A phosphorus acid triphenyl ester fluorescent quantitative rapid detection method, product and application thereof

Abstract

The application discloses a phosphorus acid triphenyl ester fluorescent quantitative rapid detection method and products and application thereof, and relates to a phosphorus acid triphenyl ester hapten, an artificial antigen, a monoclonal antibody and a preparation method thereof, and a phosphorus acid triphenyl ester fluorescent quantitative rapid detection test strip. The test strip comprises a sample pad, a label pad, a reaction membrane and a water absorption pad which are sequentially connected and fixed on a PVC bottom plate. The label pad is coated with an anti-phosphorus acid triphenyl ester monoclonal antibody which is labeled with a detectable label. The reaction membrane is provided with a T line and a C line. The T line is coated with a phosphorus acid triphenyl ester artificial antigen, and the C line is coated with a sheep anti-phosphorus acid triphenyl ester antibody. The test strip is combined with the detection method in the application, and has the advantages of high specificity, high sensitivity, short detection time, low cost, low skill requirement for an operator and the like under the premise of ensuring detection accuracy, and the detection limit reaches 0.1 ug / kg. The test strip is suitable for rapid detection of a large number of samples on site and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of rapid food safety testing, and relates to a test strip and a testing method for detecting the residual amount of triphenyl phosphate in aquatic products, animal muscle, eggs, and milk. Background Technology

[0002] Triphenyl phosphate (TPHP) is a widely used aromatic organophosphate flame retardant (aryl-OPFRs) that causes biotoxicity through accumulation in organisms via environmental media such as air and water. Most furniture, appliances, and building materials contain TPHP. Due to its volatility and difficulty in degradation, it has become a recognized emerging organic pollutant in air and dust. TPHP released into the air can enter water bodies through rainfall and surface runoff, and industrial wastewater generated during the production of TPHP and related products can also pollute water sources. TPHP is lipid-soluble, and in aquatic environments, it can enter and accumulate in aquatic organisms through contact and filter feeding. In recent years, TPHP has been found in humans, with inhalation, skin absorption, and food ingestion being common accumulation pathways. The World Health Organization has confirmed that long-term exposure to TPHP can cause allergic reactions, skin inflammation, and even adversely affect male fertility.

[0003] Currently, methods for detecting triphenyl phosphate (TPHP) include instrumental methods such as gas chromatography, gas chromatography-tandem mass spectrometry (GC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS). These methods are characterized by high specificity and sensitivity, but they are cumbersome to operate and expensive, making them unsuitable for screening and detecting large batches of samples and failing to meet on-site testing needs. Therefore, establishing efficient, sensitive, accurate, and portable analytical methods is of great significance for studying the enrichment effect of TPHP in organisms. Fluorescence immunochromatography combines the high sensitivity of ELISA with the rapid characteristics of colloidal gold assays, offering advantages such as ease of operation, high sensitivity, high throughput, and rapid detection, and has promising application prospects. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology. This invention provides a fluorescent quantitative rapid test strip for detecting triphenyl phosphate content, its preparation method and application, which can accurately and rapidly detect triphenyl phosphate residues in animal-derived foods on a large scale.

[0005] To achieve the above objectives, the present invention employs the following technical means:

[0006] The present invention provides a triphenyl phosphate hapten, the structure of which is shown below:

[0007]

[0008] The preparation method of the triphenyl phosphate hapten includes the following steps:

[0009] 680.6 mg of toluene diphenyl phosphate was added to a 50 mL reaction flask, followed by 20 mL of carbon tetrachloride. After stirring until dissolved, 50 mg of benzoyl peroxide was added. The mixture was stirred at room temperature for 10 min, and then 430 mg of N-bromosuccinimide was added. The mixture was refluxed at 80 °C and stirred for 16 h. 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. Elution was performed using petroleum ether:ethyl acetate = 10:1. The main product was collected to obtain 750 mg of methyl bromide of toluene diphenyl phosphate as an oily substance.

[0010] 250 mg of 4-piperidinecarboxylic acid was added to a 50 mL reaction flask, followed by 15 mL of N,N dimethylformamide. After stirring until dissolved, 520 mg of anhydrous potassium carbonate was added. The mixture was stirred at room temperature for 30 min, and then 650 mg of methyl bromide of toluene diphenyl phosphate was added. The mixture was stirred at 50 °C for 16 h. The salt was removed by filtration, and the filtrate was concentrated under reduced pressure. The filtrate was 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 and ethyl acetate in a 1:1 ratio. The main product was collected to obtain 585 mg of methyl-modified 4-piperidinecarboxylic acid hapten of toluene diphenyl phosphate in an oily form.

[0011] The specific synthetic route is as follows:

[0012]

[0013] The artificial antigen of triphenyl phosphate constructed based on the triphenyl phosphate hapten also falls within the scope of protection of this invention. The artificial antigen of triphenyl phosphate includes an immunogen and a coating antigen, which is obtained by coupling the above-mentioned triphenyl phosphate hapten with a macromolecular carrier protein.

[0014] Triphenyl phosphate monoclonal antibodies prepared based on triphenyl phosphate antigen are also within the scope of protection of this invention, specifically prepared by immunizing mice with triphenyl phosphate artificial antigen as an immunogen.

[0015] The amino acid sequence of the light chain of the triphenyl phosphate monoclonal antibody is: Val Asp Val Leu Ser ThrGln Pro Leu Thr Leu Ser Val Thr Ile Gly Pro GIn Ala Ser Ile Cys Ser Lys SerSer Gln Leu Lys Leu Asp Tyr Ser Asn Asp Gly Thr Leu Asn Arg Trp Leu Phe GlnPro Gly Gln Ser Pro Lys Arg Leu Ile Tyr Val Asp Val Ser Glu Leu Ser Gly ValAsp Arg Phe Gly Pro Ser Gly Ser Gly Tyr Thr Asp Thr Phe Ser Lys Ile Arg SerVal Glu Ala Glu Asp Trp Gly Val Tyr Tyr Cys Asn Trp Gly Thr Phe His Pro LysTrp Thr Phe Ala Gly Ser Thr Phe Leu Glu Gly; the amino acid sequence of the heavy chain is: Gln Leu GluAla Val Gln Ser Ser Gly Thr Val Leu Arg Pro Gly Ala Ser Val Lys Met Ser CysHis Ala Ser Gly Tyr Tyr Gln Phe His Ser Trp Leu His Trp Ile Gln Arg Pro GlyGln Lys Gly Leu Glu Trp Val Gly Gly Ile Pro Tyr Gly Asn Arg Ser Val Thr SerTyr Lys Gln Lys Phe Lys Asp Lys Thr Ala Leu Thr Ala Val Thr Ser Ala Ser ThrAla Tyr Met Glu Leu Ser Leu Thr Ser Asn Glu Asp Ser Ala Val Tyr Tyr Cys IleArg Gly Thr Tyr Asn Trp Gly Gln Gly Thr Val Thr Val Ser Thr His.

[0016] The present invention also includes a fluorescent quantitative rapid test strip for detecting triphenyl phosphate, wherein the detection area of ​​the test strip is coated with the above-mentioned triphenyl phosphate coating agent, and the quality control area is coated with goat anti-triphenyl phosphate.

[0017] The present invention also provides a method for detecting triphenyl phosphate, the method comprising the following steps:

[0018] Take an appropriate amount of the sample to be tested and drop it onto the sample pad of the above test strip. Incubate at 40℃ for 5 minutes. Use a fluorescence immunoassay analyzer to detect the sample. The excitation wavelength is 360-365nm and the detection wavelength is 610-615nm. Obtain the T-line fluorescence signal value and the C-line fluorescence signal value. Substitute the ratio of the T-line fluorescence signal value to the C-line fluorescence signal value into the standard curve to obtain the triphenyl phosphate content in the sample to be tested.

[0019] The beneficial effects of this invention are as follows: This invention designs a suitable triphenyl phosphate hapten, synthesizes an artificial antigen, and obtains an antibody with high specificity and sensitivity, providing a rapid quantitative fluorescent test strip for triphenyl phosphate. While ensuring high specificity and sensitivity and detection accuracy, it also has the advantages of short detection time, low cost, and low skill requirements for operators. It is suitable for rapid detection of a large number of samples on site and has good application prospects. Attached Figure Description

[0020] Figure 1 Mass spectrum of triphenyl phosphate hapten

[0021] Figure 2 NMR spectrum of triphenyl phosphate hapten

[0022] Figure 3 MALDI-TOF-MAS plot for BSA

[0023] Figure 4 MALDI-TOF-MAS diagram of the triphenyl phosphate-BSA complex.

[0024] Figure 5 Standard curve for rapid quantitative fluorescence detection of triphenyl phosphate test strips. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0026] Example 1: Preparation of Triphenyl Phosphate Hapten

[0027] I. Preparation of Triphenyl Phosphate Hapten

[0028] 680.6 mg of toluene diphenyl phosphate was added to a 50 mL reaction flask, followed by 20 mL of carbon tetrachloride. After stirring until dissolved, 50 mg of benzoyl peroxide was added. The mixture was stirred at room temperature for 10 min, and then 430 mg of N-bromosuccinimide was added. The mixture was refluxed at 80 °C and stirred for 16 h. 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. Elution was performed using petroleum ether:ethyl acetate = 10:1. The main product was collected to obtain 750 mg of methyl bromide of toluene diphenyl phosphate as an oily substance.

[0029] 250 mg of 4-piperidinecarboxylic acid was added to a 50 mL reaction flask, followed by 15 mL of N,N dimethylformamide. After stirring until dissolved, 520 mg of anhydrous potassium carbonate was added. The mixture was stirred at room temperature for 30 min, and then 650 mg of methyl bromide of toluene diphenyl phosphate was added. The mixture was stirred at 50 °C for 16 h. The salt was removed by filtration, and the filtrate was concentrated under reduced pressure. The filtrate was 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 and ethyl acetate in a 1:1 ratio. The main product was collected to obtain 585 mg of methyl-modified 4-piperidinecarboxylic acid hapten of toluene diphenyl phosphate in an oily form.

[0030] The hapten synthesis route is as follows:

[0031]

[0032] II. Structural Identification of Triphenyl Phosphate Hapten

[0033] 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 molecular weight of the target hapten was 467.15, and a strong peak appeared at m / z 468 in its 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.

[0034]

[0035] Example 2: Preparation and structural identification of triphenyl phosphate artificial antigen

[0036] 1. Synthesis of immunogens

[0037] (1) Dissolve 12.54 mg of triphenyl phosphate hapten in 2 mL of DMF, add 15.45 mg of EDC to dissolve, then add 9.28 mg of NHS and activate at room temperature (500 rpm) for 2-3 hours.

[0038] (2) Weigh 20 mg of BTG protein and dissolve it in 5 mL of CB buffer. Stir at 200 rpm for 10 min to fully dissolve it. Add the reaction solution from step 1 dropwise (1 mL / min) while stirring at 1000 rpm. Stir at 500 rpm for 8 h.

[0039] (3) The reaction product was placed in a dialysis bag (10cm) rinsed with distilled water, and dialyzed with 1L 0.01M PBS (1×, pH 7.2) at 4℃ with stirring (100rpm) for 3 days. The medium was changed 3 times a day (once in the morning, once in the afternoon, and once in the evening) for a total of 9 changes. The dialysis product was centrifuged at 5000rpm for 6min, aliquoted into 1.5mL / tube, and stored at -20℃ for later use.

[0040] 2. Synthesis of coating antigens

[0041] (1) Dissolve 31.37 mg of triphenyl phosphate hapten in 2 mL of DMF, add 38.63 mg of EDC to dissolve, then add 23.2 mg of NHS and stir at room temperature (500 rpm) for 2-3 hours to activate.

[0042] (2) Weigh 50mg of BSA and dissolve it in 5mL of CB buffer. Stir at 200rpm for 10min to fully dissolve it. Add the reaction solution from step 1 dropwise (1mL / min) while stirring at 1000rpm. Stir at 500rpm for 8h.

[0043] (3) The reaction product was placed in a dialysis bag (10cm) rinsed with distilled water, and dialyzed with 1L 0.01M PBS (1×, pH 7.2) at 4℃ with stirring (100rpm) for 3 days. The medium was changed 3 times a day (once in the morning, once in the afternoon, and once in the evening) for a total of 9 times. The dialyzed product was centrifuged at 5000rpm for 6min, aliquoted into 1.5mL / tube, and stored at -20℃ for later use.

[0044] 2. Identification of antigens

[0045] (1) The concentrations of the synthesized immunogen and coating antigen were determined by ultraviolet absorption spectrometry. 0.01M PBS was used as a blank control, and the OD of the protein solution was measured using a NanoDrop 2000 ultraviolet spectrophotometer. 280 nm OD 260nm The results for immunogen and coating antigen were 12.47 mg / mL and 8.25 mg / mL, respectively.

[0046] (2) The immunogen was identified using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). The identification results were obtained by... Figure 3 (Results of BSA carrier protein MALDI-TOF detection) and Figure 4 (Results of immunogenic triphenyl phosphate-BSAMALDI-TOF detection) are shown; the molar ratio of triphenyl phosphate hapten (Formula I) to bovine serum albumin (BSA) was calculated to be: R = (70390.35 - 66269.14) / 467.15 = 8.82.

[0047] Example 3: Preparation of monoclonal antibodies by immunizing animals with triphenyl phosphate artificial antigen

[0048] (1) Immunizing mice

[0049] Using the artificial antigen of triphenyl phosphate, which is based on the above-mentioned carrier protein and is a bovine serum albumin, as an immunogen, and emulsified with an equal volume of Freund's adjuvant, 6-8 week old Balb / c female mice were immunized subcutaneously in the neck and back. The immunization dose per mouse was 100 μg. On days 7, 14, and 28 after the initial immunization, an additional immunization was performed by mixing the immunogen with an equal volume of Freund's incomplete adjuvant. Three days before fusion, a final immunization was performed by administering 100 μg of the immune complex per mouse without Freund's adjuvant.

[0050] Seven days after the fourth immunization, orbital venous blood was collected from mice to detect serum titers. After being incubated at room temperature for 2 hours, the supernatant antiserum was collected after centrifugation at 4000 rpm for 10 minutes. The optimal working concentrations of the coating antigen and antibody were determined by indirect ELISA square array method, and the sensitivity of the antibody was then detected by indirect competitive ELISA method.

[0051] (2) Hybridoma cell fusion

[0052] Spleen cells from immunized mice were mixed with myeloma cells (SP2 / 0) in the logarithmic growth phase. Preheated fusion agent (PEG4000) was slowly added over 45 seconds to induce fusion. The cells were then suspended in HAT medium and a suitable amount of feeder cells were added. 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.

[0053] (3) Subcloning and screening

[0054] When the cells grew to 1 / 4 of the culture well area, hybridoma cells were screened using a stepwise screening method. The cell supernatant was tested with ELISA. Cells in wells with strong positive results were subjected to limiting dilution clonal culture. Cells in wells that were positive after three clonal culture tests were identified as hybridoma cells secreting monoclonal antibodies.

[0055] (4) Antibody Acquisition

[0056] After amplification and culture of hybridoma cells, they were inoculated into the peritoneal cavity of 8-10 week old Balb / c mice to produce antibody-containing ascites. The ascites was purified using the caprylic acid-ammonium sulfate precipitation method to obtain high-purity, high-specificity monoclonal antibodies, which were then stored at -20°C.

[0057] The amino acid sequences of the light chain variable region of the triphenyl phosphate monoclonal antibody were obtained as shown in Sequence 1, and the amino acid sequences of the heavy chain variable region of the triphenyl phosphate monoclonal antibody were obtained as shown in Sequence 2.

[0058] Sequence 1: Val Asp Val Leu Ser Thr Gln Pro Leu Thr Leu Ser Val Thr Ile GlyPro GIn Ala Ser Ile Cys Ser Lys Ser Ser Gln Leu Lys Leu Asp Tyr Ser Asn AspGly Thr Leu Asn Arg Trp Leu Phe Gln Pro Gly Gln Ser Pro Lys Arg Leu Ile TyrVal Asp Val Ser Glu Leu Ser Gly Val Asp Arg Phe Gly Pro Ser Gly Ser Gly TyrThr Asp Thr Phe Ser Lys Ile Arg Ser Val Glu Ala Glu Asp Trp Gly Val Tyr TyrCys Asn Trp Gly Thr Phe His Pro Lys Trp Thr Phe Ala Gly Ser Thr Phe Leu GluGly;

[0059] Sequence 2: Gln Leu Glu Ala Val Gln Ser Ser Gly Thr Val Leu Arg Pro Gly AlaSer Val Lys Met Ser Cys His Ala Ser Gly Tyr Tyr Gln Phe His Ser Trp Leu HisTrp Ile Gln Arg Pro Gly Gln Lys Gly Leu Glu Trp Val Gly Gly Ile Pro Tyr GlyAsn Arg Ser Val

[0060] Thr Ser Tyr Lys Gln Lys Phe Lys Asp Lys Thr Ala Leu Thr Ala Val ThrSer Ala Ser Thr Ala Tyr Met Glu Leu Ser Leu Thr Ser Asn Glu Asp Ser Ala ValTyr Tyr Cys Ile Arg Gly Thr Tyr Asn Trp Gly Gln Gly Thr Val Thr Val Ser ThrHis.

[0061] Example 4: Antibody labeling of fluorescent microspheres

[0062] (1) Add 50 μL of fluorescent microspheres to 450 μL of MES (0.05 M, pH 5.0) activation buffer and sonicate for 5 min; then add EDC and NHS solutions to the microsphere solution in sequence to make the final concentrations of both 1 mmol / L and shake at room temperature for 0.5 h.

[0063] (2) Centrifuge at 12000g for 5 min and discard the supernatant; reconstitute the precipitate with 500 μL PB (0.04M, pH 8.0), sonicate for 5 min, add 3 μL monoclonal antibody, oscillate at room temperature for 2 h, and centrifuge at 15000g for 5 min.

[0064] (3) Add 500 μL of blocking buffer (0.01 M PB, 2% BSA, pH 8.0) to the precipitate and react overnight at 4°C with shaking;

[0065] (4) Centrifuge the reaction solution at 15000g for 5min, discard the supernatant, reconstitute the microspheres with 50μL 20mM PB, sonicate for 2min, and store at 4℃ in the dark for later use to obtain fluorescent microsphere-labeled triphenyl phosphate monoclonal antibody.

[0066] Example 5: Preparation of fluorescent quantitative immunochromatographic test strips

[0067] Using a membrane coating instrument, the triphenyl phosphate coating agent obtained in Example 2 (8.25 mg / mL, 0.5 μL / cm) and the goat anti-mouse antibody (2.11 mg / mL, 0.2 μL / cm) were coated onto a nitrocellulose membrane (NC membrane) as the detection zone (T line) and the quality control zone (C line), respectively.

[0068] The fluorescent microsphere-labeled triphenyl phosphate monoclonal antibody prepared in Example 4 was sprayed onto the labeling pad at a spray volume of 1.5 μL / cm and dried in an oven at 37°C for 2 hours to obtain a labeling pad containing fluorescent microsphere-labeled triphenyl phosphate monoclonal antibody.

[0069] The sample pad, the labeled pad containing fluorescent microsphere-labeled antibody, the nitrocellulose membrane containing the detection zone T and the control zone C, and the absorbent pad are sequentially glued to a PVC base plate. One end of the base plate is the absorbent pad, and the other end is the sample pad. The two ends of the nitrocellulose membrane are overlapped (1-3 mm) with the absorbent pad and the labeled pad containing fluorescent microsphere-labeled antibody, respectively. The sample pad is pressed on the labeled pad containing fluorescent microsphere-labeled antibody (overlapped 1-3 mm). The strips are then cut into 3.90 mm wide test strips to obtain the test strips for detecting triphenyl phosphate.

[0070] Example 6: Method for quantitative detection of triphenyl phosphate

[0071] 1. Drawing the standard curve

[0072] Take triphenyl phosphate standard (LC-MS / MS detects no triphenyl phosphate), and add triphenyl phosphate to each of the seven standard working solutions. The concentrations of the solute in the seven standard working solutions are 0 μg / kg, 0.01 μg / kg, 0.03 μg / kg, 0.09 μg / kg, 0.27 μg / kg, 0.81 μg / kg, and 2.43 μg / kg, respectively.

[0073] Take 100 μL of each of the above samples and add them to the sample pad of the test strip. Incubate at 40℃ for 5 min. Use a fluorescence immunoassay analyzer to detect the samples. The excitation wavelength is 365 nm and the detection wavelength is 610 nm. Obtain the fluorescence signal values ​​of the T line and the C line.

[0074] Each concentration was tested five times, and the average value was taken. The concentration of triphenyl phosphate added to the sample was used as the X-axis, and the ratio of the fluorescence signal value of the detection area (T) to the fluorescence signal value of the control area (C) (T / C) was used as the Y-axis. A four-parameter nonlinear fitting analysis was performed using Origin 8.0 to obtain the standard curve: Y = 0.0227 + 0.9606 / (1 + (x / 0.0697)). 1.8259 ).

[0075] The results are as follows Figure 5 As shown, the fitting of the test data demonstrates that the established detection method IC 50 R is 0.070. 2 =0.999.

[0076] (2) Detection of the sample to be tested

[0077] Add 100 μL of sample solution to the sample pad of the rapid quantitative test strip for triphenyl phosphate. After reacting for 5 minutes, remove the sample and use a fluorescence immunoassay analyzer to detect the T-line fluorescence signal value and the C-line fluorescence signal value. Substitute the ratio of the T-line fluorescence signal value to the C-line fluorescence signal value into the standard curve to obtain the concentration of triphenyl phosphate in each sample.

[0078] Example 7: Performance evaluation of the method for detecting triphenyl phosphate

[0079] (1) Limit of detection and limit of quantitation of the test strip

[0080] Following the method described in "Testing of Samples", 20 samples each of aquatic products, pork, eggs, and milk that tested negative using triphenyl phosphate test strips (LC-MS / MS tests were negative) were tested. The mean and standard deviation of the triphenyl phosphate concentration were calculated respectively: the limit of detection was the mean plus 3 times the standard deviation; the limit of quantitation was the mean plus 10 times the standard deviation.

[0081] Table 1. Validation of the detection limit (μg / kg) of the rapid test strip for triphenyl phosphate.

[0082]

[0083] The verification results are shown in Table 1. To ensure the accuracy and reliability of the results, the detection limit of the rapid quantitative test strip for triphenyl phosphate fluorescence detection of the present invention is set at 0.10 μg / kg, and the quantification limit is set at 0.20 μg / kg.

[0084] (2) Method accuracy and precision

[0085] Take 20 samples each of negative aquatic products, pork, eggs, and milk, and add triphenyl phosphate to each sample. The drug concentration added to the samples is the corresponding limit of quantitation and twice the limit of quantitation. Five replicates are added for each concentration gradient. Calculate the sample recovery rate and intra-batch and inter-batch coefficients of variation.

[0086] The accuracy and precision of the established fluorescence immunochromatographic assay were analyzed based on the recovery data.

[0087] Table 2. Accuracy and precision of rapid test strips for triphenyl phosphate.

[0088]

[0089]

[0090] Recovery rate = (average value / added concentration) × 100%; Coefficient of variation = (standard deviation / average value) × 100%;

[0091] The results are shown in Table 2. The recovery rate of the test strips of the present invention was 91.64-115.66%, and the intra-batch and inter-batch coefficients of variation were both less than 15%, indicating that the method has good accuracy and precision.

[0092] (3) Test strip sample determination

[0093] The triphenyl phosphate test strip and detection method provided by this invention were used to test blind samples (20 samples each of aquatic products, pork, eggs, and milk). The results are shown in Table 3. The blind samples consisted of negative samples and positive samples at different concentration levels. The negative samples were those confirmed as negative by the reference method, and the positive samples were those confirmed as positive samples at a specified concentration by the reference method.

[0094] Table 3 Comparison of rapid test strip results and LC-MS / MS results for triphenyl phosphate (μg / kg)

[0095]

[0096] The results showed that in the blind sample detection capability validation of aquatic products, pork, eggs, and milk, blind samples with triphenyl phosphate content of 0.1 μg / kg and 0.5 μg / kg were all positive (detection rate 100%), while blind samples with triphenyl phosphate content of 0.05 μg / kg were not detected (detection rate 0%). All triphenyl phosphate negative samples were correctly identified. Therefore, the triphenyl phosphate fluorescent quantitative test strip has good accuracy and can be used for the detection of triphenyl phosphate in animal-derived food samples such as aquatic products, pork, eggs, and milk.

Claims

1. A rapid quantitative fluorescent test strip for triphenyl phosphate, characterized in that, The system includes a sample pad, a labeling pad, a reaction membrane, and an absorbent pad. The labeling pad is coated with a triphenyl phosphate monoclonal antibody. The reaction membrane has a detection zone and a control zone. The detection zone is coated with a triphenyl phosphate artificial antigen. The triphenyl phosphate artificial antigen is obtained by conjugating the carboxyl group of the triphenyl phosphate hapten shown in Formula I with bovine serum albumin. The triphenyl phosphate monoclonal antibody is prepared using a triphenyl phosphate immunogen, which is obtained by conjugating the carboxyl group of the triphenyl phosphate hapten shown in Formula I with bovine thyroglobulin. Formula I.

2. The rapid quantitative fluorescent test strip for triphenyl phosphate according to claim 1, characterized in that, The method for preparing the triphenyl phosphate hapten includes the following steps: Toluene diphenyl phosphate and carbon tetrachloride were added to a reaction flask and dissolved. Carbon tetrachloride was then added and stirred until dissolved. Benzoyl peroxide was added and N-bromosuccinimide was added. The mixture was reacted at 80°C for 16 h. After chromatographic elution, the main product was collected to obtain the methyl bromide of toluene diphenyl phosphate. 4-Piperidinic acid and N,N-dimethylformamide were added to a reaction flask and dissolved. Anhydrous potassium carbonate was added and reacted at room temperature. The methyl bromide of toluene diphenyl phosphate was added and reacted at 50°C for 16 h. After chromatographic elution, the triphenyl phosphate hapten was obtained.

3. The rapid quantitative fluorescent test strip for triphenyl phosphate according to claim 1, characterized in that, The light chain variable region of the triphenyl phosphate monoclonal antibody has the amino acid sequence shown in SEQ ID NO: 1, and the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:

2.

4. The rapid quantitative fluorescent test strip for triphenyl phosphate according to claim 1, characterized in that, The detectable marker is a fluorescent microsphere-labeled triphenyl phosphate monoclonal antibody.

5. The rapid quantitative fluorescent test strip for triphenyl phosphate according to claim 1, characterized in that, The test samples are aquatic products, pork, eggs, or milk. The detection limit for triphenyl phosphate in the above samples is 0.1 μg / kg, and the quantitation limit is 0.2 μg / kg.

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