to the test strip, the detection result can be determined within 10 minutes. It can detect triazophos residues in traditional Chinese medicine more quickly, sensitively, and conveniently; the result interpretation is vivid, intuitive, accurate, and simple; it is less prone to false positives and false negatives; it saves costs, has a wide range of applications, and is easy to promote; it has broad market prospects and significant economic and social benefits.

[0055] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A triazole hapten, characterized in that, The structural formula of the triazole hapten is shown in formula (Ⅰ): 。 2. A complete triazophos antigen, characterized in that, It is obtained by conjugating the triazole hapten of claim 1 with a carrier protein.

3. The triazophos complete antigen as described in claim 2, characterized in that, The carrier protein is one or more of bovine serum albumin, ovalbumin, thyroprotein, or human serum albumin.

4. The use of the triazophos hapten as described in claim 1 in the preparation of hybridoma cell lines that secrete triazophos monoclonal antibodies.

5. A monoclonal cell line OXY that secretes a triazophos monoclonal antibody, characterized in that, The monoclonal cell line OXY was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 12, 2025, with accession number CGMCC No. 46733.

6. A triazophos monoclonal antibody, characterized in that, The monoclonal antibody is prepared by secretion from the monoclonal cell line OXY as described in claim 5.

7. The use of the monoclonal antibody as described in claim 6 in the detection of triazophos.

8. A triazophos test strip, characterized in that, The test strip comprises the monoclonal antibody as described in claim 6.

9. The application of the triazophos test strip as described in claim 8 in any of the following aspects: (1) Preparation of triazophos detection kit; (2) Preparation of a triazophos detection device; (3) Detection of triazophos.

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