CN117783530A

CN117783530A - Preparation and application of immunoassay kit for detecting triphenyl phosphate esterase

Info

Publication number: CN117783530A
Application number: CN202311782827.8A
Authority: CN
Inventors: 秦誉; 马立才; 贾良曦; 贾晶莹; 丁亚芳; 杨柳; 邢维维; 金兴
Original assignee: Beijing Wdwk Biotechnology Co ltd
Current assignee: Beijing Wdwk Biotechnology Co ltd
Priority date: 2023-12-22
Filing date: 2023-12-22
Publication date: 2024-03-29

Abstract

The invention discloses a preparation method of triphenyl phosphate hapten, and also discloses a kit for detecting triphenyl phosphate enzyme linked immunosorbent assay, and a preparation method and application thereof. The kit comprises: the kit comprises an ELISA plate coated with triphenyl phosphate hapten and carrier protein conjugate, an antibody working solution, an enzyme labeling working solution, a washing solution, a sample dilution solution, a sample extraction solution, triphenyl phosphate standard solution with different concentrations, a substrate color development solution and a termination solution. The triphenyl phosphate detection kit provided by the invention 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 on triphenyl phosphate is 0.1 mu g/L, the recovery rate of each added concentration of the sample is between 80 and 120 percent, the intra-batch variation coefficient of each added concentration is lower than 10 percent, and the inter-batch variation coefficient is lower than 15 percent, and the kit has the advantages of simplicity and rapidness in operation, high sensitivity, strong specificity, strong accuracy and suitability for screening a large number of samples.

Description

Preparation and application of immunoassay kit for detecting triphenyl phosphate esterase

Technical Field

The invention belongs to the technical field of biology, and relates to a preparation method and application of a kit for detecting triphenyl phosphate linked immunosorbent assay.

Background

Triphenyl phosphate (Triphenyl phosphate, TPHP) is one of the common organophosphate compounds. In the chemical industry, the flame retardant and the plasticizer are widely applied to the production of high polymers such as polyvinyl chloride, rubber, cellulose resin, vinyl resin and the like. In addition, TPHP can be used as an intermediate of organophosphorus pesticides for preparing pesticides. TPHP does not form chemical bonds with the polymeric matrix, and is mainly released into environmental media through leakage and leaching of hydraulic oil, ageing and volatilizing of plastic products and industrial production processes, and particularly is enriched in sediments and natural water bodies. While TPHP in water can accumulate and migrate in aquatic organisms through food chains, and can show obvious endocrine toxicity, cardiac toxicity, neurotoxicity and reproductive and developmental toxicity. The exposure of TPHP to the human body is widespread and can be eventually enriched in the human body by the food chain, jeopardizing the physical health. It follows that the potential environmental hazards and health risks of TPHP are not negligible.

The detection of the TPHP content has important significance for guaranteeing the safety of human bodies. The extraction method of TPHP in different substances is often different: the method commonly used in liquid environments (such as seawater) is a solid phase extraction method; the common methods in the soil include Soxhlet extraction, ultrasonic extraction, rapid solvent extraction and the like; the common enrichment method in biological tissues is acetonitrile-ultrasonic extraction method and the like. After the TPHP is extracted in different media, quantitative analysis is carried out by utilizing an ultra-high performance liquid chromatography-tandem mass spectrometry or a gas chromatography-tandem mass spectrometry. However, the method has the defects of complicated detection process, long detection time, large organic solvent consumption, complex operation, expensive instrument and the like, and is difficult to popularize. Based on biological recognition materials such as monoclonal antibodies, the immune rapid screening technology (such as an enzyme-linked immunosorbent assay) has the advantages of simple operation, short detection time, no need of expensive equipment, low professional requirements, suitability for field detection and the like, and plays a great role in precise recognition and rapid screening of compounds. However, no report on the immunological detection method of triphenyl phosphate is currently available.

The main factors affecting the quality of ELISA detection are the specificity and affinity of antigen and antibody. After the antigen and the antibody are specifically combined, the detection substance can be qualitatively or quantitatively analyzed through enzyme catalysis, and the detection sensitivity is improved. The specificity and affinity of antigens and antibodies depend on the structure of the immune hapten molecules, so the molecular design and synthesis of immune hapten is the most basic and critical step for producing specific antibodies and establishing a rapid detection technology of small molecule substance residues. The invention provides a method for synthesizing triphenyl phosphate hapten, further synthesizes immunogen and coating antigen, prepares a mouse monoclonal antibody and finally develops an enzyme-linked immunosorbent assay kit for detecting triphenyl phosphate.

Disclosure of Invention

The invention aims to provide an ELISA kit for detecting triphenyl phosphate, and a detection method which has high sensitivity, strong specificity and low detection cost and is suitable for screening a large number of samples.

In order to achieve the above object, the technical scheme of the present invention is as follows:

an ELISA kit for detecting triphenyl phosphate comprises an ELISA plate, a standard substance working solution, an antibody working solution, an enzyme labeling working solution, a sample dilution solution, a sample extracting solution, a washing solution, a substrate color development solution and a termination solution.

In the kit, the ELISA plate is coated with a conjugate of triphenyl phosphate hapten and carrier protein.

The triphenyl phosphate hapten structure is shown as a formula I:

the preparation method of the triphenyl phosphate hapten comprises the following steps:

(1) 680.6mg of toluene diphenyl phosphate is put into a 50mL reaction bottle, 20mL of carbon tetrachloride is added, and stirring is carried out until the mixture is dissolved;

(2) 50mg of benzoyl peroxide is added and stirred for 10min at room temperature;

(3) 430mg of N-bromosuccinimide is added, the mixture is refluxed and stirred at 80 ℃ for reaction for 16 hours, cooled to room temperature and filtered to remove NBS and byproducts;

(4) Concentrating the filtrate under reduced pressure, adding dichloromethane for dissolving, adding 2000mg of 100-200 mesh silica gel for sample stirring after the solution is completely dissolved, performing 200-300 mesh silica gel column chromatography, and petroleum ether: ethyl acetate=10:1 elution and collection of the main product gave toluene diphenyl phosphate methyl bromide 780mg oil;

(5) 275mg of 4-methylamino butyrate is put into a 50mL reaction bottle, 15mL of N-N dimethylformamide is added, and the mixture is stirred until the mixture is dissolved, 745mg of anhydrous potassium carbonate is added, and the mixture is stirred at room temperature for 30min;

(6) 630mg of toluene diphenyl phosphate methyl bromide is added and stirred at 50 ℃ for reaction for 16 hours;

(7) Filtering to remove salt, concentrating the filtrate under reduced pressure, adding dichloromethane for dissolving, adding 2000mg of 100-200 mesh silica gel for sample mixing, and performing 200-300 mesh silica gel column chromatography, petroleum ether: ethyl acetate=1:1.5, and 550mg of the main product, i.e. the hapten compound of formula i, was collected.

The preparation method of the triphenyl phosphate antigen also belongs to the protection scope of the invention.

The triphenyl phosphate antigen is obtained by coupling the triphenyl phosphate hapten (formula I) and carrier protein through an acid amine condensation reaction.

The carrier protein may specifically be Bovine Serum Albumin (BSA), bovine Thyroglobulin (BTG), human Serum Albumin (HSA), murine serum protein (MSA), keyhole Limpet Hemocyanin (KLH), rabbit serum protein (RSA), human serum protein (HSA) or Ovalbumin (OVA).

The triphenyl phosphate hapten (formula I) is coupled with the Bovine Serum Albumin (BSA) to prepare triphenyl phosphate immunogen, and the reaction mole ratio of triphenyl phosphate to the bovine serum albumin is 8.16:1.

in the invention, the triphenyl phosphate antigen is prepared and obtained according to the following steps:

(1) Immunogens: 8.15mg of triphenyl phosphate (formula I) is dissolved in 2mL of Dimethylformamide (DMF), 10.3mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 6.18mg of N-hydroxysuccinimide (NHS) are added, and the mixture is magnetically stirred and reacted for 2 to 3 hours at the temperature of 20 to 25 ℃ to obtain a solution I; after 20mg of BTG is fully dissolved by 5mL of CB buffer, adding the solution I, and reacting overnight to obtain a solution II; the solution II was dialyzed with phosphate buffer salt solution (0.01 mol/L, pH 7.2) at 4℃with stirring for 3 days to give the triphenyl phosphate immunogen.

(2) Coating: 20.37mg of triphenyl phosphate (formula I) is added with 2mL of DMF and stirred until the triphenyl phosphate is completely dissolved, 25.75mg of EDC and 15.46mg of NHS are added, and the mixture is magnetically stirred at 20-25 ℃ for reaction for 2-3 hours to obtain a solution I (1); after 50mg of BSA protein was sufficiently dissolved in 5mL of CB solution, the solution I (1) was added, and the reaction was carried out overnight to obtain a solution II (2); and (3) stirring and dialyzing the solution II (2) with a phosphate buffer salt solution (0.01 mol/L, pH 7.2) at 4 ℃ for 3 days to obtain the triphenyl phosphate coating.

The ELISA plate is a 96-well plate coated with triphenyl phosphate coating antigen.

The preparation method of the triphenyl phosphate antibody comprises the following steps:

(1) Immunization of animals

The prepared triphenyl phosphate immunogen is injected into a mouse body according to 100 mug/mouse, the triphenyl phosphate antigen is dissolved by normal saline, the triphenyl phosphate antigen is uniformly mixed with Freund's complete adjuvant in an equal volume, the Balb/c female mouse with 6-8 weeks old is subcutaneously injected into the back of the neck, the immunogen and Freund's incomplete adjuvant are uniformly mixed in equal volume on days 7, 14 and 28 after primary immunization, the immunization is carried out once respectively, 100 mug/mouse of immune complex is carried out 3 days before fusion, and the Freund's adjuvant is not added for one more time.

(2) Cell fusion and cloning

Mixing spleen cells of immunized mice with mouse myeloma cells (SP 2/0) in logarithmic phase, slowly adding preheated fusion agent (PEG 4000) for fusion within 45s, suspending with HAT medium, adding appropriate amount of feeder cells, culturing in 96-well culture plate at 37deg.C and 5% CO ₂ Culturing in an incubator, half-changing with HT medium after 5 days, and full-changing after 9 days.

After the cells are fused, when the cells grow to 1/4 of the area of the culture hole, the step-by-step screening method is adopted to screen the hybridoma cells. The primary selection adopts an indirect ELISA method, an ELISA plate is coated with coating antigen (the optimal coating concentration and positive serum dilution are conventionally titrated in advance by a square matrix method), the culture supernatant of a tested hole is added, incubation is carried out, goat anti-mouse IgG-HRP and IgM-HRP are added after washing, and the OPD o-phenylenediamine is oxidized and then color reaction occurs. The positive holes screened according to the color reaction are screened by an indirect competition ELISA method, firstly, cell supernatant and 100 mug/mL triphenyl phosphate are mixed in equal volume, water bath is carried out for 30min at 37 ℃, and then the mixture is added into a coated ELISA plate. The control group replaced triphenyl phosphate with PBS and the rest of the procedure was the same. OD after blocking with triphenyl phosphate _450nm If the value falls below 50% of the control wells, the wells are judged to be positive, and the wells which are positive after 2-3 times of detection are immediately subcloned by a limiting dilution method.

(3) Preparation and purification of monoclonal antibodies

Amplifying and culturing hybridoma cells after 2-3 times of subcloning and strain establishment, collecting supernatant, measuring titer by using indirect ELISA, and freezing; and taking 0.5 mL/mouse of 8-10 week old Balb/c mice, injecting 1-2X 105 hybridoma cells into the abdominal cavity after 7-10 days, and extracting the ascites of the mice after 7-10 days. Purifying by octanoic acid-saturated ammonium sulfate method, collecting supernatant to obtain purified triphenyl phosphate drug monoclonal antibody.

In the kit, the solvent of the standard working solution is PBS buffer solution containing light stabilizer and bovine serum albumin, and the solute is triphenyl phosphate; the concentration of the solute in the 6 standard working solutions is 0 mug/L, 0.01 mug/L, 0.03 mug/L, 0.09 mug/L, 0.27 mug/L, 0.81 mug/L and 2.43 mug/L respectively; the PBS buffer solution and the antibody diluent PBS buffer solution have a pH value of 7.2.

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

The antibody diluent is PBS buffer solution containing bovine serum albumin, gelatin, preservative Proclin-300, tween-20 and Tris; the solvent of the antibody diluent is deionized water, and the solute is bovine serum albumin, gelatin, preservative Proclin-300, tween-20, tris, disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride and potassium chloride; the concentrations of the bovine serum albumin, the gelatin, the preservative Proclin-300, the Tween-20 and the Tris in deionized water are 2.5 percent, 0.1 percent, 0.02 percent, 1mL/L and 0.1g/L respectively; the solutes in PBS are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride and potassium chloride, and the concentration of the solutes in deionized water is 1.44g/L, 0.24g/L, 8.0g/L and 0.3g/L, respectively.

In the kit, the enzyme marker working solution is obtained by diluting horseradish peroxidase (Horseradish Peroxidase, HRP) -marked goat anti-mouse secondary antibody by 500 times with secondary antibody diluent.

The secondary antibody diluent is PBS buffer solution containing fetal calf serum, glycerol and Proclin 300; the PBS buffer solution is prepared by the same method; the concentrations of the fetal bovine serum, glycerol and Proclin 300 in the PBS buffer were 200mL/L, 50mL/L and 1mL/L, respectively.

In the kit, the sample diluent is PBS buffer solution containing light stabilizer, bovine serum albumin and surfactant; the PBS buffer solution is prepared by the same method; the concentrations of light stabilizer, bovine serum albumin and surfactant in the PBS buffer were 0.2g/L, 0.1g/L and 0.1g/L, respectively, and the pH was 7.2.

In the kit, the sample extracting solution is PBS buffer solution containing light stabilizer, bovine serum albumin and surfactant; the PBS buffer solution is prepared by the same method; the concentrations of light stabilizer, bovine serum albumin and surfactant in the PBS buffer were 0.2g/L, 0.1g/L and 0.2g/L, respectively, and the pH was 7.2.

In the kit, the washing liquid is PBS buffer solution containing Tween-20 and Proclin 300; the PBS buffer solution is prepared by the same method; the concentrations of Tween-20 and Proclin 300 in the PBS buffer were 20mL/L and 300. Mu.L/L, respectively.

In the kit, the substrate color development liquid is a mixed aqueous solution of 1.0g/L carbamide peroxide, 5.0g/L sodium acetate, 0.5g/L light stabilizer, 2.5mL/L phosphoric acid and 5.0g/L tetramethyl benzidine, and the solvent is deionized water.

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

It is another object of the present invention to provide a method for detecting triphenyl phosphate in a sample, comprising the steps of:

(1) Pretreating a sample to obtain a solution of the sample;

(2) Detecting the solution by using the kit;

(3) And analyzing the detection result.

The sample pretreatment method comprises the following steps: accurately weighing 1+/-0.1 g (or mL) of sample, adding 5mL of sample extracting solution, swirling 1min at a high speed at room temperature (25+/-2 ℃), centrifuging 5000g for 5min, taking 200 mu L of supernatant, adding 200 mu L of sample diluent, sufficiently shaking, uniformly mixing and then testing.

The detection steps of the kit are as follows: adding 50 mu L of standard working solution or sample pretreatment solution into the ELISA plate coated with the triphenyl phosphate antigen, and then adding a specific antibody solution containing triphenyl phosphate; after incubation, washing 4 times by adding a washing solution, fully beating, adding an enzyme-labeled anti-antibody, developing for 10min, adding a stop solution, and measuring the absorbance values of the solution at 450nm and 620nm by using an enzyme-labeling instrument.

The analysis process of the detection result provided by the invention comprises the following steps: the absorbance average (B) of the standard working solution at each concentration obtained was divided by the absorbance value (B0) of the first standard solution (0 ng/mL standard) and multiplied by 100%, i.e., the percent absorbance value. The calculation formula is as follows: percent absorbance (%) = (B/B0) ×100%.

And drawing a standard curve graph by taking the half-logarithmic value of the concentration (mug/L) of the triphenyl phosphate standard working solution as an X axis and the percentage absorbance value as a Y axis. The percentage absorbance value of the sample solution is calculated by the same method, and the content of the triphenyl phosphate in the sample can be read from the standard curve corresponding to the concentration of each sample.

The analysis of the detection result in the invention can also adopt a regression equation method to calculate the concentration of the sample solution.

The triphenyl phosphate hapten and the triphenyl phosphate antigen provided by the invention have the advantages of simple synthesis method, high purity and high yield, and have great value for the preparation of triphenyl phosphate antibodies and the detection of triphenyl phosphate drug residues.

The invention relates to a kit for detecting triphenyl phosphate esterase linked immunosorbent assay, which comprises the following components: and (3) specifically competing the triphenyl phosphate in the sample with the antigen immobilized on the ELISA plate, adding an ELISA secondary antibody, reacting with an antibody, developing color by using an enzyme-catalyzed color developing agent, and judging the content of triphenyl phosphate in the sample according to the color development depth. Detecting that the absorbance is inversely related to the content of the triphenyl phosphate contained in the sample, substituting the absorbance into a standard curve, and multiplying the absorbance by the corresponding dilution multiple to obtain the residual quantity of the triphenyl phosphate in the sample.

Drawings

FIG. 1 is a mass spectrum of triphenyl phosphate hapten.

FIG. 2 is a diagram showing the NMR structure of triphenyl phosphate hapten.

FIG. 3 shows a BSA mass spectrum of the carrier protein.

FIG. 4 is a mass spectrum of triphenyl phosphate antigen.

FIG. 5 is a graph of triphenyl phosphate detection criteria.

Detailed Description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The kit or the detection method can detect the residue of the triphenyl phosphate in the aquatic products and the meat, eggs and milk.

Example 1 preparation of triphenyl phosphate hapten

1. Preparation of triphenyl phosphate hapten

(7) Filtering to remove salt, concentrating the filtrate under reduced pressure, adding dichloromethane for dissolving, adding 2000mg of 100-200 mesh silica gel for sample mixing, and performing 200-300 mesh silica gel column chromatography, petroleum ether: eluting with ethyl acetate=1:1.5, and collecting main product to obtain hapten compound shown in formula I.

The reaction equation is as follows:

2. triphenyl phosphate hapten identification

Mass spectrometry detection (figure 1) is carried out on the triphenyl phosphate hapten, and the result shows that the chemical structural formula of the triphenyl phosphate hapten is shown as a formula I. The molecular weight of the target hapten is 455.45, and a strong peak appears in m/z 478 on a mass spectrum. The synthesized hapten was also identified by nuclear magnetic resonance (Nuclear Magnetic Resonance, NMR), and the results are shown in fig. 2. The results of FIGS. 1 and 2 show that the hapten structure is correct.

Example 2 preparation of triphenyl phosphate detection kit

1. Preparation of ELISA plate

The triphenyl phosphate antigen is obtained by coupling the triphenyl phosphate hapten (formula I) and the carrier protein through an acid amine condensation chemical reaction, and the specific synthesis method is as follows:

(3) Immunogens: 8.15mg of triphenyl phosphate (formula I) is dissolved in 2mL of Dimethylformamide (DMF), 10.3mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 6.18mg of N-hydroxysuccinimide (NHS) are added, and the mixture is magnetically stirred and reacted for 2 to 3 hours at the temperature of 20 to 25 ℃ to obtain a solution I; after 20mg of BTG is fully dissolved by 5mL of catechol borane solution (CB), adding the solution I, and reacting overnight to obtain solution II; the solution II was dialyzed with phosphate buffer salt solution (0.01 mol/L, pH 7.2) at 4℃with stirring for 3 days to give the triphenyl phosphate immunogen.

(4) Coating: 20.37mg of triphenyl phosphate (formula I) is added with 2mL of DMF and stirred until the triphenyl phosphate is completely dissolved, 25.75mg of EDC and 15.46mg of NHS are added, and the mixture is magnetically stirred at 20-25 ℃ for reaction for 2-3 hours to obtain a solution I (1); after 50mg of BSA protein was sufficiently dissolved in 5mL of CB solution, the solution I (1) was added, and the reaction was carried out overnight to obtain a solution II (2); and (3) stirring and dialyzing the solution II (2) with a phosphate buffer salt solution (0.01 mol/L, pH 7.2) at 4 ℃ for 3 days to obtain the triphenyl phosphate coating.

The triphenyl phosphate coating antigen is triphenyl phosphate hapten (formula I) and ovalbumin (BSA) with a molar ratio of 8.16:1 coupling preparation.

The resulting triphenyl phosphate coating was diluted to 10. Mu.g/mL with coating buffer, 100. Mu.L of antigen was added to each well, incubated at 37℃for 2h, the dilutions were discarded, the dilutions were washed 3 times with wash solution for 30s each time, 150. Mu.L of blocking solution was added to each well after the plates were dried, and the blocking solution was discarded after incubation at 37℃for 1 h. And drying to obtain an ELISA plate, and vacuum sealing and preserving the ELISA plate by using an aluminum film.

The coating buffer is citric acid buffer with pH of 6.0 and 0.1 mol/L.

The sealing liquid is a 0.2mol/L phosphate buffer solution with pH7.7 and containing 20g/L sucrose, 30g/L skimmed milk powder, 2.5g/L casein, 0.25% fetal bovine serum and 3%o sodium azide.

2. Preparation of antibody working solution

1. Preparation of triphenyl phosphate antibody

(1) Immunization of animals

The triphenyl phosphate immunogen is triphenyl phosphate hapten (formula I) and Bovine Serum Albumin (BSA) in a molar ratio of 8.16:1 coupling preparation.

(2) Cell fusion and cloning

The spleen cells of immunized mice were mixed with mouse myeloma cells (SP 2/0) in logarithmic phase and then slowly added over 45s according to conventional methodsThe preheated fusion agent (PEG 4000) is used for fusion, HAT culture medium is used for suspension uniformly, a proper amount of feeder cells are added, and the mixture is cultured in a 96-well culture plate at 37 ℃ and 5 percent CO ₂ Culturing in an incubator, half-changing with HT medium after 5 days, and full-changing after 9 days.

(3) Preparation and purification of monoclonal antibodies

Amplifying and culturing hybridoma cells after 2-3 times of subcloning and strain establishment, collecting supernatant, measuring titer by using indirect ELISA, and freezing; and taking 0.5 mL/mouse of 8-10 week old Balb/c mice to be injected with liquid paraffin in the intraperitoneal injection, and injecting hybridoma cells in the intraperitoneal injection after 7-10 days for 1-2X 10 ⁵ And/or, extracting ascites from the mice after 7-10 days. Purifying by octanoic acid-saturated ammonium sulfate method, collecting supernatant to obtain purified triphenyl phosphate drug monoclonal antibody.

The determination of the monoclonal antibody titer is carried out by using a chessboard method, and the result shows that: the titer of triphenyl phosphate monoclonal antibody is 1:27000, half inhibition (IC ₅₀ ) Is 0.091 mug/L.

Through detection, the amino acid sequence of the heavy chain variable region of the triphenyl phosphate monoclonal antibody is shown as a sequence 1 in a sequence table, and the amino acid sequence of the light chain variable region of the triphenyl phosphate monoclonal antibody is shown as a sequence 2 in the sequence table.

Sequence 1:

Glu Val Arg Leu Gln Glu Ser Gly Ser Val Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Pro Ser Arg Phe Tyr Ser Thr Trp Leu His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Thr Gly Gly Ile Tyr Arg Pro Asn Glu Val Asn Ser Tyr Lys Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Ala Val Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Ile Pro Gly Thr Gln Ile Trp Gly Gln Gly Thr Thr Val Thr Val His Arg

sequence 2:

Asp Val Asn Leu Leu Thr Gln Ser Pro Leu Thr Leu Ser Tyr Thr Ile Gly GIn Pro Ala Ser Ile Ser Cys Lys Ser Gln Leu Thr Asn Asp Arg Asp Gly Glu Thr Val Tyr Asn Trp Leu Phe Gln Arg Pro Gly Gln Ser Pro Lys Arg Leu Ile Tyr Ser Val Ser Phe Leu Asp Ser Gly Val Pro Asp Arg Phe Tyr Gly Ser Phe Ser Gly Thr Asp Phe Thr Leu Lys His Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gly Pro Thr Val Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Val

2. preparation of antibody working solution

And diluting the obtained monoclonal antibody of the triphenyl phosphate medicament by 1000 times by using an antibody diluent to obtain an antibody working solution containing the monoclonal antibody of the triphenyl phosphate medicament.

The antibody diluent is PBS buffer solution containing bovine serum albumin, gelatin, preservative Proclin-300, tween-20 and Tris; the solvent of the antibody diluent is deionized water, and the solute is fetal bovine serum albumin, gelatin, preservative 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, and the concentrations of the solution in the deionized water are 1.44g/L, 0.24g/L, 8.0g/L and 0.3g/L respectively; the concentrations of bovine serum albumin, gelatin, preservative Proclin-300, tween-20 and Tris in deionized water are 2.5%, 0.1%, 0.01%, 1mL/L and 0.1g/L respectively.

2. Preparation of enzyme-labeled working fluid

The enzyme marker working solution is obtained by diluting horseradish peroxidase (Horseradish Peroxidase, HRP) marked goat anti-mouse secondary antibody by 500 times with secondary antibody diluent.

3. Preparation of standard working solution

In the kit, the solvent of the standard working solution is PBS buffer solution containing light stabilizer and bovine serum albumin, and the solute is triphenyl phosphate; the concentration of the solute in the 7 standard working solutions is 0 mug/L, 0.01 mug/L, 0.03 mug/L, 0.09 mug/L, 0.27 mug/L, 0.81 mug/L and 2.43 mug/L respectively; the PBS buffer solution and the antibody diluent PBS buffer solution have a pH value of 7.2.

4. Preparation of other reagents

The kit may further comprise a sample diluent and/or a sample extract and/or a washing solution and/or a substrate color developing solution and/or a stop solution.

In the kit, the substrate color development liquid is a solution containing carbamide peroxide, sodium acetate, a light stabilizer, phosphoric acid and tetramethyl benzidine; the substrate color development liquid solvent is deionized water, and solutes are carbamide peroxide, sodium acetate, light stabilizer, phosphoric acid and tetramethyl benzidine, and the solute concentrations are 1.0g/L, 5.0g/L, 0.5g/L, 2.5mL/L and 5.0g/L respectively.

The stop solution is 0.05mol/L sulfuric acid aqueous solution.

Example 3, method of Using the kit of example 2

1. Pretreatment of samples

Raw milk: taking 100 mu L of mixed raw milk for detection.

Animal meat: 1.00+/-0.05 g of the homogenized sample is weighed into a 10mL centrifuge tube (egg sample is stirred and mixed uniformly, and 1+/-0.05 mL of the sample is taken); 0.8ml of 0.3M NaOH is added and vortexed for 1min; adding 4mL of ethyl acetate, whirling for 3min, and centrifuging for 5min with 4000 g; transferring 500 mu L of supernatant into a 5mL centrifuge tube, and drying by nitrogen at 60 ℃; and adding 400 mu L of sample diluent into the blow-dried centrifuge tube, shaking uniformly, and waiting to be detected. The sample diluent is 29.01g of disodium hydrogen phosphate dodecahydrate, 2.96g of sodium dihydrogen phosphate dihydrate and 5.84g of sodium chloride, the volume is fixed to 1L by pure water, and the pH value is adjusted to 8.0 by 1mol/L sodium hydroxide.

2. Detection Using the example 1 kit

1. The ELISA plate is inserted into the ELISA plate frame, the positions of each standard substance and each sample are recorded, each sample is parallel to 3, and the unused ELISA plate strips are immediately stored in an environment of 2-8 ℃ after being sealed by a self-sealing bag;

2. adding 50 mu L of each standard working solution or sample solution into the corresponding standard or sample hole respectively;

3. add 50. Mu.L of antibody working fluid to each plate well;

4. covering a cover plate film, lightly oscillating the ELISA plate for 10s, fully and uniformly mixing, and carrying out light-shielding reaction at room temperature (25+/-2 ℃) for 30min;

5. uncovering the cover plate film, pouring out the liquid in the plate holes, adding 260 mu L of washing working solution (the washing solution is diluted by 20 times by deionized water) into each hole, fully washing for 3-4 times, and soaking for 15-30 s each time; the method comprises the steps of carrying out a first treatment on the surface of the

6. Pouring out the liquid in the plate hole, inverting the ELISA plate on the absorbent paper, and drying;

7. adding 100 mu L of enzyme marker working solution into each plate hole; covering the cover plate film, slightly oscillating the ELISA plate for 10s, fully and uniformly mixing, and carrying out light-shielding reaction for 30min at room temperature (25+/-2 ℃);

8. repeating the steps 5-6;

9. immediately adding 100 mu L of substrate developing solution A, B mixed solution (the substrate developing solution A and the substrate developing solution B are mixed according to the volume of 1:1) into each hole, covering a cover plate film, and carrying out light-proof reaction for 15min;

10. uncovering the cover plate film, adding 50 mu L of stop solution into each plate hole, gently oscillating the ELISA plate for 10s, and fully and uniformly mixing;

11. and (5) reading the absorbance value of the ELISA plate at the dual wavelength of 420nm and 630nm within 5min after termination by using an ELISA reader.

3. Analyzing the detection result

1. Calculation of the percent absorbance values

The average absorbance value of each standard (or sample to be measured) is divided by the absorbance value of the zero standard (standard with the concentration of 0 mug/L), and the absorbance value is multiplied by 100%, so that the percentage of the absorbance corresponding to each standard (or sample to be measured), namely the percentage absorbance value, can be obtained.

Absorbance percentage = B/b0×100%

Wherein: average absorbance value of B-standard (or sample); average absorbance value of B0-standard at 0. Mu.g/L.

2. Making a standard curve

Drawing a standard curve graph by taking the percentage absorbance value of each standard substance as an ordinate and the concentration (mug/L) of triphenyl phosphate in each standard substance working solution as an abscissa, and carrying out nonlinear fitting analysis by using origin8.0 (origin Lab Corp., northampton, MA, USA) to form a four-parameter fitting curve:

y＝D+(A-D)/[1+(x/C)^B]

wherein y is the percentage of absorbance; x is the concentration of the substance to be detected; a, B, C and D are four parameters of the standard curve.

By test data, the standard curve equation for triphenyl phosphate is: y=7.06+ (100.06-7.06)/[ 1+ (x/0.091) ] 0.83]Linear correlation R ² 0.99.

The standard graph is shown in fig. 5.

3. Calculating the content of triphenyl phosphate in the sample

Substituting the percentage absorbance value of the sample to be measured into a standard curve to obtain the corresponding residual concentration of the sample to be measured, and multiplying the residual concentration by the dilution multiple of the corresponding sample to obtain the actual content of triphenyl phosphate in the original sample to be measured.

Example 4 specificity, limit of detection, accuracy, precision detection of the kits of example 2

1. Specificity test of the kit:

the specificity of the triphenylesterase linked immunosorbent assay kit is determined by performing a cross-reaction test with the corresponding substance.

Triphenyl phosphate and its related analogues, tris (2-chloroethyl) phosphate (Tris (2-chloroethyl) phosphate, TCEP), tris (2-chloroisopropyl) phosphate (Tris (1-chloro-2-propyl) phosphate, TCPP), tris (1, 3-dichloro-2-propyl) phosphate (Tris (1, 3-dichloro-2-propyl) phosphate, TDCPP) and t-butyldiphenyl phosphate (tert-Butylphenyldiphenyl phodphate, MDPP) were serially diluted, respectively, and a standard curve was prepared by substituting the "triphenyl phosphate standard working solution" with a serial dilution of triphenyl phosphate and its analogues, and finding out the respective 50% Inhibitory Concentration (IC) on the curve ₅₀ ) The specific method comprises the following steps: the corresponding triphenyl phosphate concentration (. Mu.g/L) with an ordinate value equal to 50%, i.e.IC, was obtained ₅₀ Values. The cross-reactivity of the kit to triphenyl phosphate and various common organophosphorus and carbamate pesticides was calculated using the following formula:

cross-reaction rate (%) = (concentration of triphenyl phosphate causing 50% inhibition/concentration of triphenyl phosphate analog causing 50% inhibition) ×100%.

The results are shown in Table 1.

Table 1 specificity of the kit

Experiments show that the kit has better specificity on triphenyl phosphate, namely the kit can detect triphenyl phosphate.

2. Detection limit determination of kit

According to the detection index, fish, shrimp, pork, mutton, egg and milk blank samples (LC-MS/MS detection negative) were taken, detected according to the method of example 3, the measurement values were obtained according to the standard curve, the average value was calculated, and the minimum detection Limit (LOD) was obtained by adding 3 times of standard deviation. The results are shown in Table 2.

TABLE 2 measurement results of blank sample

The result shows that the detection limit of the kit on triphenyl phosphate in aquatic products, meat, eggs and milk can be defined to be 0.1 mug/L for preventing false positive.

3. Accuracy and precision test of kit

Accuracy refers to the degree of agreement between measured and actual values, accuracy is often expressed in terms of recovery, and precision is often expressed in terms of coefficient of variation. The water-extracted product, meat, egg, and milk blank sample (negative in LC-MS/MS detection) were pretreated as described in step one of example 3, and then triphenyl phosphate standard was added to the required concentration of 0.1. Mu.g/kg, 0.2. Mu.g/kg, respectively, to obtain a detection sample solution.

The detection was performed with 3 different batches of kits, each set of detection was repeated 6 times, and the coefficient of variation was calculated separately. The results are shown in Table 3.

The calculation method of the intra-batch variation coefficient comprises the following steps: intra-lot coefficient of variation = coefficient of variation for each parallel sample in the same assay.

The calculation method of the variation coefficient between batches comprises the following steps: inter-lot coefficient of variation = coefficient of variation of the same sample measured in different lots, and the average value is taken.

TABLE 3 sample addition recovery accuracy and precision

The results show that the recovery rate of each addition concentration of all samples is between 80 and 120 percent. The intra-batch variation coefficient of each additive concentration is lower than 10% and the inter-batch variation coefficient is lower than 15%.

4. The detection of the kit is compared with the detection result of LC-MS/MS

Samples of aquatic products, meat, eggs, and milk were tested as in example 3, and the results of the tests were confirmed and compared with LC-MS/MS, respectively.

And (3) drawing a scatter diagram by taking the concentration of triphenyl phosphate measured by the kit as an X axis and the concentration of triphenyl phosphate medicament measured by LC-MS/MS as a Y axis. The measurement results of the two methods are subjected to linear analysis, and a regression equation is as follows: y=0.022+0.997x, correlation coefficient R ² 0.999, it is demonstrated that the established method of the invention has good consistency with LC-MS/MS detection results.

5. Shelf life test of kit

The storage conditions of the kit of example 2 were 2 to 8 ℃, and the maximum absorbance (zero standard), 50% inhibition concentration and actual measurement value of triphenyl phosphate addition of the kit were all within the normal range after 12 months of measurement. Considering that abnormal preservation conditions appear in the transportation and use processes, the kit is placed for 8 days at 37 ℃ for accelerated aging test, and the result shows that the indexes of the first to fourth steps of the kit completely meet the requirements. Considering the occurrence of the freezing condition of the kit, the kit is placed for 8 days at the temperature of-20 ℃, and all indexes of the steps one to four completely meet the requirements. From the above results, the kit of example 1 can be stored at 2 to 8℃for at least 12 months.

Claims

1. An enzyme-linked immunosorbent assay kit for detecting triphenyl phosphate, comprising: the kit comprises an ELISA plate coated with triphenyl phosphate antigen, an antibody working solution, an enzyme labeling working solution, a sample dilution solution, a sample extraction solution, triphenyl phosphate standard solution containing different concentrations, a washing solution, a substrate color development solution and a termination solution; the triphenyl phosphate antigen is obtained by coupling triphenyl phosphate hapten shown in the formula I and carrier protein;formula I.

2. An enzyme-linked immunosorbent assay kit for detecting triphenyl phosphate according to claim 1, wherein: the preparation method of the triphenyl phosphate hapten comprises the following steps: 680.6 Adding 20mL carbon tetrachloride to mg of toluene diphenyl phosphate, stirring until the mixture is dissolved, adding 50mg benzoyl peroxide, stirring at room temperature for 10min, adding 430mg of N-bromosuccinimide, refluxing and stirring at 80 ℃ for reaction for 16 hours, cooling to room temperature, filtering to remove NBS and byproducts, concentrating the filtrate under reduced pressure, dissolving with dichloromethane, adding 2000mg of 100-200 mesh silica gel, stirring, performing 200-300 mesh silica gel column chromatography, and petroleum ether: eluting with ethyl acetate=10:1, and collecting main product to obtain toluene diphenyl phosphate methyl bromide; 50 275mg of 4-methylamino butyrate is put into a mL reaction bottle, N-N dimethylformamide 15mL is added, anhydrous potassium carbonate 745mg is added after stirring until the mixture is dissolved, 630mg toluene diphenyl phosphate methyl bromide is added after stirring for 30min at room temperature, stirring is carried out for 16 hours at 50 ℃, salt is removed by filtration, the filtrate is concentrated under reduced pressure, dichloromethane is dissolved, 2000mg of 100-200 mesh silica gel is added for sample mixing, 200-300 mesh silica gel column chromatography and petroleum ether are carried out: eluting with ethyl acetate=1:1.5, and collecting main product to obtain triphenyl phosphate hapten.

3. An enzyme-linked immunosorbent assay kit for detecting triphenyl phosphate according to claim 1, wherein: the carrier protein can be bovine serum albumin BSA, bovine thyroglobulin BTG, human serum albumin HSA, murine serum protein MSA, keyhole limpet hemocyanin KLH, rabbit serum protein RSA, human serum protein HSA or egg serum albumin OVA.

4. An enzyme-linked immunosorbent assay kit for detecting triphenyl phosphate according to claim 1, wherein:

the antibody working solution is obtained by diluting a triphenyl phosphate monoclonal antibody with an antibody diluent by 1000 times; the triphenyl phosphate monoclonal antibody is prepared through the steps of animal immunization, cell fusion, cloning, purification and the like;

the enzyme-labeled working solution is obtained by diluting a commercial goat anti-mouse secondary antibody by 500 times with a secondary antibody diluent;

the working solution concentrations of the standard substances are 0 mug/L, 0.01 mug/L, 0.03 mug/L, 0.09 mug/L, 0.27 mug/L, 0.81 mug/L and 2.43 mug/L respectively;

the sample diluent is PBS buffer solution containing light stabilizer, bovine serum albumin and surfactant, wherein the concentrations of the light stabilizer, the bovine serum albumin and the surfactant in the PBS buffer solution are respectively 0.2g/L, 0.1g/L and 0.1g/L, and the pH value is 7.2;

the sample extracting solution is PBS buffer solution containing light stabilizer, bovine serum albumin and surfactant, the concentrations of the light stabilizer, the bovine serum albumin and the surfactant in the PBS buffer solution are respectively 0.2g/L, 0.1g/L and 0.2g/L, and the pH value is 7.2;

the washing solution is PBS buffer solution containing Tween-20 and Proclin 300, and the concentrations of the Tween-20 and Proclin 300 in the PBS buffer solution are respectively 20mL/L and 300 mu L/L;

the substrate color development liquid solvent is deionized water, and solutes are carbamide peroxide, sodium acetate, light stabilizer, phosphoric acid and tetramethyl benzidine, and the solute concentrations are 1.0g/L, 5.0g/L, 0.5g/L, 2.5mL/L and 5.0g/L respectively;

the stop solution is 0.05mol/L sulfuric acid aqueous solution.

5. The antibody working fluid of claim wherein: is PBS buffer solution containing bovine serum albumin, gelatin, preservative Proclin-300, tris and Tween-20; the solvent of the antibody diluent is deionized water, and the solute is fetal bovine serum albumin, gelatin, preservative Proclin-300, tween-20, disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride and Tris, and the concentration of the solute in the deionized water is 2.5%, 0.1%, 0.01%, 1mL/L, 1.44g/L, 0.24g/L, 8.0g/L, 0.3g/L and 0.1g/L respectively.

6. The enzyme-labeled working fluid according to claim 4, wherein: the secondary antibody diluent is PBS buffer solution containing fetal calf serum, glycerol and Proclin 300; the concentrations of the fetal bovine serum and Proclin 300 in the PBS buffer were 200mL/L, 50mL/L and 1mL/L, respectively.

7. The application method of the ELISA kit for detecting triphenyl phosphate comprises the following steps:

pretreating a sample to be detected;

detecting by using a kit;

and (5) analyzing a detection result.

8. The method for using the kit for detecting triphenyl phosphate according to claim 8, wherein: the kit can detect the content of triphenyl phosphate in a sample, the detection limit is 0.1 mug/kg, the inter-batch variation coefficient is less than 10%, and the intra-batch variation coefficient is less than 15%.