CN102590493A - Kit for diethyl phthalate fluorescence polarization immunoassay - Google Patents

Abstract

A diethyl phthalate fluorescent polarization immunoassay kit, including fluorescent marker stock solution, DEP standard solution, DEP polyclonal antibody serum and concentrated phosphate (PBS) buffer solution, which are respectively packaged in reagent tubes, used To detect the concentration of DEP in the sample. The detection method adopts fluorescence polarization technology, and obtains a standard curve by measuring the fluorescence polarization of a series of DEP solutions with known concentrations, and uses the standard curve to calculate the concentration of DEP in the mixture. The advantages of the present invention are: the detection method combines the high specificity of the antibody-antigen reaction with the high sensitivity of the fluorescent label, the operation is simple and fast, no separation and other operations are required during detection, the detection accuracy is high, and the maximum detection range of DEP It is 10-200ng/mL, and the sensitivity can reach 40.4ng/mL. It plays an important role in the rapid detection of residual DEP in actual systems such as water, juice drinks and capsules.

Description

A kind of diethyl phthalate fluorescence polarization immunoassay kit

【Technical field】

The present invention relates to a food inspection immunoassay technology, in particular to a diethyl phthalate fluorescence polarization immunoassay kit.

【Background technique】

In April 2011, Taiwan's relevant departments found that "Jingyuan Probiotics" contained plasticizer di(2-ethyl)hexyl phthalate (DEHP) during routine sampling of food, and the concentration was as high as 600ppm (parts per million). one concentration). On May 23, Taiwan discovered that Yushen Fragrance Company had illegally added toxic plasticizers to the food additive "clouding agent" (that is, emulsifier), which resulted in contamination of 9 well-known sports drinks, fruit juices, and enzyme drinks. Taiwan was affected by the incident. Nearly 200 manufacturers were involved. As of June 6, 2011, the General Administration of Quality Supervision, Inspection and Quarantine announced the latest "List of Problem Enterprises and Products Polluted by Plasticizers in Taiwan". . Foods contaminated by plasticizers include sports drinks; fruit juice drinks; tea drinks; jam, pulp or jelly; capsule powdered food; additives and other six categories.

Plasticizer is a term in Taiwan, while it is generally called plasticizer in mainland China. It is an accompanying product of plastic products. Adding plasticizer can reduce the glass transition temperature of plastics and make hard and rigid plastics soft and flexible. It is an industrial It is a widely used polymer material additive in the world. Adding this substance in plastic processing can make it more flexible, easy to process, and can be legally used for industrial purposes.

There are many kinds of plasticizers. In the research and development stage, there were more than 1,000 kinds of plasticizers. There are only more than 200 kinds of plasticizers produced as commodities, and most of them are phthalates. Phthalates are esters formed from dicarboxylic phthalic acid and alcohols, and have good water and oil repellency. At present, there are fourteen kinds used in industry, and it is also a harmful ingredient in hormone toys and toxic cosmetics. Such plasticizers are not food or food additives and are toxic. Because of its molecular instability, it will be continuously released from materials over time and volatilized into the atmosphere, soil and water. Therefore, human beings will inhale it through breathing, eating and other ways. The plasticizers ingested through normal channels are very small, and they will basically be excreted by the human body in a short period of time. Since May 2011, six phthalate plasticizers including DEHP, DINP, DNOP, DBP, DMP, and DEP have been detected in food in Taiwan, and DEP and DIDP have been detected in pharmaceuticals.

Announcement No. 16 of 2011 of the Ministry of Health of the People's Republic of China stipulates that non-edible substances and easily abused food additives phthalates (sixth batch) that may be illegally added to food include: Base) hexyl ester (DEHP), dibutyl phthalate (DBP), diisononyl phthalate (DINP), diphenyl phthalate, dimethyl phthalate (DMP), Diethylphthalate (DEP), Dipentylphthalate (DPP), Dihexylphthalate (DHXP), Dinonylphthalate (DNP), Diisophthalate Butyl phthalate (DIBP), dicyclohexyl phthalate (DCHP), di-n-octyl phthalate (DNOP), butyl benzyl phthalate (BBP), bis(2) phthalate -Methoxy)ethyl ester (DMEP), bis(2-ethoxy)ethyl phthalate (DEEP), bis(2-butoxy)ethyl phthalate (DBEP), diphthalate Bis(4-methyl-2-pentyl) formate (BMPP), etc. The types of food that may be added include: emulsifier food additives, other food additives or foods that use emulsifiers, etc.

On June 3, 2011, the State Food and Drug Administration officially issued an emergency notice, requiring all localities to suspend the production and sales of two health foods containing phthalates, namely Xiehe brand Ganoderma lucidum spore powder tablets (Guoshi Jianzi G 20070306) Hemeizhongqingsu brand multiple amino acid tablets (Guoshi Jianzi G 20100217), and requested that the two products that are currently on the market be removed from the shelves immediately. The notice also emphasized that health food companies should carry out self-inspection, and immediately suspend production of health food products containing phthalates in their formulations, immediately recall products that are currently on the market, and promptly report to the local food and drug supervision and management department . In addition, various capsules such as Rouyan Capsules have been detected to contain phthalate plasticizers.

Among the pharmaceutical excipients in my country, there are three kinds of phthalates that can be used. They are dimethyl phthalate, diethyl phthalate, and dibutyl phthalate. The above-mentioned substances are mainly used as excipients in the process of pharmaceutical production, and are mostly used in tablet coating, capsules, etc. The 2010 edition of the Chinese Pharmacopoeia clearly lists "diethyl phthalate" as an available pharmaceutical excipient in the catalog of pharmaceutical excipients. According to the Pharmacopoeia, this substance category belongs to pharmaceutical excipients, plasticizers and coating materials, etc. However, the "Pharmacopia" does not restrict or prompt the dosage and safety of "diethyl phthalate". Although the United States Pharmacopoeia also allows phthalate analogs to be used as pharmaceutical excipients, it also stipulates the upper limit of the total daily intake of its homologues.

The website of the Ministry of Health pointed out that the health impact of phthalates depends on their intake, and occasional consumption of small amounts of phthalates will not cause health hazards. It is understood that taking an adult with a body weight of 60kg as an example, the World Health Organization, the US Food and Drug Administration and the European Union respectively believe that the daily intake of phthalates of 1.5mg, 2.4mg, 3.0mg and below per person is safe.

The testing standards for plasticizers in various places are as follows:

1. Taiwan:

According to Taiwan's "Hygienic Standards for Food Utensils, Containers and Packaging", the dissolution limit of DEHP plasticizer is 1.5ppm, and DBP is 0.3ppm. Related reports: The revised hygiene standard for food utensils, containers and packaging in Taiwan has been implemented. The mainland standard "GB 9685-2008 Hygienic Standards for the Use of Additives for Food Containers and Packaging Materials" stipulates that the specific migration amount or maximum residual amount of DBP in plastics and rubber is 0.3mg/kg

2. Mainland China:

On July 8, 2011, the National Certification and Accreditation Administration Committee formulated the industry standard "Determination of Phthalates in Food", which can detect 22 kinds of phthalates at one time (including international highly concerned DINP, DIDP and DAP, etc.), detection limit (0.01-0.5mg/kg).

3. International regulations:

According to the usual practice, the current tolerable daily intake of 60kg adults in various countries ranges from 1.2-8.4mg. Within this content standard, the human body will metabolize it out of the body in the form of urine and feces.

4. Hong Kong plasticizer inspection - stricter than international standards:

The Hong Kong Special Administrative Region Government has established a maximum limit of 1.5ppm for the plasticizer "diester phthalate" (DEHP) in food and drugs, and included plasticizers in the scope of constant monitoring, and strengthened random inspections of imported products. If the DEHP in the product is found to exceed the upper limit, it is strictly prohibited to be sold in Hong Kong according to the Food Safety Law, and the sold product must also be recalled and destroyed. The detection of terephthalate plasticizers in China usually adopts instrumental methods, and high performance liquid chromatography and gas chromatography are commonly used.

The national standard adopts the GC-MS method of GB/T21911-2008. The principle is: all kinds of food are extracted and purified, and then measured by gas chromatography-mass spectrometry. Using the feature-selected ion monitoring scanning mode (SIM), the fragment abundance ratio is used for qualitative detection, and the standard sample quantitative ion external standard method is used for quantitative detection. The oil-free substances were extracted with n-hexane, and the oil-containing substances were extracted with ethyl acetate and cyclohexane. After purification by gel permeation chromatography (GPC), liquid chromatography-mass spectrometry was used for detection and analysis. In this standard method, the detection limit of each phthalate compound in oil-containing samples is 1.5 mg/kg, and the detection limit of each phthalate compound in oil-free samples is 0.05 mg/kg.

The Joint Research Center (JRC) of the European Commission announced on July 29, 2011 that three plasticizer detection methods were developed for the "Taiwan plasticizer incident" research, mainly for the detection of diisooctyl phthalate (DEHP ), using gas chromatography-mass spectrometry (GC-MS), ultra-high liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC -MS) method. The above three methods are also extensively collecting feedback from method users to continuously improve and improve the applicability and stability of the method. The scope of application of these three methods: detection of DEHP in the concentration range of 3-100mg/L in sports drinks.

The illegal addition of harmful substances in food and medicine is closely related to people's health. Establishing fast and accurate detection methods and controlling food quality can better provide protection for people's health. On the other hand, my country's trade with the world is becoming increasingly close, and the illegal addition of harmful substances in food and medicine is an important factor affecting my country's exports. If there is no fast and accurate detection method to check, the quality of exported products will not meet the requirements of the exporting country, which will cause huge economic losses in my country's export trade and damage China's image in the world.

【Content of invention】

The purpose of the present invention is to overcome the above-mentioned deficiencies existing in the prior art, and to provide a diethyl phthalate (DEP) fluorescence polarization immunoassay kit that is easy and fast to operate and has high detection accuracy.

Technical scheme of the present invention:

A diethyl phthalate fluorescent polarization immunoassay kit, including fluorescent marker stock solution, DEP standard solution, DEP polyclonal antibody serum and concentrated phosphate (PBS) buffer solution and respectively packaged in reagent tubes, fluorescent The marker stock solution is a solution obtained by dissolving fluorescein isothiocyanate (FITC)-labeled DEP fluorescent conjugates in 200 μL methanol and packaging it in a reagent tube with a volume of 1.5 mL; DEP standard solution is as follows: DEP in methanol solvent The different concentrations of DEP were set and packaged in seven reagent tubes with a volume of 1.5mL, and the solvent was methanol, 1mL in each tube; DEP polyclonal antibody serum was 600μL and packaged in a reagent tube with a volume of 1.5mL; concentrated PBS buffer The solution is packaged in two reagent tubes with a volume of 10 mL, and each reagent tube contains 9 mL of liquid.

The preparation method of the fluorescent marker stock solution is to react 1 mg of 4-amino-diethyl phthalate and 1 mg of FITC in 500 μL of methanol with a mass percentage content of 25% triethylamine at a temperature of 4° C. After 8-10 hours, take 50 μL of the above-mentioned mixed solution, draw a band on the bottom of the thin-layer chromatography silica gel sheet, and put the thin-layer chromatography silica gel sheet into a developer with a volume ratio of chloroform and methanol of 4:1 for chromatography. Then scrape off the band with Rf of 0.5 on the thin-layer chromatography silica gel sheet, dissolve it in 200mL methanol and filter, and take the filtrate as the fluorescent marker stock solution.

The concentration that described DEP standard solution is set is 0ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 75ng/mL, 100ng/mL and 200ng/mL, methanol is solvent, every tube 1mL; 0.2mg DEP standard was dissolved in 1mL methanol to prepare a 200ng/mL standard solution, and then the 200ng/mL standard solution was diluted with methanol to 100ng/mL, 75ng/mL, 50ng/mL, 20ng/mL and 10ng/mL mL standard solution, take 1mL methanol as 0ng/mL blank control standard solution.

The preparation method of the DEP polyclonal antibody serum is that the amino group of the DEP hapten transformation product 4-amino-diethyl phthalate is coupled with bovine serum albumin to prepare the DEP immune antigen, and the immune antigen is purified and freeze-dried , injected into New Zealand white rabbits in 5 times, and the heart blood was collected and centrifuged to obtain polyclonal antibody serum, which was stored at -20°C after aliquoting.

The concentration of each component in the concentrated phosphate (PBS) buffer solution is 27mmol/L KCl, 1380mmol/L NaCl, 70mmol/L Na ₂ HPO ₄ and 15mmol/L KH ₂ PO ₄ .

Detection principle of the present invention:

The detection method of the kit adopts fluorescence polarization technology, extracts DEP from the sample with a solvent, and obtains a mixture by mixing the extract, fluorescent marker and DEP polyclonal antibody, and the fluorescent marker can be combined with DEP polyclonal antibody to produce a Detection of fluorescence polarization changes. The fluorescent marker is obtained by coupling a suitable fluorophore with DEP, and the artificial immunogen for preparing the antibody is made by coupling DEP with bovine serum albumin. Measure the fluorescence polarization of the mixture. A standard curve is obtained by measuring the fluorescence polarization of a series of DEP solutions with known concentrations, and then the fluorescence polarization of the mixture is measured, and the concentration of DEP in the mixture is calculated by using the standard curve.

Advantage and positive effect of the present invention:

The kit combines the high specificity of antibody-antigen reaction with the high sensitivity of fluorescent labeling, and uses the change of fluorescence polarization after antigen-antibody binding to detect the concentration of the sample. High, the maximum detection range for DEP is 10-200ng/mL, and the sensitivity can reach 40.4ng/mL. It plays an important role in the rapid detection of residual DEP in water, fruit juice drinks and capsules and other actual systems.

【Description of drawings】

Figure 1 is the inhibition rate curve of the DEP antibody of the present invention.

Fig. 2 is the standard curve of the present invention.

【Detailed ways】

Example:

A diethyl phthalate fluorescent polarization immunoassay kit, including fluorescent marker stock solution, DEP standard solution, DEP polyclonal antibody serum and concentrated phosphate (PBS) buffer solution and respectively packaged in reagent tubes, fluorescent The marker stock solution is a solution obtained by dissolving fluorescein isothiocyanate (FITC)-labeled DEP fluorescent conjugates in 200 μL methanol and packaging it in a reagent tube with a volume of 1.5 mL; DEP standard solution is as follows: DEP in methanol solvent The different concentrations of DEP were set and packaged in seven reagent tubes with a volume of 1.5mL, and the solvent was methanol, 1mL in each tube; DEP polyclonal antibody serum was 600μL and packaged in a reagent tube with a volume of 1.5mL; concentrated PBS buffer The solution is packaged in two reagent tubes with a volume of 10 mL, and each reagent tube contains 9 mL of liquid.

The preparation of described solution:

1) The preparation method of the fluorescent marker stock solution is to react 1 mg of 4-amino-diethyl phthalate and 1 mg of FITC in 500 μL of methanol with a mass percentage of 25% triethylamine at a temperature of 4°C After 8-10 hours, take 50 μL of the above-mentioned mixed solution, draw a band on the bottom of the thin-layer chromatography silica gel sheet, and put the thin-layer chromatography silica gel sheet into a developer with a volume ratio of chloroform and methanol of 4:1 for chromatography. Then scrape off the band with Rf of 0.5 on the thin-layer chromatography silica gel sheet, dissolve it in 200mL methanol and filter, and take the filtrate as the fluorescent marker stock solution.

2) The concentration of the DEP standard solution is 0ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 75ng/mL, 100ng/mL and 200ng/mL, methanol is the solvent, and each tube is 1mL; the preparation method is to 0.2mg DEP standard was dissolved in 1mL methanol to prepare a 200ng/mL standard solution, and then the 200ng/mL standard solution was diluted with methanol to 100ng/mL, 75ng/mL, 50ng/mL, 20ng/mL and 10ng/mL mL standard solution, take 1mL methanol as 0ng/mL blank control standard solution.

3) The preparation method of the DEP polyclonal antibody serum is that the amino group of the DEP hapten transformation product 4-amino-diethyl phthalate is coupled with bovine serum albumin to obtain the DEP immune antigen, and the immune antigen is purified and freeze-dried , injected into New Zealand white rabbits in 5 times, and the heart blood was collected and centrifuged to obtain polyclonal antibody serum, which was stored at -20°C after aliquoting.

4) The concentration of each component in the concentrated phosphate buffer solution (PBS) is 27mmol/L KCl, 1380mmol/LNaCl, 70mmol/L Na ₂ HPO ₄ and 15mmol/L KH ₂ PO ₄ .

Specific implementation steps:

1) Synthesis of immune antigen

Weigh 0.05328g (0.24mmol) of diethyl 4-aminophthalate, mix with 0.1mL concentrated HCI and 3mL H ₂ O, heat to dissolve; place in an ice bath to cool below 5°C, and drop into 4mL under stirring at low temperature 0.0110g NaNO ₂ aqueous solution, continue to react for 30min; add 40mL solution of 160mg BSA sodium borate buffer solution (pH value 9.2) dropwise to the above diazonium salt, an orange-red solution gradually appears, continue to react for 2h, and obtain the crude antigen Reaction solution: put the reaction solution into a dialysis bag and dialyze in PBS buffer at 4°C for 2 days, then in PBS buffer at 4°C for 6 days, and freeze-dry to obtain an orange-red solid powder of DEP immune antigen.

2) Preparation of DEP polyclonal antibody serum

New Zealand white rabbits were used as immunized animals, and the conjugate of DEP hapten transformation and bovine serum albumin was used as the immune antigen. Complete adjuvant made into an emulsifier, injected at multiple points on the back of the neck, after immunization, take half the dose of immune antigen dissolved in equal volume of normal saline and Freund's incomplete adjuvant, mix and emulsify, the interval between the first immunization and the second immunization is 20 days, and later The mice were immunized every two weeks for a total of five times, and the last time was without adjuvant. Seven days later, blood was collected from the heart, and the DEP polyclonal antibody serum was obtained by centrifugation.

3) Preparation of fluorescent marker stock solution

The reaction to prepare the fluorescent marker stock solution is as follows:

Dissolve 1mg of 4-amino-diethylphthalate and 1mg of FITC in 500μL of methanol containing 25% triethylamine, and react overnight at 4°C for 8 hours; Draw a band at the bottom, and point a small amount of FITC and 4-amino-diethyl phthalate on the left and right sides of this band as a control; put this thin-layer chromatography silica gel into chloroform:methanol=(4:1) After about 5 minutes, the developer reaches a distance of about 1 cm from the top of the thin-layer chromatography silica gel sheet, and is taken out. The plate presents two main bands, one is the band of Rf=0.5, and the other is the band of Rf=0.3; scrape off the band of Rf=0.5 on the thin-layer chromatography silica gel sheet, dissolve it in 200 μL of methanol and filter it, and take the filtrate as a fluorescent marker stock solution.

4) Establishment of fluorescence polarization detection method

① Optimum dilution of fluorescent marker stock solution:

Dilute the fluorescent marker stock solution by 500, 1000, 2000, 4000, and 8000 times, respectively, take 1 mL and add it to a test tube, and read it on a fluorescence polarimeter (read the value of the working buffer PBS as blank); the fluorescence value is the working The value of 5 times of the buffer solution is the most sensitive and accurate, and the final dilution of the fluorescent marker is 1:1000.

②Optimized polyclonal antibody serum dilution:

Dilute the polyclonal antibody serum by 50, 100, 200, 400, 800, and 1600 times, respectively, add it to a test tube containing 0.5 mL of the above-mentioned preferred dilution of the fluorescent marker, vortex and mix, and read on a fluorescence polarizer; At this time, the fluorescence value and the mP value are different. The more concentrated the antibody, the greater the mP value. According to experience, the mP value is 0.7 times the maximum mP value.

③Determination of antibody sensitivity and establishment of standard curve:

According to the above-mentioned optimized experiments on the concentration of antibodies and fluorescent markers, the dilution ratio of the polyclonal antibody serum was selected and determined to be 1:300, and the dilution ratio of the fluorescent marker stock solution was 1:1000, and the fluorescent marker working solution and the antibody working solution were prepared respectively. Solution, carry out the determination of the sensitivity of the antibody, the specific steps are as follows:

Adding samples: Add 495 μL of DEP fluorescent marker working solution to a 4 mL fluorescent polarization detection small test tube, add 10 μL of DEP standard solutions of different concentrations, and vortex to mix;

Incubation: Then add 495 μL of DEP antibody working solution, vortex and mix for 0.5 min, and incubate at room temperature (25°C) for 13.5 min;

Detection: Measure the fluorescence polarization intensity of each tube with a fluorescence polarimeter;

The detection result is calculated by inhibition rate: % inhibition rate=%mP/mP ₀ , mP is the fluorescence polarization value of different concentrations of drugs as competitors, and mP ₀ is the fluorescence polarization value of no drug added.

Fig. 1 is the inhibitory rate curve of the DEP antibody of the present invention, the log value of the concentration of the added drug (DEP) is on the abscissa, and the fluorescence polarization value ratio mP/ _mP0 of the blank control is plotted on the ordinate, and each point It is the average value of 5 repeated test results, and the concentration of the drug when calculating the 50% inhibition rate is the sensitivity of the antibody. The dilution ratio of the fluorescent marker stock solution is 1:1000, and the dilution ratio of the polyclonal antibody serum is 1/300. At this time, the IC ₅₀ and the linear range are the best, and the sensitivity of the method is the best.

Fig. 2 is the standard curve of the present invention, draws by intercepting the linear portion of the inhibition rate curve (the part where the DEP concentration is within the range of 10-200ng mL ^-1 ), the linear equation corresponding to the standard curve is y=-0.35067x+1.16876, R ² =0.99942 (R is a linear correlation coefficient), and the concentration of DEP in the sample to be tested is calculated according to the linear equation of the standard curve. The figure shows that the linearity between the ratio mP/mP ₀ of the fluorescence polarization value and the logarithm value of the drug concentration within the range of DEP concentration from 10 ng/mL to 200 ng/mL is good.

Detection application of DEP immunoassay kit:

1) Preparation of reagents

: Working buffer solution: Dilute the concentrated PBS buffer solution provided in the kit 10 times with distilled water before use.

Fluorescent marker working solution: Dilute the fluorescent marker stock solution to 1000 times with the working buffer solution before use.

Antibody working solution: Dilute polyclonal antibody serum to 1000 times with working buffer solution before use.

2) Sample pretreatment

Bottled drinking water: Take 600mL bottled drinking water in a separatory funnel, add 30mL of n-hexane, shake and separate the liquid, separate and obtain 25mL of the upper layer extract, spin evaporate to dryness, add 1.25mL of methanol, vortex for 3min and pass 0.22μm filter membrane, take the clear night to get the sample to be tested.

Juice drink: Weigh 60mL sample, add 24mL n-hexane, shake and separate the liquid, separate to obtain 20mL of the upper layer extract, spin evaporate to dryness, add 10.0mL methanol, vortex for 3min, pass through a 0.22μm filter membrane to take the clear night to obtain the test sample.

Capsules: Weigh 4.6g of enteric-coated capsules, open and remove the drug content, add the shell of the capsules to 200mL of water, heat in a water bath at 70°C for 40min, dissolve the capsules, add 46mL of n-hexane, shake and separate the liquids, separate to obtain 15mL of the upper layer extract, and spin evaporate After drying, add 10.0 mL of methanol, vortex for 3 min, pass through a 0.22 μm filter membrane to take the clear night, and dilute the solution to 80 times to obtain the sample to be tested.

3) Detection steps

Adding samples: Add 495 μL of DEP fluorescent marker working solution to a 4mL fluorescent polarization detection test tube, add 10 μL of the sample solution to be tested, and vortex to mix;

Incubation: Then add 495 μL of DEP antibody working solution, vortex and mix for 0.5 min, and incubate at room temperature (25°C) for 13.5 min;

Detection: Measure the fluorescence polarization intensity of each tube with a fluorescence polarimeter.

4) The test results of the samples to be tested and the performance identification of the kit

Sample inhibition rate (%)=DEP solution or sample fluorescence polarization value (or sample)/sample fluorescence polarization value without DEP×100%

Put the value of the inhibition rate of the sample (as the y value) into the linear equation y=0.35067x+1.16876 corresponding to the standard curve (Figure 2), and calculate the DEP concentration value (ie, the x value) in the sample to be tested.

Precision and Accuracy Tests:

Taking the detection of fruit juice drinks as an example, the fruit juice drinks containing DEP were moderately diluted to 1/50 times according to the maximum allowable addition amount, and the DEP addition amount of three kinds of concentration drinks were detected, and the detection recovery rate of DEP was calculated. The intra-assay coefficient of variation was calculated five times on the same day. The measurement was repeated 5 times a day for 3 days, and the coefficient of variation between batches was calculated (a total of 45 samples were detected). The quantitative calculation of the recovery rate is calculated according to the linear equation of the standard curve, and the formula is as follows:

% Recovery = % [measured value of spiked sample (ppm) - measured value of control solution (ppm)]/added concentration (ppm)

The test results of the kit are shown in Table 1:

Table 1. Kit precision and accuracy test results

The test results showed that the coefficient of variation was lower than 17.0%, and the recovery rate was between 80% and 120%. It shows that the kit has good repeatability and accuracy, and is a diethyl phthalate (DEP) fluorescence polarization immunoassay kit with simple and fast operation and high detection accuracy.

Claims (5)

1. a diethyl phthalate fluorescence polarization immunoassay kit, is characterized in that: comprise fluorescent marker stock solution, DEP standard solution, DEP polyclonal antibody serum and concentrated phosphate (PBS) buffer solution and package respectively In the reagent tube, the fluorescent marker stock solution is a solution obtained by dissolving fluorescein isothiocyanate (FITC)-labeled DEP fluorescent conjugate in 200 μL methanol and packaging it in a reagent tube with a volume of 1.5 mL; DEP standard The solution is set according to different concentrations of DEP in methanol solvent and packaged in seven reagent tubes with a volume of 1.5mL. The solvent is methanol, 1mL in each tube; DEP polyclonal antibody serum is 600μL and packaged in a reagent tube with a volume of 1.5mL tube; the concentrated PBS buffer solution is packaged in two reagent tubes with a volume of 10 mL, and each reagent tube contains 9 mL of liquid. 2. according to the described diethyl phthalate fluorescent polarization immunoassay kit of claim 1, it is characterized in that: the preparation method of described fluorescent marker stock solution is, with 1mg 4-amino-phthalic acid diethyl The ester and 1 mg FITC were reacted in 500 μL of methanol with a mass percentage of 25% triethylamine at a temperature of 4°C for 8-10 hours. Take 50 μL of the above mixed solution, draw a band on the bottom of a thin-layer chromatography silica gel sheet, and place Put this thin-layer chromatography silica gel sheet into a developer with a volume ratio of chloroform and methanol of 4:1 for chromatography, then scrape off the band with Rf of 0.5 on the thin-layer chromatography silica gel sheet, dissolve it in 200mL methanol and filter, and take the filtrate As a fluorescent marker stock solution. 3. according to the described diethyl phthalate fluorescence polarization immunoassay kit of claim 1, it is characterized in that: the concentration that described DEP standard solution is set is 0ng/mL, 10ng/mL, 20ng/mL, 50ng/mL , 75ng/mL, 100ng/mL and 200ng/mL, methanol as solvent, 1mL per tube; the preparation method is to dissolve 0.2mg DEP standard substance in 1mL methanol to prepare a 200ng/mL standard solution, and then dilute 200ng/mL with methanol /mL standard solution gradient dilution to 100ng/mL, 75ng/mL, 50ng/mL, 20ng/mL and 10ng/mL standard solution, take 1mL methanol as 0ng/mL blank control standard solution. 4. according to the described diethyl phthalate fluorescence polarization immunoassay kit of claim 1, it is characterized in that: the preparation method of described DEP polyclonal antibody serum is, the DEP hapten transformant 4-amino-o-phenyl The amino group of diethyl diformate is coupled with bovine serum albumin to prepare the DEP immune antigen. After the immune antigen is purified and freeze-dried, it is injected into New Zealand white rabbits in 5 times. After the heart blood is collected and centrifuged, the polyclonal antibody serum is obtained and packaged. Then store at -20°C. 5. according to the described diethyl phthalate fluorescence polarization immunoassay kit of claim 1, it is characterized in that: in described concentrated phosphate (PBS) buffer solution, each component concentration is 27mmol/L KCl, 1380mmol/L NaCl, 70 mmol/L Na ₂ HPO ₄ and 15 mmol/L KH ₂ PO ₄ .

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