Cyromazine enzyme-linked immunosorbent assay kit and application thereof

Abstract

The invention provides a cyromazine enzyme-linked immunosorbent assay kit which comprises a 96-hole elisa plate, an enzyme-labeled antibody working solution, a cyromazine standard substance, a 20 * concentrated washing solution, a substrate solution A, a substrate solution B and a stop buffer, the 96-hole elisa plate is coated with a cyromazine hapten-carrier protein conjugate, and the 96-hole elisa plate is coated with a cyromazine hapten-carrier protein conjugate. The enzyme-labeled antibody is a specific cyromazine antibody labeled by horse radish peroxidase. The invention also provides application of the cyromazine enzyme-linked immunosorbent assay kit in detection of mutton and chicken samples. The cyromazine enzyme-linked immunosorbent assay kit disclosed by the invention is high in detection sensitivity, relatively high in detection accuracy, precision and other technical indexes and long in reagent storage life, and also has the advantages of simple structure, easiness in operation, short detection time and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology detection, and specifically relates to a cyromazine enzyme-linked immunosorbent rapid detection kit and application thereof. Background Art

[0002] Cyromazine, also known as "fly miticide" and "mosquito maggot cleaner", is a triazine compound. In recent years, cyromazine has been widely added to animal feed to control the growth and development of fly maggots in livestock and poultry houses and the surrounding environment, and to improve the growth environment of livestock and poultry. Cyromazine remaining in livestock and poultry can cause harm to human health through enrichment in the food chain.

[0003] Currently, many countries in the world have set upper limits for the residues of cyromazine in animal foods.

[0004] Currently, the methods for detecting cyromazine reported at home and abroad mainly include high performance liquid chromatography (HPLC), gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), etc. Although the instrumental methods have high accuracy and sensitivity in detection results, the instrumental detection methods have the disadvantages of complex sample pretreatment procedures, expensive instrument detection costs, and the need for professional technicians to operate, which is not conducive to the rapid screening of large quantities of samples. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, one of the objects of the present invention is to provide a cyromazine enzyme-linked immunosorbent assay kit with high detection sensitivity and accuracy, short detection reaction time and easy operation, which can meet the needs of rapid screening of large quantities of samples.

[0006] Technical solution of the present invention:

[0007] The cyclopropane enzyme-linked immunosorbent assay kit of the invention comprises a 96-well enzyme-labeled plate, an enzyme-labeled antibody, a cyclopropane standard substance, a 20× concentrated washing solution, a substrate A solution, a substrate B solution and a stop solution, wherein the 96-well enzyme-labeled plate is coated with a cyclopropane hapten-OVA conjugate, the enzyme-labeled antibody is a cyclopropane specific antibody labeled with horseradish peroxidase, and a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid solution containing 10% trehalose, 0.1% sodium ethylenediaminetetraacetate, 0.05% L-methionine, 1% polyethylene glycol 6000, 0.1% diimidazoles urea and 0.05 mol / L pH 7.5 is added to the enzyme-labeled antibody as an enzyme-labeled antibody stabilizer, thereby further improving the stability of the antibody reagent.

[0008] Among them, the cyromazine standards are 0μg / L, 1μg / L, 3μg / L, 9μg / L, 27μg / L, and 81μg / L.

[0009] The composition of the 20× concentrated washing solution is 2% Tween-20, 0.1 mol / L pH 7.0 phosphate buffer.

[0010] Among them, substrate A liquid is hydrogen peroxide, and substrate B liquid is TMB.

[0011] Among them, the stop solution is 2mol / LH2SO4.

[0012] The cyromazine-specific antibody is prepared by using the cyromazine hapten-BSA conjugate as an immunogen.

[0013] The specific preparation steps of the cyromazine hapten-carrier protein conjugate are as follows:

[0014] Take 10 mg of cyromazine and dissolve it with 1 mL of DMF, add 50 μL of 50% glutaraldehyde, and stir at room temperature for 30 minutes to obtain an activated cyromazine hapten solution. Take 50 mg of BSA or OVA and dissolve it with 5 mL of 0.1 mol / L PBS, slowly add the activated cyromazine hapten solution to the carrier protein solution, stir at room temperature for 15 hours, continue to add 10 μL of 5 mol / L sodium cyanoborohydride, react at room temperature for 2 hours, and then dialyze with 0.01 mol / L PBS for three days, change the liquid three times a day, and package after the dialysis to obtain the cyromazine hapten-carrier protein conjugate.

[0015] Another object of the present invention is to provide an application of a cyromazine enzyme-linked immunosorbent assay kit in detecting mutton and chicken samples. The specific steps of the cyromazine enzyme-linked immunosorbent assay kit of the present invention are: processing mutton and chicken samples; detecting with the cyromazine enzyme-linked immunosorbent assay kit of the present invention; and analyzing the detection results.

[0016] Beneficial effects of the present invention:

[0017] The present invention establishes an ELISA method for detecting cyromazine drug residues. The detection sensitivity of the cyromazine enzyme-linked immunosorbent assay kit of the present invention to cyromazine is as high as 1 μg / L, the recovery rate of mutton and chicken samples is between 79.2% and 108.4%, and the detection coefficient of variation is less than 8%. The cyromazine enzyme-linked immunosorbent assay kit established by the method of the present invention can have a shelf life of more than 18 months under the condition of 2 to 8°C.

[0018] The cyclopropane-enzyme-linked immunosorbent assay kit of the technical solution of the present invention has a simple structure, high detection sensitivity, good technical parameters such as precision, accuracy, stability, etc., low detection cost, low requirements on detection personnel, can be used for rapid screening of a large number of samples, and is an ideal cyclopropane-enzyme-linked immunosorbent assay product. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1Standard curve of cyromazine enzyme-linked immunosorbent assay kit. DETAILED DESCRIPTION

[0020] 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.

[0021] Embodiment 1

[0022] 1. The cyromazine ELISA kit contains the following components:

[0023] (1) a 96-well ELISA plate coated with a cyromazine hapten-OVA conjugate;

[0024] (2) Horseradish peroxidase-labeled cyromazine polyclonal antibody, 7 mL / bottle;

[0025] (3) Cyromazine standard: 0 μg / L, 1 μg / L, 3 μg / L, 9 μg / L, 27 μg / L, 81 μg / L, 1 mL / bottle.

[0026] (4) 20× concentrated detergent, containing 1% hexadecyltrimethylammonium bromide, 0.5 mol / L pH 7.2 borate buffer, 30 mL / bottle, percentages are weight to volume ratio. When used, dilute with deionized water at a ratio of 1:19.

[0027] (5) Substrate A solution is hydrogen peroxide, and substrate B solution is TMB, both are 7 mL / bottle.

[0028] (6) The stop solution is 2 mol / L H2SO4, 7 mL / bottle.

[0029] 2. Preparation of main components:

[0030] 2.1 Synthesis of coating and immunogen

[0031] The cyclopropane hapten-BSA conjugate is an immunogen, and the preparation process is as follows: 10 mg of cyclopropane is dissolved in 1 mL of DMF, and then 50 μL of 50% glutaraldehyde is added, and the mixture is stirred at room temperature for 30 min; 50 mg of BSA is dissolved in 5 mL of 0.1 mol / L PBS; the activated cyclopropane hapten solution is slowly added to the dissolved BSA solution, and the mixture is stirred and reacted at room temperature for 15 h, and 10 μL of 5 mol / L sodium cyanoborohydride is continuously added, and the mixture is reacted at room temperature for 2 h, and then dialyzed with 0.01 mol / L PBS for three days, with the liquid being changed three times a day, and the mixture is packaged after the dialysis is completed.

[0032] The preparation method of the coating source is the same as the preparation method of the cyromazine hapten-BSA conjugate, except that the carrier protein is OVA.

[0033] 2.2 Preparation and specificity determination of cyromazine polyclonal antibodies

[0034] 2.2.1. Cyromazine hapten-BSA conjugate was mixed with Freund's complete adjuvant in a 1:1 ratio and then immunized New Zealand white rabbits. The immunization dose was 400 μg and multiple injections were given subcutaneously at the back of the neck. Immunization was performed every three weeks. For the second and third immunizations, Freund's complete adjuvant was replaced with Freund's incomplete adjuvant. The dose was the same as the first immunization. After the third immunization, the interval between each immunization was 2 weeks, for a total of 5 immunizations. Blood was collected 7 days after the last immunization and purified cyromazine polyclonal antibodies were obtained by graded precipitation with ammonium sulfate.

[0035] 2.2.2 Specificity determination of cyromazine antibodies

[0036] 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:

[0037] Cross-reaction rate (%) = (Cyromazine IC 50 / Analog IC 50 )×100%

[0038] It is measured that the cross-reaction rate of the cyclopropanezine enzyme-linked immunosorbent assay kit of the present invention to cyclopropanezine is 100%, and there is no cross-reaction to penicillin, amoxicillin, cephalexin, streptomycin, gentamicin, kanamycin, neomycin, spectinomycin, chlortetracycline, etc., and the cyclopropanezine in the sample can be specifically detected.

[0039] 2.3 Preparation of enzyme-labeled antibodies

[0040] The enzyme-labeled antibody is a horseradish peroxidase-labeled cyclopropane polyclonal antibody, which is obtained by reacting horseradish peroxidase with the cyclopropane polyclonal antibody using a sodium periodate method. The prepared horseradish peroxidase-labeled cyclopropane polyclonal antibody is added with 10% trehalose, 0.1% sodium ethylenediaminetetraacetate, 0.05% L-methionine, 1% polyethylene glycol 6000, 0.1% diimidazole lignourea, and 0.05 mol / L pH 7.5 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid as an enzyme-labeled antibody stabilizer, and the percentage is a mass volume ratio.

[0041] 2.4. ELISA plate

[0042] Cyromazine hapten-OVA was diluted to 0.1 μg / mL with 0.05 mol / L, pH 9.6 carbonate coating solution, 100 μL was added to each well, incubated at 37°C for 2 hours, washed twice with washing solution, patted dry, and then 250 μL of 0.02 mol / L, pH 7.2 PBS buffer containing 5% skimmed milk powder was added to each well for blocking, incubated at 37°C for 2 hours, the liquid in the well was poured off, and the well was vacuum sealed with aluminum film after drying for storage. 5% skimmed milk powder was added for blocking during the preparation process of the ELISA plate of the present invention, which effectively reduced background interference and improved the accuracy of the test results of the kit. The percentage is the mass percentage.

[0043] The assembled test kit may also include instructions, a cover film, a ziplock bag, and a certificate of conformity. After being tested at 2 to 8° C. for 18 months, the maximum absorbance value (zero standard), 50% inhibition concentration, and actual measured value of the cyromazine standard added to the test kit are all within the normal range, and the shelf life of the test kit is more than 18 months.

[0044] 3. Cyromazine ELISA Kit Detection Principle:

[0045] Utilizing the property of specific binding between cyclopropane antibody and cyclopropane antigen, the cyclopropane-carrier protein coupled antigen is first pre-coated on the ELISA plate, and then the standard substance or sample and the enzyme-labeled cyclopropane antibody are added. Cyromazine competes with the antigen fixed on the ELISA plate for the enzyme-labeled cyclopropane antibody, and finally the substrate is added to catalyze the color development. At this time, the color development depth is inversely proportional to the content of cyclopropane in the standard substance or sample.

[0046] 4. Specific detection steps of the kit

[0047] (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.

[0048] (2) Add 50 μL of the standard / sample to the corresponding microwells, then add 50 μL / well of the horseradish peroxidase-labeled cyromazine polyclonal antibody working solution, gently shake for 5 seconds to mix, cover the plate and place it in a dark environment at 25°C for 30 minutes.

[0049] (3) Remove the cover film, shake off the liquid in the wells, add 250 μL / well of washing solution, wash three times, pour out the washing solution, and pat dry with absorbent paper.

[0050] (4) Add 100 μL of substrate colorimetric solution (50 μL of hydrogen peroxide in solution A and 50 μL of tetramethylbenzidine in solution B, mix before use) and mix well. Cover the plate and place in a dark environment at 25°C for 15 min.

[0051] (5) Add 50 μL / well of stop solution and mix well. When the substrate solution turns from blue to yellow, read the absorbance at 450 nm and 630 nm using a microplate reader.

[0052] 5. Analysis of test results

[0053] Assume that the average absorbance of each standard (or sample) is B, the zero standard is a standard with a concentration of 0 μg / L, and the average absorbance B0 is (B / B0)×100% as the percentage of the absorbance corresponding to each standard (or sample): use a semi-logarithmic system to substitute the percentage of the absorbance corresponding to the standard, and fit the standard curve with the concentration of the standard. Substitute the percentage of the 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 cyromazine content in the sample.

[0054] Experimental Example: Detection of Cyromazine in Lamb and Chicken Samples

[0055] 1. Tissue sample pretreatment method

[0056] Weigh 2±0.05g homogenized fresh tissue sample into a 10mL centrifuge tube; add 3.6mL deionized water and 400μL sample extractant in turn, vortex thoroughly for 3min, and centrifuge at 4000rpm for 10min at room temperature; aspirate 600μL supernatant, add 200μL sample diluent, mix thoroughly; take 50μL for analysis. The sample extractant is 50% acetonitrile + 1% potassium carbonate. The sample diluent is 0.05mol / L pH7.5 potassium dihydrogen phosphate-sodium hydroxide buffer.

[0057] 2. Standard curve fitting

[0058] The standard curve consists of 6 concentrations (0 μg / L, 1 μg / L, 3 μg / L, 9 μg / L, 27 μg / L, 81 μg / L). Each of the 6 standard points was measured twice to determine the working concentration range and IC 50 The standard curve was prepared with the logarithmic value of the cyromazine standard concentration as the horizontal axis and the inhibition rate (B / B0%) of each corresponding concentration as the vertical axis. 50 The results are shown in Figure 1 .

[0059] 3. Sample precision and accuracy test

[0060] Accuracy is expressed as recovery rate, and precision is expressed as coefficient of variation. Cyromazine was added to mutton and chicken samples for recovery, so that the final concentration was 9.0 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.

[0061] Table 1 Recovery rate and coefficient of variation determination

[0062]

[0063] The results showed that when 9.0 μg / kg cyromazine was added, the recovery rates of mutton samples were between 86.4% and 108.4%, and the coefficient of variation was less than 8%; the recovery rates of chicken samples were between 79.2% and 100.2%, and the coefficient of variation was less than 6%.

[0064] Example 2

[0065] The difference from Example 1 is that the enzyme-labeled antibody is a monoclonal antibody labeled with horseradish peroxidase. Preparation of cyromazine monoclonal antibody:

[0066] Animal immunization: Purebred female Balb / c mice aged 4 to 6 weeks were selected and injected subcutaneously at multiple points with cyclopropane hapten-BSA conjugate containing an equal amount of Freund's complete adjuvant. The immunization dose was 200 μg / mouse. In the third week, they were immunized with an immunogen containing an equal amount of Freund's incomplete adjuvant. Thereafter, they were immunized once every three weeks. 3 booster immunizations were performed with the same immunization dose as the first time. The spleen was collected and fused three days after the last immunization.

[0067] Cell fusion and monoclonalization: SP2 / 0 myeloma cells were cultured in RPMI-1640 medium, and the logarithmically growing myeloma cells were centrifuged; the spleen of mice was taken three days after booster immunization, ground, and passed through a cell sieve, and the spleen cell suspension was filtered through a 200-mesh stainless steel gauze to make a spleen cell suspension, which was counted and used for later use. Myeloma cells and spleen cells were mixed at a ratio of 1:10, fused under the action of PEG4000, and the cell supernatant was measured by indirect competitive ELISA to screen positive wells. The positive wells were monoclonalized using the limiting dilution method until a hybridoma cell line that stably secreted cyromazine monoclonal antibodies was obtained.

[0068] Cell freezing and thawing: The cyromazine monoclonal hybridoma cell line was made into a cell suspension and stored in liquid nitrogen. When thawing, take out the cryotube, immediately put it in a 37°C water bath to thaw quickly, transfer it to a conical flask, add 15mL of complete culture medium, and culture it in a CO2 incubator.

[0069] Mass production and purification of monoclonal antibodies: Balb / c female mice were intraperitoneally injected with 0.5 mL of paraffin oil per mouse. Ten days later, 10% of cyromazine monoclonal hybridoma cell line was intraperitoneally injected. 6 The ascites was collected after 7 days. The antibody was purified by caprylic acid-saturated ammonium sulfate method and stored at -20℃ after purification.

[0070] 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 cyclopropane-enzyme immunoassay kit, comprising a 96-well ELISA plate, an enzyme-labeled antibody, a cyclopropane-enzyme standard, a 20× concentrated washing solution, a substrate A solution, a substrate B solution, and a stop solution, wherein the 96-well ELISA plate is coated with a cyclopropane-hapten-carrier protein conjugate, and the enzyme-labeled antibody is a cyclopropane-specific antibody labeled with horseradish peroxidase.

2. The cyromazine enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The steps for preparing the cyromazine hapten-carrier protein conjugate are as follows: 1) Dissolve 10 mg of cyromazine in 1 mL of DMF, mix with 50 μL of 50% glutaraldehyde, and stir at room temperature for 30 min; 2) Dissolve 50 mg of OVA or BSA in 5 mL of 0.1 mol / L PBS; 3) slowly adding the solution obtained in step 1) into the solution obtained in step 2), stirring at room temperature for 15 hours; 4) Add 10 μL of 5 mol / L sodium cyanoborohydride to the solution obtained in step 3), react at room temperature for 2 h, and after the reaction, dialyze with 0.01 mol / L PBS to prepare the cyromazine hapten-carrier protein conjugate.

3. The cyromazine enzyme-linked immunosorbent assay kit according to claim 1 or 2, characterized in that: The concentrations of the cyromazine standard are 0 μg / L, 1 μg / L, 3 μg / L, 9 μg / L, 27 μg / L, and 81 μg / L, respectively.

4. The cyromazine enzyme-linked immunosorbent assay kit according to claim 1 or 2, characterized in that: The substrate A solution is hydrogen peroxide, the substrate B solution is TMB, and the stop solution is 2 mol / L H2SO4.

5. The cyromazine enzyme-linked immunosorbent assay kit according to claim 1 or 2, characterized in that: The concentrated washing solution contains 1% hexadecyltrimethylammonium bromide and 0.5 mol / L pH 7.2 borate buffer, and the percentages are weight-volume ratios.

6. The cyromazine enzyme-linked immunosorbent assay kit according to claim 1 or 2, characterized in that: The cyromazine-specific antibody is prepared by using a cyromazine hapten-carrier protein conjugate as an immunogen.

7. The cyromazine enzyme-linked immunosorbent assay kit according to claim 1 or 2, characterized in that: The enzyme-labeled antibody is added with 10% trehalose, 0.1% sodium ethylenediaminetetraacetate, 0.05% L-methionine, 1% polyethylene glycol 6000, 0.1% diimidazole urea, and 0.05 mol / L pH 7.5 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid as an enzyme-labeled antibody stabilizer, and the percentage is mass volume ratio.

8. A method of using the cyromazine enzyme-linked immunosorbent assay kit according to any one of claims 1 to 7 to detect cyromazine residues in mutton and chicken.

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