Preparation and application of a detection kit for phosphotriphenyl esterase

Abstract

The application discloses a preparation method of a triphenyl phosphate hapten, and further discloses a triphenyl phosphate enzyme-linked immunoassay kit, a preparation method and application thereof. The kit comprises an enzyme-labeled plate coated with a triphenyl phosphate hapten and a carrier protein conjugate, antibody working solution, enzyme-labeled working solution, washing solution, sample diluent, sample extraction solution, different concentrations of triphenyl phosphate standard solution, substrate color developing solution and termination solution. The triphenyl phosphate detection kit provided by the application adopts an enzyme-linked immunosorbent assay (ELISA) method and can be used for detecting triphenyl phosphate in aquatic products, meat, eggs and milk. The detection limit of the kit for triphenyl phosphate is 0.1 µg / L, the recovery rate of each added concentration of sample is between 80 and 120%, the batch variation coefficient of each added concentration is less than 10%, the batch variation coefficient is less than 15%, and the kit has the advantages of simple operation, rapidness, high sensitivity, strong specificity, high accuracy and suitability for screening of a large number of samples.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for preparing and applying an enzyme-linked immunosorbent assay kit for detecting triphenyl phosphate. Background Technology

[0002] Triphenyl phosphate (TPHP) is a common organophosphate compound. In the chemical industry, it is widely used as a flame retardant and plasticizer in the production of polymers such as polyvinyl chloride, rubber, cellulose resins, and vinyl resins. Furthermore, TPHP serves as an intermediate in the preparation of organophosphate pesticides. TPHP does not form chemical bonds with the polymer matrix and is mainly released into the environment through hydraulic oil leakage and leaching, aging and volatilization of plastic products, and industrial production processes, particularly accumulating in sediments and natural water bodies. In aquatic bodies, TPHP can accumulate and migrate through the food chain in aquatic organisms, exhibiting significant endocrine toxicity, cardiotoxicity, neurotoxicity, and reproductive and developmental toxicity. Exposure to TPHP in humans is widespread, and it can ultimately accumulate in the human body through the food chain, harming health. Therefore, the potential environmental hazards and health risks of TPHP cannot be ignored.

[0003] Detecting TPHP levels is crucial for ensuring human safety. Extraction methods for TPHP vary depending on the substance: solid-phase extraction is commonly used in liquid environments (such as seawater); Soxhlet extraction, ultrasonic extraction, and rapid solvent extraction are common methods in soil; and acetonitrile-ultrasonic extraction is commonly used for enrichment in biological tissues. After extraction in different media, TPHP is quantitatively analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) or gas chromatography-tandem mass spectrometry (GC-MS / MS). However, these methods suffer from drawbacks such as cumbersome detection processes, long detection times, large amounts of organic solvents used, complex operations, and expensive equipment, hindering widespread adoption. Based on biorecognition materials such as monoclonal antibodies, rapid immunoassay techniques (such as enzyme-linked immunosorbent assay) offer advantages such as ease of operation, short detection times, no need for expensive equipment, low professional requirements, and suitability for on-site testing, playing a significant role in accurate compound identification and rapid screening. However, no immunological detection methods for triphenyl phosphate have been reported to date.

[0004] The main factors affecting the quality of ELISA detection are the specificity and affinity of antigens and antibodies. After specific binding between antigens and antibodies, enzymatic catalysis allows for qualitative or quantitative analysis of the detected substance, improving detection sensitivity. The specificity and affinity of antigens and antibodies depend on the structure of the immune hapten molecule; therefore, the molecular design and synthesis of immune haptens are the most fundamental and crucial steps in generating specific antibodies and establishing rapid detection technologies for small molecule residues. This invention provides a method for synthesizing a triphenyl phosphate hapten, and further synthesizes immunogens and coating antigens, prepares mouse monoclonal antibodies, and ultimately develops an enzyme-linked immunosorbent assay kit for detecting triphenyl phosphate. Summary of the Invention

[0005] The purpose of this invention is to provide an enzyme-linked immunosorbent assay (ELISA) kit for detecting triphenyl phosphate, and to provide a detection method that is highly sensitive, specific, low in cost, and suitable for screening large batches of samples.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] An enzyme-linked immunosorbent assay (ELISA) kit for detecting triphenyl phosphate comprises an ELISA plate, standard working solution, antibody working solution, enzyme-labeled working solution, sample diluent, sample extraction solution, washing solution, substrate chromogenic solution, and stop solution.

[0008] In the kit, the enzyme-labeled plate is coated with a conjugate of triphenyl phosphate hapten and carrier protein.

[0009] The structure of the triphenyl phosphate hapten is shown in Formula I:

[0010]

[0011] The method for preparing the triphenyl phosphate hapten is as follows:

[0012] (1) Add 680.6 mg of toluene diphenyl phosphate to a 50 mL reaction flask, add 20 mL of carbon tetrachloride, and stir until dissolved;

[0013] (2) Add 50 mg of benzoyl peroxide and stir at room temperature for 10 min;

[0014] (3) Add 430 mg of N-bromosuccinimide, reflux at 80 °C and stir for 16 hours, cool to room temperature and filter to remove NBS and byproducts;

[0015] (4) After the filtrate is concentrated under reduced pressure, dichloromethane is added to dissolve it. After complete dissolution, 2000 mg of 100-200 mesh silica gel is added and mixed. The sample is then packed into a column of 200-300 mesh silica gel for chromatography. The elution is carried out with petroleum ether: ethyl acetate = 10:1. The main product is collected to obtain 780 mg of toluene diphenyl phosphate methyl bromide in oil.

[0016] (5) Add 275 mg of 4-methylaminobutyrate salt to a 50 mL reaction flask, add 15 mL of NN dimethylformamide, stir until dissolved, then add 745 mg of anhydrous potassium carbonate and stir at room temperature for 30 min.

[0017] (6) Add 630 mg of toluene diphenyl phosphate methyl bromide and stir at 50 °C for 16 hours;

[0018] (7) Filter to remove salt, concentrate the filtrate under reduced pressure, add dichloromethane to dissolve, add 2000mg of 100-200 mesh silica gel and mix, pack into a 200-300 mesh silica gel column for chromatography, elute with petroleum ether: ethyl acetate = 1:1.5, and collect 550mg of the main product, which is the hapten compound described in Formula I.

[0019] The method for preparing the triphenyl phosphate antigen is also within the scope of protection of this invention.

[0020] The triphenyl phosphate antigen is obtained by coupling the triphenyl phosphate hapten (Formula I) with a carrier protein via an acid-amine condensation reaction.

[0021] The carrier protein may specifically be bovine serum albumin (BSA), bovine thyroglobulin (BTG), human serum albumin (HSA), mouse serum albumin (MSA), keyhole hemocyanin (KLH), rabbit serum albumin (RSA), human serum albumin (HSA), or ovalbumin (OVA).

[0022] The triphenyl phosphate hapten (Formula I) was coupled with bovine serum albumin (BSA) to prepare the triphenyl phosphate immunogen, with the molar ratio of triphenyl phosphate to bovine serum albumin being 8.16:1.

[0023] In this invention, the triphenyl phosphate antigen is prepared according to the following steps:

[0024] (1) Immunogen: 8.15 mg of the triphenyl phosphate (Formula I) was dissolved in 2 mL of dimethylformamide (DMF), and 10.3 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 6.18 mg of N-hydroxysuccinimide (NHS) were added. The mixture was stirred magnetically at 20-25 °C for 2-3 h to obtain solution I. 20 mg of BTG was fully dissolved in 5 mL of CB buffer and added to solution I. The mixture was reacted overnight to obtain solution II. Solution II was dialyzed with phosphate buffer solution (0.01 mol / L, pH 7.2) at 4 °C for 3 days to obtain the triphenyl phosphate immunogen.

[0025] (2) Coating agent: 20.37 mg of the triphenyl phosphate (Formula I) was added to 2 mL of DMF and stirred until completely dissolved. 25.75 mg of EDC and 15.46 mg of NHS were added and the mixture was magnetically stirred at 20-25 °C for 2-3 h to obtain solution I (1). 50 mg of BSA protein was fully dissolved in 5 mL of CB solution and then added to solution I (1). The mixture was reacted overnight to obtain solution II (2). Solution II (2) was dialyzed at 4 °C for 3 days with phosphate buffer solution (0.01 mol / L, pH 7.2) to obtain the triphenyl phosphate coating agent.

[0026] The enzyme-labeled plate is a 96-well plate coated with triphenyl phosphate-coated antigen.

[0027] The preparation steps for the triphenyl phosphate antibody are as follows:

[0028] (1) Animal Immunization

[0029] The prepared triphenyl phosphate immunogen was injected into mice at a dose of 100 μg / mouse. The triphenyl phosphate antigen was dissolved in physiological saline and mixed with an equal volume of Freund's complete adjuvant. The mice were then subcutaneously injected into the neck and back of 6-8 week old Balb / c female mice. On days 7, 14, and 28 after the initial immunization, the immunogen was mixed with an equal volume of Freund's incomplete adjuvant and administered as a booster immunization. Three days before fusion, a booster immunization was administered with 100 μg / mouse of the immune complex without Freund's adjuvant.

[0030] (2) Cell fusion and cloning

[0031] Following standard procedures, spleen cells from immunized mice were mixed with myeloma cells (SP2 / 0) in the logarithmic growth phase. Preheated fusion agent (PEG 4000) was then slowly added over 45 seconds for fusion. The cells were then suspended in HAT medium and homogenized. An appropriate amount of feeder cells was added, and the mixture was cultured in 96-well plates at 37°C in a 5% CO2 incubator. After 5 days, the medium was partially replaced with HT medium, and after 9 days, the medium was completely replaced.

[0032] After cell fusion, when the cells reached 1 / 4 of the culture well area, hybridoma cells were screened using a stepwise screening method. Initial selection was performed using an indirect ELISA method. The ELISA plate was coated with a coating antigen (pre-titrated using a checkerboard method to determine the optimal coating concentration and positive serum dilution). Culture supernatant from the test wells was added, and the plates were incubated. After washing, goat anti-mouse IgG-HRP and IgM-HRP were added. OPD (o-phenylenediamine) was oxidized, resulting in a colorimetric reaction. Positive wells selected based on the colorimetric reaction were then screened using an indirect competitive ELISA method. Cell supernatant was first mixed with an equal volume of 100 μg / mL triphenyl phosphate, incubated at 37°C for 30 min, and then added to the coated ELISA plate. The control group used PBS instead of triphenyl phosphate, with the remaining steps the same. If OD200 after triphenyl phosphate blockade...450nm If the value drops to below 50% of that of the control well, it is considered positive. Wells that are positive after 2 to 3 tests should be immediately subcloned using the limiting dilution method.

[0033] (3) Preparation and purification of monoclonal antibodies

[0034] Hybridoma cells, after 2-3 subclonings, were expanded and cultured. The supernatant was collected, and the titer was determined by indirect ELISA. The cells were then frozen. Eight- to ten-week-old Balb / c mice were intraperitoneally injected with 0.5 mL of liquid paraffin per mouse. Seven to ten days later, hybridoma cells (1-2 × 10⁵ per mouse) were injected intraperitoneally. Seven to ten days later, ascites fluid was collected from the mice. The cells were purified using the caprylic acid-saturated ammonium sulfate method, and the purified monoclonal antibody against triphenyl phosphate was obtained from the supernatant.

[0035] In the kit, the solvent of the standard working solution is PBS buffer containing a light stabilizer and bovine serum albumin, and the solute is triphenyl phosphate; the concentrations of the solute in the six standard working solutions are 0 μg / L, 0.01 μg / L, 0.03 μg / L, 0.09 μg / L, 0.27 μg / L, 0.81 μg / L and 2.43 μg / L, respectively; the PBS buffer is the same as the antibody dilution PBS buffer, with a pH of 7.2.

[0036] In the kit, the working solution of the triphenyl phosphate antibody is obtained by diluting a specific monoclonal antibody of triphenyl phosphate 9000 times with antibody diluent.

[0037] The antibody diluent is a PBS buffer containing bovine serum albumin, gelatin, preservatives Proclin-300, Tween-20, and Tris; the solvent of the antibody diluent is deionized water, and the solutes are bovine serum albumin, gelatin, preservatives Proclin-300, Tween-20, Tris, disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride; the concentrations of bovine serum albumin, gelatin, preservatives Proclin-300, Tween-20, and Tris in deionized water are 2.5%, 0.1%, 0.02%, 1 mL / L, and 0.1 g / L, respectively; the solutes in the PBS are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride, and the concentrations of these solutes in deionized water are 1.44 g / L, 0.24 g / L, 8.0 g / L, and 0.3 g / L, respectively.

[0038] In the kit, the enzyme-labeled working solution is obtained by diluting horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody 500 times with secondary antibody dilution buffer.

[0039] The secondary antibody diluent is a PBS buffer containing fetal bovine serum, glycerol, and Proclin 300; the PBS buffer is prepared in the same way as above; the concentrations of fetal bovine serum, glycerol, and Proclin 300 in the PBS buffer are 200 mL / L, 50 mL / L, and 1 mL / L, respectively.

[0040] In the kit, the sample diluent is a PBS buffer containing a light stabilizer, bovine serum albumin, and a surfactant; the PBS buffer is prepared as described above; the concentrations of the light stabilizer, bovine serum albumin, and surfactant in the PBS buffer are 0.2 g / L, 0.1 g / L, and 0.1 g / L, respectively, and the pH value is 7.2.

[0041] In the kit, the sample extraction solution is a PBS buffer containing a light stabilizer, bovine serum albumin, and a surfactant; the PBS buffer is prepared in the same manner as above; the concentrations of the light stabilizer, bovine serum albumin, and surfactant in the PBS buffer are 0.2 g / L, 0.1 g / L, and 0.2 g / L, respectively, and the pH value is 7.2.

[0042] In the kit, the washing solution is a PBS buffer containing Tween-20 and Proclin 300; the PBS buffer is prepared as described above; the concentrations of Tween-20 and Proclin 300 in the PBS buffer are 20 mL / L and 300 μL / L, respectively.

[0043] In the kit, the substrate chromogenic solution is a mixed aqueous solution of 1.0 g / L urea peroxide, 5.0 g / L sodium acetate, 0.5 g / L light stabilizer, 2.5 mL / L phosphoric acid, and 5.0 g / L tetramethylbenzidine, with deionized water as the solvent.

[0044] In the kit, the stop solution is a 0.05 mol / L aqueous sulfuric acid solution.

[0045] Another object of the present invention is to provide a method for detecting triphenyl phosphate in a sample, comprising the following steps:

[0046] (1) Pretreatment of the sample to obtain a solution of the sample;

[0047] (2) Use the above kit to test the solution;

[0048] (3) Analyze the test results.

[0049] The sample pretreatment method is as follows: accurately weigh 1±0.1g (or mL) of sample and add 5mL of sample extract, vortex at high speed for 1min at room temperature (25±2℃), centrifuge at 5000g for 5min, take 200μL of supernatant and add 200μL of sample diluent, shake and mix thoroughly before testing.

[0050] The detection steps of the kit are as follows: Add 50 μL of standard working solution or sample pretreatment solution to the ELISA plate coated with the triphenyl phosphate antigen, and then add the specific antibody solution containing triphenyl phosphate; after incubation, wash 4 times with washing buffer, pat dry thoroughly, add enzyme-labeled anti-antibody, develop color for 10 min, add stop solution, and use an ELISA reader to measure the absorbance of the solution at 450 nm and 620 nm.

[0051] The analysis process for the detection results provided by this invention is as follows: Divide the average absorbance (B) of the obtained standard working solution of each concentration by the absorbance value (B0) of the first standard solution (0 ng / mL standard), and then multiply by 100% to obtain the percentage absorbance value. The calculation formula is: Percent absorbance value (%) = (B / B0) × 100%.

[0052] A standard curve was plotted with the half-logarithm of the concentration (μg / L) of the triphenyl phosphate standard working solution as the X-axis and the percentage absorbance as the Y-axis. The percentage absorbance of the sample solutions was calculated using the same method, and the triphenyl phosphate content in each sample could be read from the standard curve.

[0053] The analysis of the detection results in this invention can also be performed using the regression equation method to calculate the concentration of the sample solution.

[0054] The triphenyl phosphate hapten and the triphenyl phosphate antigen provided by this invention have simple synthesis methods, high purity, and high yield, and are of great value for the preparation of triphenyl phosphate antibodies and the detection of triphenyl phosphate drug residues.

[0055] The principle of the enzyme-linked immunosorbent assay (ELISA) kit for detecting triphenyl phosphate described in this invention is as follows: Triphenyl phosphate in the sample competes specifically with the antigen fixed on the ELISA plate. Enzyme-labeled secondary antibody is added, reacting with the antibody. Color development is achieved through enzyme catalysis and a chromogenic agent. The content of triphenyl phosphate in the sample is determined based on the intensity of the color development. The absorbance is negatively correlated with the triphenyl phosphate content in the sample. Substituting the absorbance into the standard curve and multiplying it by the corresponding dilution factor yields the residual amount of triphenyl phosphate in the sample. Attached Figure Description

[0056] Figure 1 This is the mass spectrum of the triphenyl phosphate hapten.

[0057] Figure 2 This is the NMR structure diagram of the triphenyl phosphate hapten.

[0058] Figure 3 This is the mass spectrum of the carrier protein BSA.

[0059] Figure 4 This is the mass spectrum of the triphenyl phosphate antigen.

[0060] Figure 5 This is a standard curve for the detection of triphenyl phosphate. Detailed Implementation

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0062] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0063] The kit or detection method of this invention can detect triphenyl phosphate residues in aquatic products and meat, eggs and dairy products.

[0064] Example 1: Preparation of Triphenyl Phosphate Hapten

[0065] I. Preparation of Triphenyl Phosphate Hapten

[0066] (1) Add 680.6 mg of toluene diphenyl phosphate to a 50 mL reaction flask, add 20 mL of carbon tetrachloride, and stir until dissolved;

[0067] (2) Add 50 mg of benzoyl peroxide and stir at room temperature for 10 min;

[0068] (3) Add 430 mg of N-bromosuccinimide, reflux at 80 °C and stir for 16 hours, cool to room temperature and filter to remove NBS and byproducts;

[0069] (4) After the filtrate is concentrated under reduced pressure, dichloromethane is added to dissolve it. After complete dissolution, 2000 mg of 100-200 mesh silica gel is added and mixed. The sample is then packed into a column of 200-300 mesh silica gel for chromatography. The elution is carried out with petroleum ether: ethyl acetate = 10:1. The main product is collected to obtain 780 mg of toluene diphenyl phosphate methyl bromide in oil.

[0070] (5) Add 275 mg of 4-methylaminobutyrate salt to a 50 mL reaction flask, add 15 mL of NN dimethylformamide, stir until dissolved, then add 745 mg of anhydrous potassium carbonate and stir at room temperature for 30 min.

[0071] (6) Add 630 mg of toluene diphenyl phosphate methyl bromide and stir at 50 °C for 16 hours;

[0072] (7) Filter to remove salt, concentrate the filtrate under reduced pressure, add dichloromethane to dissolve, add 2000mg of 100-200 mesh silica gel and mix, pack into a 200-300 mesh silica gel column for chromatography, elute with petroleum ether: ethyl acetate = 1:1.5, and collect the main product to obtain the hapten compound described in Formula I.

[0073] The reaction equation is as follows:

[0074]

[0075] II. Identification of Triphenyl Phosphate Hapten

[0076] The obtained triphenyl phosphate hapten was detected by mass spectrometry. Figure 1 The results showed that its chemical structure was as shown in Formula I, which is the triphenyl phosphate hapten. The target hapten has a molecular weight of 455.45, and a strong peak appears at m / z 478 in the mass spectrum. Furthermore, the synthesized hapten was identified using nuclear magnetic resonance (NMR), and the results are as follows: Figure 2 As shown. By Figure 1 and Figure 2 The results show that the hapten structure is correct.

[0077]

[0078] Example 2: Preparation of Triphenyl Phosphate Detection Kit

[0079] I. Preparation of ELISA Plates

[0080] The enzyme-labeled plate is a 96-well plate coated with triphenyl phosphate-coated antigen.

[0081] The triphenyl phosphate antigen is obtained by coupling the triphenyl phosphate hapten (Formula I) with the carrier protein through an acid-amine condensation chemical reaction. The specific synthesis method is as follows:

[0082] (3) Immunogen: 8.15 mg of the triphenyl phosphate (Formula I) was dissolved in 2 mL of dimethylformamide (DMF), 10.3 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 6.18 mg of N-hydroxysuccinimide (NHS) were added, and the mixture was magnetically stirred at 20-25 °C for 2-3 h to obtain solution I; 20 mg of BTG was fully dissolved in 5 mL of catechol borane solution (CB), and then added to solution I and reacted overnight to obtain solution II; solution II was dialyzed at 4 °C for 3 days with phosphate buffer solution (0.01 mol / L, pH 7.2) to obtain the triphenyl phosphate immunogen.

[0083] (4) Coating agent: 20.37 mg of the triphenyl phosphate (Formula I) was added to 2 mL of DMF and stirred until completely dissolved. 25.75 mg of EDC and 15.46 mg of NHS were added and the mixture was stirred magnetically at 20-25 °C for 2-3 h to obtain solution I (1). 50 mg of BSA protein was fully dissolved in 5 mL of CB solution and then added to solution I (1). The mixture was reacted overnight to obtain solution II (2). Solution II (2) was dialyzed at 4 °C for 3 days with phosphate buffer solution (0.01 mol / L, pH 7.2) to obtain the triphenyl phosphate coating agent.

[0084] The phenyl phosphate coating was prepared by coupling phenyl phosphate hapten (Formula I) and ovalbumin (BSA) in a molar ratio of 8.16:1.

[0085] The obtained triphenyl phosphate coating antigen was diluted to 10 μg / mL with coating buffer. 100 μL of antigen was added to each well, and the plate was incubated at 37°C for 2 h. The diluent was discarded, and the plate was washed three times with washing buffer for 30 s each time. After drying, 150 μL of blocking buffer was added to each well, and the plate was incubated at 37°C for 1 h. The blocking buffer was then discarded. After drying, the ELISA plate was obtained and vacuum-sealed with aluminum foil for storage.

[0086] The coating buffer is a 0.1 mol / L citrate buffer solution with a pH of 6.0.

[0087] The blocking solution is a 0.2 mol / L pH 7.7 phosphate buffer solution containing 20 g / L sucrose, 30 g / L skim milk powder, 2.5 g / L casein, 0.25% fetal bovine serum, and 3‰ sodium azide.

[0088] II. Preparation of Antibody Working Solution

[0089] 1. Preparation of Triphenyl Phosphate Antibody

[0090] (1) Animal Immunization

[0091] The prepared triphenyl phosphate immunogen was injected into mice at a dose of 100 μg / mouse. The triphenyl phosphate antigen was dissolved in physiological saline and mixed with an equal volume of Freund's complete adjuvant. The mice were then subcutaneously injected into the neck and back of 6-8 week old Balb / c female mice. On days 7, 14, and 28 after the initial immunization, the immunogen was mixed with an equal volume of Freund's incomplete adjuvant and administered as a booster immunization. Three days before fusion, a booster immunization was administered with 100 μg / mouse of the immune complex without Freund's adjuvant.

[0092] The triphenyl phosphate immunogen was prepared by coupling triphenyl phosphate hapten (Formula I) with bovine serum albumin (BSA) in a molar ratio of 8.16:1.

[0093] (2) Cell fusion and cloning

[0094] Following standard procedures, spleen cells from immunized mice were mixed with myeloma cells (SP2 / 0) in the logarithmic growth phase. Preheated fusion agent (PEG 4000) was then slowly added over 45 seconds for fusion. The cells were then suspended in HAT medium and homogenized. An appropriate amount of feeder cells was added, and the mixture was cultured in 96-well plates at 37°C in a 5% CO2 incubator. After 5 days, the medium was partially replaced with HT medium, and after 9 days, the medium was completely replaced.

[0095] After cell fusion, when the cells reached 1 / 4 of the culture well area, hybridoma cells were screened using a stepwise screening method. Initial selection was performed using an indirect ELISA method. The ELISA plate was coated with a coating antigen (pre-titrated using a checkerboard method to determine the optimal coating concentration and positive serum dilution). Culture supernatant from the test wells was added, and the plates were incubated. After washing, goat anti-mouse IgG-HRP and IgM-HRP were added. OPD (o-phenylenediamine) was oxidized, resulting in a colorimetric reaction. Positive wells selected based on the colorimetric reaction were then screened using an indirect competitive ELISA method. Cell supernatant was first mixed with an equal volume of 100 μg / mL triphenyl phosphate, incubated at 37°C for 30 min, and then added to the coated ELISA plate. The control group used PBS instead of triphenyl phosphate, with the remaining steps the same. If OD200 after triphenyl phosphate blockade... 450nm If the value drops to below 50% of that of the control well, it is considered positive. Wells that are positive after 2 to 3 tests should be immediately subcloned using the limiting dilution method.

[0096] (3) Preparation and purification of monoclonal antibodies

[0097] Hybridoma cells obtained after 2-3 subclonings were cultured extensively, and the supernatant was collected for titer determination by indirect ELISA and then frozen. Eight- to ten-week-old Balb / c mice were intraperitoneally injected with 0.5 mL of liquid paraffin per mouse, and 7-10 days later, 1-2 × 10⁶ hybridoma cells were injected intraperitoneally. 5 Ascites fluid was collected from each mouse 7–10 days later. The fluid was purified using the caprylic acid-saturated ammonium sulfate method, and the supernatant was used to obtain the purified monoclonal antibody against triphenyl phosphate.

[0098] The checkerboard method was used to determine the titer of the monoclonal antibody. The results showed that the titer of the triphenyl phosphate monoclonal antibody was 1:27000, and the half-maximal inhibitory concentration (IC50) was [missing value]. 50 The concentration was 0.091 μg / L.

[0099] The amino acid sequences of the heavy chain variable region of the triphenyl phosphate monoclonal antibody were shown in Sequence 1 of the sequence listing, and the amino acid sequences of the light chain variable region of the triphenyl phosphate monoclonal antibody were shown in Sequence 2 of the sequence listing.

[0100] Sequence 1:

[0101] Glu Val Arg Leu Gln Glu Ser Gly Ser Val Leu Ala Arg Pro Gly Ala SerVal Lys Met Ser Cys Lys Ala Ser Pro Ser Arg Phe Tyr Ser Thr Trp Leu His TrpIle Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Thr Gly Gly Ile Tyr Arg Pro AsnGlu Val Asn Ser Tyr Lys Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Ala Val ThrSer Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala ValTyr Tyr Cys Ile Pro Gly Thr Gln Ile Trp Gly Gln Gly Thr Thr Val Thr Val HisArg

[0102] Sequence 2:

[0103] Asp Val Asn Leu Leu Thr Gln Ser Pro Leu Thr Leu Ser Tyr Thr Ile GlyGIn Pro Ala Ser Ile Ser Cys Lys Ser Gln Leu Thr Asn Asp Arg Asp Gly Glu ThrVal Tyr Asn Trp Leu Phe Gln Arg Pro Gly Gln Ser Pro Lys Arg Leu Ile Tyr SerVal Ser Phe Leu Asp Ser Gly Val Pro Asp Arg Phe Tyr Gly Ser Phe Ser Gly ThrAsp Phe Thr Leu Lys His Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr CysTrp Gly Pro Thr Val Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Val

[0104] 2. Preparation of antibody working solution

[0105] The obtained monoclonal antibody against triphenyl phosphate was diluted 1000 times with antibody diluent to obtain an antibody working solution containing the monoclonal antibody against triphenyl phosphate.

[0106] The antibody diluent is a PBS buffer containing bovine serum albumin, gelatin, preservatives Proclin-300, Tween-20, and Tris; the solvent of the antibody diluent is deionized water, and the solutes are fetal bovine serum albumin, gelatin, preservatives Proclin-300, Tween-20, Tris, disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride; the PBS solution is deionized water, and the solvents are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride, with concentrations of 1.44 g / L, 0.24 g / L, 8.0 g / L, and 0.3 g / L, respectively; the concentrations of bovine serum albumin, gelatin, preservatives Proclin-300, Tween-20, and Tris in deionized water are 2.5%, 0.1%, 0.01%, 1 mL / L, and 0.1 g / L, respectively.

[0107] II. Preparation of enzyme-labeled working solutions

[0108] The enzyme-labeled working solution was obtained by diluting horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody 500 times with secondary antibody dilution buffer.

[0109] The secondary antibody diluent is a PBS buffer containing fetal bovine serum, glycerol, and Proclin 300; the PBS buffer is prepared in the same way as above; the concentrations of fetal bovine serum, glycerol, and Proclin 300 in the PBS buffer are 200 mL / L, 50 mL / L, and 1 mL / L, respectively.

[0110] III. Preparation of Standard Working Solution

[0111] In the kit, the solvent of the standard working solution is PBS buffer containing a light stabilizer and bovine serum albumin, and the solute is triphenyl phosphate; the concentrations of the solute in the seven standard working solutions are 0 μg / L, 0.01 μg / L, 0.03 μg / L, 0.09 μg / L, 0.27 μg / L, 0.81 μg / L and 2.43 μg / L, respectively; the PBS buffer is the same as the antibody dilution PBS buffer, with a pH of 7.2.

[0112] IV. Preparation of other reagents

[0113] The kit may also contain sample diluent and / or sample extraction solution and / or washing solution and / or substrate color development solution and / or stop solution.

[0114] In the kit, the sample diluent is a PBS buffer containing a light stabilizer, bovine serum albumin, and a surfactant; the PBS buffer is prepared as described above; the concentrations of the light stabilizer, bovine serum albumin, and surfactant in the PBS buffer are 0.2 g / L, 0.1 g / L, and 0.1 g / L, respectively, and the pH value is 7.2.

[0115] In the kit, the sample extraction solution is a PBS buffer containing a light stabilizer, bovine serum albumin, and a surfactant; the PBS buffer is prepared in the same manner as above; the concentrations of the light stabilizer, bovine serum albumin, and surfactant in the PBS buffer are 0.2 g / L, 0.1 g / L, and 0.2 g / L, respectively, and the pH value is 7.2.

[0116] In the kit, the washing solution is a PBS buffer containing Tween-20 and Proclin 300; the PBS buffer is prepared as described above; the concentrations of Tween-20 and Proclin 300 in the PBS buffer are 20 mL / L and 300 μL / L, respectively.

[0117] In the kit, the substrate chromogenic solution is a solution containing urea peroxide, sodium acetate, a light stabilizer, phosphoric acid, and tetramethylbenzidine; the solvent of the substrate chromogenic solution is deionized water, and the solutes are urea peroxide, sodium acetate, a light stabilizer, phosphoric acid, and tetramethylbenzidine, with solute concentrations of 1.0 g / L, 5.0 g / L, 0.5 g / L, 2.5 mL / L, and 5.0 g / L, respectively.

[0118] The terminating solution is a 0.05 mol / L aqueous solution of sulfuric acid.

[0119] Instructions for use of the reagent kits in Example 3 and Example 2

[0120] I. Sample Pretreatment

[0121] Raw milk: Take 100 μL of the mixed raw milk for testing.

[0122] Animal meat: Weigh 1.00±0.05g of homogenized sample into a 10mL centrifuge tube (for egg samples, mix thoroughly and take 1±0.05mL of sample); add 0.8mL of 0.3M NaOH, vortex for 1min; add 4mL of ethyl acetate, vortex for 3min, centrifuge at 4000g for 5min; transfer 500μL of the supernatant into a 5mL centrifuge tube, and dry under nitrogen at 60℃; add 400μL of sample diluent to the dried centrifuge tube, shake well, and prepare for testing. The sample diluent consists of 29.01g of disodium hydrogen phosphate dodecahydrate, 2.96g of sodium dihydrogen phosphate dihydrate, and 5.84g of sodium chloride, diluted to 1L with pure water. Adjust the pH to 8.0 with 1mol / L sodium hydroxide.

[0123] II. Detection using the kit from Example 1

[0124] 1. Insert the ELISA plate into the ELISA plate holder and record the position of each standard and sample. Perform 3 replicates for each sample. Seal unused ELISA strips in a self-sealing bag and store them immediately at 2-8°C.

[0125] 2. Add 50 μL of each standard working solution or sample solution to the corresponding standard or sample well;

[0126] 3. Add 50 μL of antibody working solution to each well;

[0127] 4. Cover the plate with the protective film, gently shake the plate for 10 seconds to mix thoroughly, and incubate at room temperature (25±2℃) in the dark for 30 minutes.

[0128] 5. Remove the cover film, pour out the liquid in the wells, add 260μL of washing working solution to each well (the washing solution is diluted 20 times with deionized water), and wash thoroughly 3-4 times, soaking for 15-30 seconds each time;

[0129] 6. Pour out the liquid from the wells, invert the ELISA plate onto absorbent paper, and pat it dry;

[0130] 7. Add 100 μL of enzyme-labeled working solution to each well; cover the plate with the membrane, gently shake the plate for 10 seconds to mix thoroughly, and incubate at room temperature (25±2℃) in the dark for 30 minutes.

[0131] 8. Repeat steps 5-6;

[0132] 9. Immediately add 100 μL of substrate color development solution A and B mixture (substrate color development solution A and substrate color development solution B are mixed in a 1:1 volume ratio) to each well, cover with the cover film, and react in the dark for 15 min.

[0133] 10. Remove the cover film, add 50 μL of stop solution to each well, and gently shake the ELISA plate for 10 seconds to mix thoroughly.

[0134] 11. Within 5 minutes after termination, use an ELISA reader to read the absorbance value of the ELISA plate at dual wavelengths of 420nm and 630nm.

[0135] III. Analysis of Test Results

[0136] 1. Calculate the percentage absorbance value

[0137] The percentage of absorbance of each standard (or sample to be tested) can be obtained by dividing the average absorbance value of each standard (or sample to be tested) by the absorbance value of the zero standard (standard with a concentration of 0 μg / L) and multiplying by 100%.

[0138] Absorbance percentage = B / B0 × 100%

[0139] Wherein: B - average absorbance value of standard (or sample); B0 - average absorbance value of standard with a concentration of 0 μg / L.

[0140] 2. Create a standard curve

[0141] A standard curve was plotted with the percentage absorbance of each standard on the ordinate and the concentration of triphenyl phosphate (μg / L) in the working solution of each standard on the abscissa. Nonlinear fitting analysis was performed using Origin 8.0 (OriginLab Corp., Northampton, MA, USA) to generate a four-parameter fitting curve.

[0142] y = D + (AD) / [1 + (x / C)^B]

[0143] Where y is the absorbance percentage; x is the concentration of the analyte; and A, B, C, and D are the four parameters of the standard curve.

[0144] Based on the test data, the standard curve equation for triphenyl phosphate is: y = 7.06 + (100.06 - 7.06) / [1 + (x / 0.091)^0.83], with a linear correlation R0.83. 2 It is 0.99.

[0145] The standard curve is attached. Figure 5 As shown.

[0146] 3. Calculate the content of triphenyl phosphate in the sample.

[0147] By substituting the percentage absorbance value of the sample into the standard curve, the residual concentration of the sample can be obtained. Multiplying this by the dilution factor of the sample yields the actual content of triphenyl phosphate in the original sample.

[0148] Example 4: Specificity, detection limit, accuracy, and precision of the kits from Example 2.

[0149] I. Specificity test of the kit:

[0150] The specificity of the triphenyl phosphate enzyme-linked immunosorbent assay kit is determined by cross-reactivity testing with the corresponding substance.

[0151] Triphenyl phosphate and its analogues, including tris(2-chloroethyl) phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCPP), tris(1,3-dichloro-2-propyl) phosphate (TDCPP), and tert-Butylphenyldiphenyl phosphate (MDPP), were serially diluted according to Example 3, replacing the "triphenyl phosphate standard working solution" with the serial dilutions of triphenyl phosphate and its analogues. Standard curves were prepared, and the 50% inhibition concentration (IC50) of each was determined from the curves. 50 The specific method is as follows: Obtain the triphenyl phosphate concentration (μg / L) corresponding to a vertical axis value equal to 50%, i.e., IC50. 50 Value. Calculate the cross-reactivity of the kit with triphenyl phosphate and various commonly used organophosphates and carbamate pesticides using the following formula:

[0152] Cross-reactivity rate (%) = (concentration of triphenyl phosphate causing 50% inhibition / concentration of triphenyl phosphate analog causing 50% inhibition) × 100%.

[0153] The results are shown in Table 1.

[0154] Table 1. Specificity of the kit

[0155]

[0156] Experiments show that the kit of the present invention has good specificity for triphenyl phosphate, that is, the kit of the present invention can detect triphenyl phosphate.

[0157] II. Determination of the detection limit of the kit

[0158] According to the detection indicators, blank samples (negative LC-MS / MS tests) of fish, shrimp, pork, mutton, eggs, and milk were taken and tested according to the method in Example 3. The measured values ​​were obtained from the standard curve, and the average value was calculated. Adding three times the standard deviation gave the limit of detection (LOD). The results are shown in Table 2.

[0159] Table 2. Results of blank sample determination

[0160]

[0161] The results showed that, to prevent false positives, the detection limit of this kit for triphenyl phosphate in aquatic products, meat, eggs, and milk can be set at 0.1 μg / L.

[0162] III. Accuracy and Precision Tests of the Reagent Kit

[0163] Accuracy refers to the degree of agreement between the measured value and the true value. Accuracy is often expressed as recovery rate, while precision is often expressed as coefficient of variation. Blank samples (LC-MS / MS negative) of aquatic products, meat, eggs, and milk were pretreated according to the method described in step one of Example 3, and then triphenyl phosphate standard was added to the required concentrations of 0.1 μg / kg and 0.2 μg / kg to obtain the test sample solutions.

[0164] The tests were performed using three different batches of reagent kits, with each test repeated six times. The coefficient of variation was calculated for each batch. The results are shown in Table 3.

[0165] The calculation method for intra-batch coefficient of variation: Intra-batch coefficient of variation = coefficient of variation of each parallel sample in the same determination.

[0166] The calculation method of inter-batch coefficient of variation: Inter-batch coefficient of variation = the coefficient of variation of the same sample measured in different batches, and take the average value.

[0167] Table 3. Accuracy and precision of sample addition and recovery

[0168]

[0169]

[0170] The results showed that the recoveries of all samples at all spiked concentrations were between 80% and 120%. The intra-batch coefficient of variation for all spiked concentrations was less than 10%, and the inter-batch coefficient of variation was less than 15%.

[0171] IV. Comparison of detection results from this kit with those from LC-MS / MS

[0172] The method described in Example 3 was used to test samples of aquatic products, meat, eggs, and milk, and the results were compared with those obtained by LC-MS / MS for confirmation.

[0173] A scatter plot was drawn with the concentration of triphenyl phosphate measured by this kit as the X-axis and the concentration of triphenyl phosphate measured by LC-MS / MS as the Y-axis. Linear analysis was performed on the results of the two methods, and the regression equation was: y = 0.022 + 0.997x, with a correlation coefficient R0. 2 The value of 0.999 indicates that the method established in this invention has good consistency with the LC-MS / MS detection results.

[0174] V. Shelf life test of the reagent kit

[0175] Example 2 kit was stored at 2–8°C. After 12 months of testing, the maximum absorbance (zero standard), 50% inhibition concentration, and actual measured values ​​of triphenyl phosphate addition were all within the normal range. Considering the possibility of abnormal storage conditions during transportation and use, the kit was placed at 37°C for 8 days for accelerated aging testing. The results showed that all indicators in steps one to four of the above steps fully met the requirements. Considering the possibility of freezing, the kit was placed at -20°C for 8 days, and all indicators in steps one to four of the above steps fully met the requirements. From the above results, it can be concluded that the kit of Example 1 can be stored at 2–8°C for at least 12 months.

Claims

1. An enzyme-linked immunosorbent assay (ELISA) kit for detecting triphenyl phosphate, comprising: The enzyme-labeled plate coated with a triphenyl phosphate coating agent, antibody working solution, enzyme-labeled working solution, sample dilution solution, sample extraction solution, standard solutions containing different concentrations of triphenyl phosphate, washing solution, substrate chromogenic solution, and stop solution; the triphenyl phosphate coating agent is obtained by conjugating the carboxyl group of the triphenyl phosphate hapten shown in Formula I with bovine thyroglobulin; the antibody working solution contains a triphenyl phosphate monoclonal antibody; the triphenyl phosphate monoclonal antibody is prepared by animal immunization with a triphenyl phosphate immunogen; the triphenyl phosphate immunogen is obtained by conjugating the carboxyl group of the triphenyl phosphate hapten shown in Formula I with bovine serum albumin. Formula I.

2. The enzyme-linked immunosorbent assay (ELISA) kit for detecting triphenyl phosphate according to claim 1, characterized in that: The preparation method of the triphenyl phosphate hapten is as follows: 680.6 mg of toluene diphenyl phosphate was added to 20 mL of carbon tetrachloride and stirred until dissolved. Then, 50 mg of benzoyl peroxide was added and stirred at room temperature for 10 min. 430 mg of N-bromosuccinimide was added, and the mixture was refluxed at 80 °C for 16 hours. The mixture was cooled to room temperature and filtered to remove NBS and byproducts. The filtrate was concentrated under reduced pressure, dissolved in dichloromethane, and mixed with 2000 mg of 100-200 mesh silica gel. The mixture was then packed into a 200-300 mesh silica gel column for chromatography, eluted with petroleum ether:ethyl acetate = 10:1, and the main product was collected to obtain the methyl bromide of toluene diphenyl phosphate. 275 mg of 4-methylaminobutyrate was added to a 50 mL reaction flask, followed by 15 mL of N,N-dimethylformamide. After stirring until dissolved, 745 mg of anhydrous potassium carbonate was added and stirred at room temperature for 30 min. 630 mg of N-bromosuccinimide was added. The methyl bromide of toluene diphenyl phosphate was stirred at 50°C for 16 hours. The salt was removed by filtration, the filtrate was concentrated under reduced pressure, dissolved in dichloromethane, and 2000 mg of 100-200 mesh silica gel was added and mixed. The mixture was then packed into a 200-300 mesh silica gel column for chromatography. The elution was performed with petroleum ether:ethyl acetate = 1:1.

5. The main product was collected to obtain the triphenyl phosphate hapten.

3. The enzyme-linked immunosorbent assay (ELISA) kit for detecting triphenyl phosphate according to claim 1, characterized in that: The antibody working solution is obtained by diluting the triphenyl phosphate monoclonal antibody 1000 times with antibody diluent to obtain a triphenyl phosphate monoclonal antibody working solution; the triphenyl phosphate monoclonal antibody is prepared through animal immunization, cell fusion and cloning and purification steps; The enzyme-labeled working solution was obtained by diluting commercially available goat anti-mouse secondary antibody 500 times with secondary antibody dilution buffer. The concentrations of the standard solutions were 0 μg / L, 0.01 μg / L, 0.03 μg / L, 0.09 μg / L, 0.27 μg / L, 0.81 μg / L, and 2.43 μg / L, respectively. The sample diluent was a PBS buffer containing a light stabilizer, bovine serum albumin, and a surfactant. The concentrations of the light stabilizer, bovine serum albumin, and surfactant in the PBS buffer were 0.2 g / L, 0.1 g / L, and 0.1 g / L, respectively, and the pH value was 7.

2. The sample extraction solution was a PBS buffer containing a light stabilizer, bovine serum albumin, and a surfactant. The concentrations of the light stabilizer, bovine serum albumin, and surfactant in the PBS buffer were 0.2 g / L, 0.1 g / L, and 0.2 g / L, respectively, and the pH value was 7.

2. The washing solution is a PBS buffer containing Tween-20 and Proclin 300, with concentrations of 20 mL / L and 300 μL / L for Tween-20 and Proclin 300, respectively. The substrate colorimetric solution is prepared using deionized water as the solvent and urea peroxide, sodium acetate, a light stabilizer, phosphoric acid, and tetramethylbenzidine as the solutes, with concentrations of 1.0 g / L, 5.0 g / L, 0.5 g / L, 2.5 mL / L, and 5.0 g / L, respectively. The terminating solution is a 0.05 mol / L aqueous solution of sulfuric acid.

4. The enzyme-linked immunosorbent assay (ELISA) kit for detecting triphenyl phosphate according to claim 3, characterized in that: The solvent for the antibody diluent is deionized water, and the solutes are bovine serum albumin, gelatin, preservatives Proclin-300, Tween-20, disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, and Tris. The concentrations of the solutes in deionized water are 2.5%, 0.1%, 0.01%, 1 mL / L, 1.44 g / L, 0.24 g / L, 8.0 g / L, 0.3 g / L, and 0.1 g / L, respectively.

5. The enzyme-linked immunosorbent assay (ELISA) kit for detecting triphenyl phosphate according to claim 3, characterized in that: The secondary antibody dilution solution is a PBS buffer containing fetal bovine serum, glycerol, and Proclin 300; the concentrations of fetal bovine serum, glycerol, and Proclin 300 in the PBS buffer are 200 mL / L, 50 mL / L, and 1 mL / L, respectively.

6. The method of using the enzyme-linked immunosorbent assay kit for detecting triphenyl phosphate according to claim 1, characterized in that... Includes the following steps: 1) Sample pretreatment; 2) Use a kit for testing; 3) Analysis of test results.

7. The method of using the enzyme-linked immunosorbent assay kit for detecting triphenyl phosphate according to claim 6, characterized in that: The kit can detect the content of triphenyl phosphate in samples, with a detection limit of 0.1 μg / kg, an intra-batch coefficient of variation of less than 10%, and an inter-batch coefficient of variation of less than 15%.

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