Diclofenac enzyme-linked immunosorbent assay kit and application

Abstract

The invention provides a diclofenac enzyme-linked immunosorbent assay kit. The diclofenac enzyme-linked immunosorbent assay kit comprises an enzyme-labeled microwell plate, a diclofenac antibody working solution, a diclofenac standard substance, a horse radish peroxidase-labeled anti-antibody working solution, a 20 * concentrated washing solution, a substrate solution A, a substrate solution B and a stop solution, the enzyme-labeled microwell plate is coated with a diclofenac hapten-carrier protein conjugate; a PBS buffer solution containing 2% of glycerin, 2.5% of trehalose, 2.5% of peptone, 1% of sorbitol, 0.01% of Proclin300 and 0.01 mol / L and having a pH value of 7.4 is added into the diclofenac antibody working solution as an antibody stabilizer; a 50mmol / LTris buffer solution containing 6% of gelatin, 0.05% of potassium sorbate, 1.5% of bovine serum albumin and 0.5% of Tween 80 is added into the horse radish peroxidase labeled anti-antibody working solution as an enzyme conjugate stabilizer. The diclofenac enzyme-linked immunosorbent assay kit is good in detection sensitivity, accuracy and precision parameters, excellent in stability performance index and capable of being stored for a long time, and is an ideal diclofenac detection product.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology detection, and in particular relates to a diclofenac enzyme-linked immunosorbent assay kit and an application thereof in detecting diclofenac drug residues in animal tissues. Background Art

[0002] Diclofenac is a non-steroidal anti-inflammatory drug with anti-inflammatory, analgesic and antipyretic effects. It can be used for rheumatoid arthritis, cancer, post-traumatic pain and fever caused by various inflammations. In veterinary clinical practice, the main medicinal form is diclofenac sodium, which is used for analgesia and antipyretic effects on small and medium-sized animals, and is toxic to poultry. The residual marker of diclofenac sodium in animals is diclofenac.

[0003] Diclofenac sodium has been approved as a new veterinary drug in China, used for pain and fever caused by trauma surgery and inflammation in pigs, and there are also reports of trials for cattle treatment. Both China and the European Union stipulate that the maximum residue limits in the muscles, livers, and kidneys of cattle and pigs are 5, 5, and 10 μg / kg, respectively. Eating animal tissues containing diclofenac will be harmful to human health.

[0004] At present, the detection methods of diclofenac are mainly instrumental detection methods, such as high performance liquid chromatography, liquid chromatography-tandem mass spectrometry, etc. Although the instrumental analysis method has the advantages of high detection accuracy and good repeatability, it has the disadvantages of complicated pre-treatment procedures, long measurement time, high instrument cost, and high professional requirements for detection personnel. It is not conducive to the screening of large quantities of samples, nor is it conducive to the effective supervision of the use of diclofenac drugs. Enzyme-linked immunosorbent assay is a rapid screening method based on the specific binding reaction between antigens and antibodies. It uses enzymes as reaction media. It has the characteristics of simple and rapid detection, large detection sample volume, and low requirements for detection personnel. Summary of the invention

[0005] The invention aims to provide a diclofenac enzyme-linked immunosorbent assay kit with strong detection stability, high detection sensitivity, strong specificity and short detection time.

[0006] Technical solution of the present invention:

[0007] A diclofenac enzyme-linked immunosorbent assay kit comprises a microporous ELISA plate coated with a diclofenac hapten-hemocyanin conjugate, a diclofenac antibody working solution, a diclofenac standard, a horseradish peroxidase-labeled anti-antibody working solution, a 20× concentrated washing solution, a substrate solution A, a substrate solution B, and a stop solution. The diclofenac hapten-carrier protein conjugate is coated on the microporous ELISA plate, a PBS buffer solution containing 2% glycerol, 2.5% trehalose, 2.5% peptone, 1% sorbitol, 0.01% Proclin 300, and 0.01 mol / L pH 7.4 is added to the diclofenac antibody working solution as an antibody stabilizer, and a 50 mmol / L Tris buffer solution containing 6% gelatin, 0.05% potassium sorbate, 1.5% bovine serum albumin, and 0.5% Tween 80 is added to the horseradish peroxidase-labeled anti-antibody working solution as an enzyme conjugate stabilizer.

[0008] Furthermore, the diclofenac hapten-carrier protein conjugate is prepared by the following steps: 10 mg of diclofenac sodium is weighed and dissolved in 0.5 mL of DMF to obtain a hapten diluent; 10 mg of EDC and 10 mg of NHS are weighed and dissolved in 0.2 mL of deionized water, and then added to the hapten diluent, and stirred at room temperature for 0.5 h to obtain an activated hapten diluent; 40 mg of carrier protein bovine serum albumin or hemocyanin is weighed and fully dissolved in 4 mL of 0.01 mol / L PBS; the activated hapten diluent is added to the dissolved carrier protein solution, and the reaction is stirred at room temperature for 16 h; and the diclofenac hapten-carrier protein conjugate is prepared by dialysis with 0.01 mol / L PBS.

[0009] The diclofenac antibody is prepared by immunizing mice or rabbits with diclofenac hapten-bovine serum albumin conjugate as an immunogen.

[0010] Furthermore, the concentrated washing solution contains 1% Tween-20 and 0.1 mol / L PBS buffer.

[0011] Furthermore, the substrate A liquid is hydrogen peroxide, and the substrate B liquid is TMB, which are mixed within 10 minutes before use.

[0012] Furthermore, the stop solution was 1 mol / L H 2 SO 4 .

[0013] The diclofenac enzyme-linked immunosorbent assay kit may further include a sample reconstitution solution when detecting a specific sample. The sample reconstitution solution is a 0.05 mol / L citric acid-sodium citrate buffer solution, pH 6.0.

[0014] Another object of the present invention is to provide the use of the above-mentioned diclofenac enzyme-linked immunosorbent assay kit in detecting diclofenac in pork samples.

[0015] Beneficial effects of the present invention:

[0016] The diclofenac enzyme-linked immunosorbent assay kit of the present invention coats the coupled antigen on a microplate, uses an antibody working solution to compete with a sample reaction for the coating, then adds an enzyme-labeled secondary antibody for detection and develops color through a substrate, analyzes and determines the diclofenac residue, and establishes an indirect competitive ELISA method. When preparing the diclofenac enzyme-linked immunosorbent assay kit, the present invention uses a stabilizer buffer to seal the enzyme-labeled plate in the process of preparing the enzyme-labeled plate, adds an antibody stabilizer to the antibody working solution, and adds a horseradish peroxidase conjugate stabilizer to the anti-antibody labeled with horseradish peroxidase, thereby effectively improving the stability of the detection kit of the present invention, greatly prolonging the shelf life of the kit, avoiding the inaccurate detection result caused by the instability of the reagent, and causing the phenomenon of waste of reagents. The diclofenac enzyme-linked immunosorbent assay kit of the present invention has a detection sensitivity of up to 0.05 μg / kg for diclofenac, a determination recovery rate between 80% and 106%, an intra-plate coefficient of variation between 6.2% and 7.4%, and an inter-plate coefficient of variation of 7.6%. The kit of the present invention has a simple structure, high detection sensitivity, high stability, good accuracy and precision detection data, and low detection cost, and is suitable for rapid screening of diclofenac in large batches of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Kit standard curve graph. DETAILED DESCRIPTION

[0018] In order to further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention. Unless otherwise specified, the reagents of the present invention are all conventional reagents and can be purchased from the market.

[0019] Embodiment 1

[0020] 1. The diclofenac ELISA kit contains the following components:

[0021] (1) A 96-well ELISA plate coated with a diclofenac hapten-hemocyanin coupled antigen.

[0022] (2) Diclofenac antibody working solution, specifically diclofenac monoclonal antibody, 7 mL / bottle. PBS buffer containing 2% glycerol, 2.5% trehalose, 2.5% peptone, 1% sorbitol, 0.01% Proclin 300, 0.01 mol / L pH 7.4 was added to the antibody working solution as an antibody stabilizer, and the percentages are by mass.

[0023] (3) Diclofenac standard: 0 μg / L, 0.05 μg / L, 0.15 μg / L, 0.45 μg / L, 1.35 μg / L, 4.05 μg / L, 1 mL / bottle.

[0024] (4) Horseradish peroxidase-labeled goat anti-mouse anti-antibody working solution, 12 mL / bottle, to which 50 mmol / L Tris buffer containing 6% gelatin, 0.05% potassium sorbate, 1.5% bovine serum albumin, and 0.5% Tween 80 was added as an enzyme conjugate stabilizer.

[0025] (5) 20× concentrated washing solution, containing 1% Tween-20, 0.1 mol / L pH 7.2 PBS buffer, 30 mL / bottle, diluted with deionized water at a ratio of 1:19 when used.

[0026] (6) Sample reconstitution solution: 0.05 mol / L citric acid-sodium citrate buffer, pH 6.0, 8 mL / bottle.

[0027] (7) Substrate A solution is hydrogen peroxide, and substrate B solution is TMB, both at 7 mL / bottle.

[0028] (8) The stop solution is 1 mol / L H 2 SO 4 , 7mL / bottle.

[0029] 2. Preparation of main components:

[0030] 2.1. Synthesis of immunogen and coating

[0031] The immunogen is diclofenac hapten-bovine serum albumin, and the coating is diclofenac hapten-hemocyanin. The preparation process is as follows:

[0032] Weigh 10 mg of diclofenac sodium and dissolve it in 0.5 mL of DMF to obtain a hapten dilution solution; weigh 10 mg of EDC and 10 mg of NHS and dissolve them in 0.2 mL of deionized water, then add them to the hapten dilution solution and stir at room temperature for 0.5 h to obtain an activated hapten dilution solution; weigh 40 mg of carrier protein bovine serum albumin or hemocyanin and fully dissolve it in 4 mL of 0.01 mol / L PBS; add the activated hapten dilution solution drop by drop into the prepared carrier protein solution, stir at room temperature for 16 h, dialyze with 0.01 mol / L PBS, with a molecular weight cutoff of 8000-14000u, dialyze at 4°C for 3 days, change the dialysate 3 times a day, and store it in aliquots at -20°C after preparation.

[0033] 2.2 Preparation of diclofenac monoclonal antibody

[0034] Six-week-old female Balb / c mice were selected and subcutaneously injected with diclofenac hapten-bovine serum albumin conjugate containing Freund's complete adjuvant at an immunization dose of 100 μg / mouse. Immunizations were performed at weeks 2, 4, and 8. Boost immunizations were performed with diclofenac hapten-bovine serum albumin conjugate containing incomplete Freund's adjuvant. The last immunization was at week 12. Splenocytes were collected three days after the last boost immunization.

[0035] SP2 / 0 myeloma cells were cultured in RPMI-1640 medium to make the cells in the logarithmic growth phase, and SP2 / 0 myeloma cell suspension was prepared; the spleen of the immunized mouse was taken, crushed, and filtered through a 200-mesh stainless steel gauze to prepare a spleen cell suspension, which was counted and used for later use. SP2 / 0 myeloma cells and spleen cells were fused and cultured at a ratio of 1:5, and positive wells were screened by indirect competitive ELISA. The positive wells were monocloned by limiting dilution method until a hybridoma cell line that stably secreted diclofenac monoclonal antibodies was obtained.

[0036] The diclofenac monoclonal hybridoma cell line was made into a cell suspension and stored in liquid nitrogen. When thawing, the cryopreserved tube was taken out and immediately placed in a 37°C water bath for rapid thawing, then transferred to a conical flask, added with complete culture medium, and heated with CO 2 Culture in incubator.

[0037] Healthy Balb / c female mice were intraperitoneally injected with paraffin oil (0.5 mL / mouse). Ten days later, 10% of diclofenac monoclonal hybridoma cell lines were intraperitoneally injected. 7 After 7 days, the ascites was collected and centrifuged at 4000 rpm for 10 min, the supernatant was collected, and the purified antibodies were stored at -20°C.

[0038] 2.3 Specificity of diclofenac monoclonal antibodies

[0039] Antibody specificity refers to the ability of an antibody to bind to antigenic determinants of different structures, which is expressed by the cross-reaction rate. A small cross-reaction rate can prove that the antibody has high specificity. The 50% inhibition concentration (IC 50 ). Use the following formula to calculate the cross-reaction rate of the kit to other drugs:

[0040] Cross-reaction rate (%) = (diclofenac IC 50 / Diclofenac Analogs IC 50 )×100%

[0041] It is measured that the cross-reaction rate of the diclofenac enzyme-linked immunosorbent assay kit of the present invention to diclofenac is 100%, and the cross-reaction rate to sodium pentachlorophenate is less than 0.1%, and the content of diclofenac in the sample can be specifically detected.

[0042] 2.4. Preparation of goat anti-mouse antibody labeled with horseradish peroxidase

[0043] The goat anti-mouse anti-antibody labeled with horseradish peroxidase is obtained by coupling horseradish peroxidase with goat anti-mouse anti-antibody by using sodium periodate method.

[0044] 2.5. ELISA plate

[0045] Dilute the diclofenac hapten-hemocyanin conjugate to 2μg / mL with 0.05mol / L, pH9.6 carbonate buffer, add 100μL to each well, incubate at 37℃ for 2h, wash twice, and spin dry; then add 250μL of coating blocking solution to each well for blocking, incubate at 37℃ for 2h, pour out the liquid in the well, pat dry with absorbent paper, and then vacuum seal with aluminum film for storage. The coating blocking solution is a PBS buffer containing 3% sucrose, 0.5% bovine serum albumin, and 0.1mol / L pH7.2. The percentage is the mass percentage. The coating blocking solution can play a blocking role, reduce the background effect of the reagent well, and improve the detection accuracy of the kit.

[0046] The coated ELISA plate was placed in a 37°C thermostat with other reagents of the present invention for aging test. On the 25th day, the absorbance value was measured and the decrease of the A value was still less than 10%.

[0047] The stability of the ELISA plate and reagents is a key indicator for measuring product quality and an important guarantee for the safety and effectiveness of the kit. In order to significantly improve the stability of the kit of the present invention. In the process of developing the kit of the present invention, a PBS buffer containing 2% glycerol, 2.5% trehalose, 2.5% peptone, 1% sorbitol, 0.01% Proclin300, and 0.01 mol / L pH 7.4 is added to the diclofenac antibody working solution as a stabilizer, and a 50 mmol / L Tris buffer containing 6% gelatin, 0.05% potassium sorbate, 1.5% bovine serum albumin, and 0.5% Tween 80 is added to the goat anti-mouse anti-antibody labeled with horseradish peroxidase as an enzyme conjugate stabilizer, which significantly improves the stability of the kit of the present invention.

[0048] The assembled test kit may also include instructions, a cover film, a ziplock bag, a certificate of conformity, etc. The shelf life of the test kit can reach 2 years under conditions of 2 to 8°C.

[0049] 3. Detection principle of diclofenac ELISA kit:

[0050] The method utilizes the property of specific binding between diclofenac antibody and diclofenac antigen. The antigen is first pre-coated on the ELISA plate, and then the standard or sample and diclofenac antibody are added. Diclofenac competes with the antigen fixed on the ELISA plate for the diclofenac antibody, and then goat anti-mouse antibody labeled with horseradish peroxidase is added for signal amplification and detection. Finally, substrate is added to catalyze color development. At this time, the color development depth is inversely proportional to the diclofenac content in the standard or sample.

[0051] 4. Specific detection steps of the kit

[0052] (1) Take out the required number of ELISA strips and insert them into the ELISA plate rack, and record the positions of each standard and sample. To reduce the fluctuation of the test value, perform a double-well parallel experiment.

[0053] (2) Add 50 μL of the standard / sample to the corresponding microwells, then add 50 μL / well of the diclofenac antibody working solution, gently shake for 5 seconds to mix, cover the plate and place it in a dark environment at 25°C for 20 minutes.

[0054] (3) Remove the cover film, shake off the liquid in the wells, add 250 μL / well of washing solution, wash twice, pour out the washing solution, and pat dry with absorbent paper.

[0055] (4) Add 100 μL of enzyme-labeled goat anti-mouse antibody to each microwell, shake gently for 5 seconds to mix, cover the plate and place in a dark environment at 25°C for 20 minutes.

[0056] (5) Remove the cover film, shake off the liquid in the wells, add 250 μL / well of washing solution, wash twice, pour out the washing solution, and pat dry with absorbent paper.

[0057] (6) Add 100 μL / well of substrate colorimetric solution, mix well, cover the plate and place in a dark environment at 25°C for 15 min. The substrate colorimetric solution is a mixture of substrate A solution and substrate B solution in a 1:1 volume ratio. The mixture should be used within 10 min.

[0058] (7) Add 50 μL / well of stop solution and mix well. When the substrate solution turns from blue to yellow, set the microplate reader at dual wavelengths of 450 nm and 630 nm to measure the OD value.

[0059] 5. Analysis of test results

[0060] Assume that the average absorbance of each standard (or sample) is B, and the zero standard, that is, the standard with a concentration of 0 μg / L, has an absorbance average of B 0 (B / B 0 )×100% is the percentage of absorbance corresponding to each standard (or sample): Use the semi-logarithmic system to substitute the percentage of absorbance corresponding to the standard, and fit the standard curve with the concentration of the standard. Substitute the percentage of absorbance of the sample to be tested into the fitted standard curve equation to obtain the concentration corresponding to the sample, and finally multiply it by the corresponding dilution factor of the sample to obtain the content of the test substance in the sample.

[0061] Experimental Example Detection of Diclofenac in Pork Samples

[0062] 1. Sample pretreatment method

[0063] Weigh 2±0.05g of pork homogenized sample into a 15mL polystyrene centrifuge tube; add 2mL of deionized water, 5mL of ethyl acetate, and 1mL of n-hexane, vortex for 2min, and centrifuge at 4000rpm for 10min at room temperature; take 1mL of supernatant into a centrifuge tube, blow dry with nitrogen in a 50-60℃ water bath; add 1mL of sample reconstitution solution, vortex for 1min; take 50μL for analysis.

[0064] The sample reconstitution solution is 0.05 mol / L citric acid-sodium citrate buffer, pH 6.0, and the percentages are mass percentages.

[0065] 2. Standard curve fitting

[0066] The standard curve consists of 6 concentrations (0 μg / L, 0.05 μg / L, 0.15 μg / L, 0.45 μg / L, 1.35 μg / L, 4.05 μg / L). Each of the 6 standard points was measured twice to determine the working concentration range and IC 50 The logarithmic value of the concentration of diclofenac standard is used as the horizontal axis, and the inhibition rate (B / B 0 %) as the ordinate, and a standard curve was prepared. Figure 1 . Kit IC 50It is 0.175μg / L.

[0067] 3. Sample precision and accuracy test

[0068] Accuracy is expressed as recovery rate, and precision is expressed as coefficient of variation. Diclofenac was added to the pork sample for recovery, so that the final concentration was 0.5 μg / kg. Five parallels were drawn for each concentration, and different kits were used for determination. The same sample was determined 3 times, and the sample recovery rate and coefficient of variation were calculated respectively. The results are shown in Table 1.

[0069] Table 1 Recovery rate and coefficient of variation determination

[0070]

[0071] The results showed that for pork samples supplemented with 0.5 μg / kg diclofenac, the recovery rate was between 80-106%, the intra-plate coefficient of variation was between 6.2-7.4%, and the inter-plate coefficient of variation was 7.6%.

[0072] Embodiment 2

[0073] (1) A 96-well ELISA plate coated with a diclofenac hapten-hemocyanin conjugate.

[0074] (2) Diclofenac polyclonal antibody working solution, 7 mL / bottle.

[0075] (3) Diclofenac standard: 0 μg / L, 0.05 μg / L, 0.15 μg / L, 0.45 μg / L, 1.35 μg / L, 4.05 μg / L, 1 mL / bottle.

[0076] (4) Horseradish peroxidase-labeled goat anti-rabbit antibody, 12 mL / bottle.

[0077] (5) 20× concentrated washing solution, containing 1% Tween-20, 0.1 mol / L pH 7.2 PBS buffer, 30 mL / bottle, diluted with deionized water at a ratio of 1:19 when used.

[0078] (6) Sample reconstitution solution: 0.05 mol / L citric acid-sodium citrate buffer, pH 6.0, 8 mL / bottle.

[0079] (7) Substrate A solution is hydrogen peroxide, and substrate B solution is TMB, both at 7 mL / bottle.

[0080] (8) The stop solution is 1 mol / L H 2 SO 4 , 7mL / bottle.

[0081] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A diclofenac enzyme-linked immunosorbent assay kit, comprising a microporous ELISA plate, a diclofenac antibody working solution, a diclofenac standard, a horseradish peroxidase-labeled anti-antibody working solution, a 20× concentrated washing solution, a substrate solution A, a substrate solution B, and a stop solution; the microporous ELISA plate is coated with a diclofenac hapten-carrier protein conjugate; a PBS buffer solution containing 2% glycerol, 2.5% trehalose, 2.5% peptone, 1% sorbitol, 0.01% Proclin 300, and 0.01 mol / L pH 7.4 is added to the diclofenac antibody working solution as an antibody stabilizer; and a 50 mmol / L Tris buffer solution containing 6% gelatin, 0.05% potassium sorbate, 1.5% bovine serum albumin, and 0.5% Tween 80 is added to the horseradish peroxidase-labeled anti-antibody working solution as an enzyme conjugate stabilizer.

2. The diclofenac ELISA kit according to claim 1, characterized in that: The diclofenac hapten-carrier protein conjugate is prepared by the following steps: 1) Weigh 10 mg of diclofenac sodium and dissolve it in 0.5 mL of DMF to obtain a hapten dilution solution; 2) Weigh 10 mg EDC and 10 mg NHS and dissolve them in 0.2 mL deionized water; 3) mixing the solution obtained in step 2) with the hapten solution obtained in step 1), stirring at room temperature for 0.5 h to obtain an activated hapten dilution solution; 4) Weigh 40 mg of carrier protein bovine serum albumin or hemocyanin and fully dissolve it in 4 mL 0.01 mol / L PBS; 5) The activated hapten dilution prepared in step 3) was added dropwise to the solution prepared in step 4), and the mixture was stirred at room temperature for 16 h. The diclofenac hapten-carrier protein conjugate was prepared by dialyzing with 0.01 mol / L PBS.

3. The diclofenac ELISA kit according to claim 1 or 2, characterized in that: The concentrated washing solution contains 1% Tween-20, 0.1 mol / L PBS buffer, and has a pH of 7.

2.

4. The diclofenac ELISA kit according to claim 1 or 2, characterized in that: The substrate A liquid A is hydrogen peroxide, and the substrate B liquid is TMB.

5. The diclofenac ELISA kit according to claim 1 or 2, characterized in that: The stop solution is 1 mol / L H2SO4.

6. The diclofenac ELISA kit according to claim 1 or 2, characterized in that: The kit also includes a sample reconstitution solution, which is a 0.05 mol / L citric acid-sodium citrate buffer solution with a pH of 6.

0.

7. The diclofenac ELISA kit according to claim 1 or 2, characterized in that: The diclofenac antibody is prepared by using a diclofenac hapten-carrier protein conjugate as an immunogen.

8. The diclofenac ELISA kit according to claim 1 or 2, characterized in that: The microporous ELISA plate is blocked by adding 3% sucrose, 0.5% bovine serum albumin, and 0.1 mol / L PBS buffer solution with pH 7.2 during coating of the diclofenac hapten-carrier protein conjugate.

9. Use of the diclofenac enzyme-linked immunosorbent assay kit according to any one of claims 1 to 8 in detecting diclofenac drug residues in animal tissues.

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