CN109061168B - Enzyme linked immunosorbent assay kit for detecting diclazuril and application thereof - Google Patents
Enzyme linked immunosorbent assay kit for detecting diclazuril and application thereof Download PDFInfo
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Abstract
The invention provides an enzyme linked immunosorbent assay kit for detecting diclazuril, which comprises: the kit comprises an ELISA plate coated with diclazuril coupling antigen, a diclazuril monoclonal antibody, an enzyme-labeled anti-antibody, a diclazuril standard solution, a substrate developing solution, a stop solution, a washing solution and a compound solution. The invention also discloses a method for detecting diclazuril by applying the enzyme linked immunosorbent assay kit, which comprises the following steps: firstly, preprocessing a sample, then detecting by using a kit, and finally analyzing a detection result. The enzyme linked immunosorbent assay kit provided by the invention can be used for detecting the content of diclazuril in animal tissues, is simple and convenient to operate, low in cost, high in sensitivity, capable of being monitored on site and suitable for screening a large number of samples.
Description
Technical Field
The invention relates to an enzyme-linked immunoassay technology, in particular to an enzyme-linked immunoassay kit for detecting diclazuril, which is particularly suitable for detecting the content of diclazuril in animal tissues.
Background
Diclazuril is a non-polyether chemical synthesis anticoccidial drug developed and researched by the pharmaceutical company of Yankee Seisan in Belgium in the 80 th century, belongs to triazine benzyl cyanide compounds, has the characteristics of high efficiency, low toxicity and broad spectrum, and is often used for preventing and treating anticoccidial diseases in poultry and rabbit farming. Diclazuril has inhibiting or killing effect on aquatic animal sporozoon, and can be used for preventing and treating sporozoon diseases such as Cyprinus myxosporidium, asodia, tail spore, tetrapolar, and homopolar in aquaculture. Due to the characteristics of low toxicity, broad spectrum, small dosage and the like, diclazuril has the conditions of drug abuse, non-compliance with withdrawal period and the like in the using process, thereby bringing about a series of veterinary drug residue problems and having potential harm to human health.
The residual marker of diclazuril is itself. The maximum residual limit of dibrels in foods of animal origin (MRLs) was established by various governments and international organizations. The Ministry of agriculture in China announces No. 235 that the MRLs of the Kezhuli in the muscle, fat, liver and kidney tissues of sheep/birds/rabbits are 500, 1000, 3000 and 2000 mu g/kg respectively.
At present, methods for detecting the residual amount of diclazuril mainly comprise a gas chromatography-mass spectrometry method, a high performance liquid chromatography, a liquid chromatography-tandem mass spectrometry method and a high resolution mass spectrometry method. The methods can simultaneously carry out multi-residue qualitative and quantitative analysis on similar drugs such as diclazuril and the like, but expensive instruments and special technicians are needed, the pretreatment process of the samples is complex, high in cost and long in time, and the requirements of rapid detection on a large number of samples and field samples are difficult to meet. Enzyme-linked immunosorbent assay (ELISA) has the characteristics of simplicity, rapidness, specificity, sensitivity, large sample capacity and low analysis cost, can simplify or even omit the sample purification step, has unique advantages in rapid screening and detection of a large number of samples and field samples, can better meet the requirements of detection work of food enterprises, government functional supervision departments and the like in China, and has great development potential.
Disclosure of Invention
The invention aims to provide an enzyme linked immunosorbent assay kit for diclazuril detection, which has the advantages of simple structure, convenient use, low price and convenient carrying, and provides a qualitative and quantitative detection method which is efficient, accurate, simple and convenient and is suitable for screening large-batch samples.
The kit of the invention comprises: an ELISA plate coated with diclazuril coupling antigen, a diclazuril monoclonal antibody, an enzyme-labeled anti-antibody, a diclazuril standard solution, a substrate color development solution, a stop solution, a washing solution and a redissolution; the diclazuril monoclonal antibody is prepared by taking a diclazuril coupling antigen as an immunogen, the diclazuril coupling antigen is obtained by coupling a diclazuril hapten and a carrier protein, the carrier protein is mouse serum protein, thyroid protein, bovine serum albumin, rabbit serum protein, human serum albumin, ovalbumin, hemocyanin or fibrinogen, the diclazuril hapten is obtained by reacting diclazuril with glacial acetic acid and hydrobromic acid, and the molecular structural formula is as follows:
the anti-antibody of the enzyme-labeled anti-antibody is a goat anti-mouse anti-antibody.
The labeled enzyme of the enzyme-labeled anti-antibody is horseradish peroxidase; the enzyme-labeled anti-antibody is obtained by coupling a labeled enzyme and the anti-antibody by a glutaraldehyde method or a sodium periodate method.
In order to facilitate field monitoring and large-scale sample screening, the kit further comprises a diclazuril standard solution, a substrate developing solution, a stop solution, a washing solution and a compound solution.
The concentration of the diclazuril standard substance solution is 0 mug/L, 1 mug/L, 3 mug/L, 9 mug/L and 27 mug/L respectively in 5 bottles.
The substrate color developing solution consists of a substrate solution A and a substrate solution B, the substrate solution A is hydrogen peroxide or carbamide peroxide, the substrate solution B is o-phenylenediamine or tetramethylbenzidine, and the stop solution is 1-2 mol/L sulfuric acid solution.
The washing solution is preferably phosphate buffer solution with the pH value of 7.4, and contains 0.5-1.0% of Tween-20, 0.01-0.03% of sodium azide and 0.1-0.3 mol/L.
The redissolution is preferably a phosphate buffer solution with the pH value of 7.0 and 0.1 mol/L.
Wherein the coating buffer solution used in the preparation process of the ELISA plate is carbonate buffer solution with the pH value of 9.6 and 0.05mol/L, and the confining solution is phosphate buffer solution with the pH value of 7.1-7.5, containing 1-3% of casein and 0.1-0.3 mol/L.
The preparation process of the ELISA plate comprises the following steps: diluting the coating source to 20 mu g/mL by using a coating buffer solution, adding 100 mu L into each hole, incubating for 2h in the dark at 37 ℃ or overnight at 4 ℃, pouring off liquid in the holes, washing for 2 times by using a washing solution, shaking to dry for 30s each time, then adding 150-200 mu L of a sealing solution into each hole, incubating for 1-2 h in the dark at 37 ℃, pouring off liquid in the holes, shaking to dry, drying, and then sealing and storing in vacuum by using an aluminum film.
The detection principle of the invention is as follows:
pre-coating diclazuril coupling antigen on a micropore strip, adding a sample solution or a standard solution, then adding a diclazuril monoclonal antibody solution, adding an enzyme-labeled anti-antibody for amplification, developing with a developing solution, wherein the absorbance value of the sample is in negative correlation with the content of diclazuril, and comparing with a standard curve to obtain the residual amount of diclazuril in the sample; meanwhile, according to the shade of the color on the ELISA plate, the concentration range of the diclazuril residue in the sample can be roughly judged by comparing the color with the color of the standard solution with the series of concentrations.
The invention also provides a method for detecting the residual quantity of diclazuril by applying the enzyme linked immunosorbent assay kit, which comprises the following steps:
(1) Sample pretreatment;
(2) Detecting by using the kit;
(3) And analyzing the detection result.
The enzyme linked immunosorbent assay kit for detecting the diclazuril mainly adopts an indirect competitive ELISA method to qualitatively or quantitatively detect the residual amount of the diclazuril in a sample; the pretreatment requirement on the samples is low, the pretreatment process of the samples is simple, and large-batch samples can be detected quickly at the same time; the main reagent is provided in the form of working solution, and the detection method is convenient and easy to implement and has the characteristics of high specificity, high sensitivity, high precision, high accuracy and the like. The enzyme linked immunosorbent assay kit has the advantages of simple structure, convenient use, low price, convenient carrying, high efficiency, accuracy, simplicity and convenience of the detection method, and is suitable for qualitative and quantitative detection of large-scale sample screening.
Drawings
FIG. 1: diclazuril hapten synthesis roadmap
FIG. 2 is a schematic diagram: standard curve diagram of reagent kit
Detailed Description
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
EXAMPLE 1 preparation of kit Components
1. Synthesis of diclazuril hapten (the synthetic route is shown in figure 1)
1g of diclazuril is taken, 5mL of glacial acetic acid is added for dissolution and clarification, the aqueous solution of hydrobromic acid is added with 20mL, the mixture is heated at 100 ℃, and the mixture is stirred and reacts for 2 hours. Stopping the reaction, adding 100mL of water, adding 1mol/L NaOH to neutralize the solution to pH 6, adding 200mL of ethyl acetate multiplied by 3, extracting the solution for three times, combining organic phases, drying the organic phases by 20g of anhydrous sodium sulfate, evaporating the dried organic phases to dryness, applying to a silica gel column, and eluting and separating ethyl acetate/petroleum ether (v/v, 1/1) to obtain 0.91g of the carboxyl diclazuril hapten product with the yield of 87.5%.
2. Synthesis and identification of diclazuril coupled antigen
Immunogen preparation-diclazuril hapten is coupled with Bovine Serum Albumin (BSA) to obtain the immunogen.
Dissolving carboxyl diclazuril hapten 16mg in 0.5mL of N, N-dimethylformamide, adding N-hydroxysuccinimide (NHS) 6mg and carbodiimide (EDC) 12mg, and stirring for 2h to obtain hapten activating solution A; taking 50mg of BSA, adding 3mL of 0.1mol/L sodium bicarbonate solution, and dissolving and clarifying to obtain solution B; slowly dripping the solution A into the solution B, stirring for 4h at 4 ℃, dialyzing and purifying for 3d by 0.02mol/L PB buffer solution, changing the solution for 3 times every day, subpackaging to obtain the diclazuril hapten-BSA immunogen, and storing at-20 ℃ for later use.
Preparation of coating antigen-coupling diclazuril hapten and Ovalbumin (OVA) to obtain the coating antigen.
Dissolving 9mg of carboxyl diclazuril hapten by adding 0.4mL of N, N-dimethylformamide, adding NHS 5mg and EDC 9mg, and stirring for 2 hours to obtain hapten activating solution A; adding 3mL of 0.1mol/L sodium bicarbonate solution into 50mg of OVA, and dissolving and clarifying to obtain solution B; slowly dripping the solution A into the solution B, stirring for 4h at 4 ℃, dialyzing and purifying for 3d by 0.02mol/L PB buffer solution, changing the solution for 3 times per day, and subpackaging to obtain diclazuril hapten-OVA coating antigen, and storing at-20 ℃ for later use.
According to the proportion of hapten, carrier protein and coupling product used in the synthetic diclazuril coupling antigen reaction, ultraviolet (200 nm-400 nm) scanning measurement is carried out, and the combination ratio is calculated by comparing the light absorption values of the hapten, the carrier protein and the coupling product at 260nm and 280nm respectively. Compared with the maximum absorption peaks of the diclazuril hapten and the carrier protein, the maximum absorption peak of the conjugate diclazuril hapten-carrier protein is obviously changed, which indicates that the synthesis of the diclazuril hapten-carrier protein is successful.
3. Preparation of diclazuril monoclonal antibody
(1) Obtaining hybridoma cells
1) First immunization: the diclazuril hapten-BSA conjugate (immunogen) and an equal amount of Freund complete adjuvant are fully emulsified, and are injected with 0.2mL of 6-week-old Balb/c mice each subcutaneously;
2) Two booster immunizations: from the first immunization, boosting once every two weeks, and replacing Freund's complete adjuvant with Freund's incomplete adjuvant in the same method and dosage as the first immunization;
3) The titer and inhibition were measured in the fundus venous blood one week after the last booster immunization, and the following last immunization was performed when the titer reached 1 or more 10000: injecting 0.1mL of immunogen solution without any adjuvant into the abdominal cavity, killing the mouse after three days, and fusing the spleen with myeloma cells;
4) And (3) measuring cell supernatant by adopting an indirect competitive enzyme-linked immunoassay method, and screening positive holes. Cloning the positive hole by using a limiting dilution method to obtain and establish a hybridoma cell strain which stably secretes the diclazuril monoclonal antibody, preparing the hybridoma cells in the logarithmic growth period into cell suspension by using a freezing medium, subpackaging in a freezing tube, and storing in liquid nitrogen for a long time.
(2) Preparation of monoclonal antibodies
1) Cell recovery: taking out the cryo-preservation tube of the diclazuril monoclonal antibody hybridoma cell strain, immediately putting the cryo-preservation tube into a water bath at 37 ℃ for fast thawing, centrifugally removing a frozen stock solution, and transferring the frozen stock solution into a culture bottle for culture;
2) Preparing ascites and purifying antibodies: injecting 0.5mL of sterilized paraffin oil into the abdominal cavity of Balb/c mice (8 weeks old) by adopting an in vivo induction method, and injecting 5 multiplied by 10 hybridoma cells into the abdominal cavity after 7 days 5 Ascites were collected 7 days later. Purifying by an octanoic acid-saturated ammonium sulfate method to obtain a diclazuril monoclonal antibody solution (stored at the temperature of minus 20 ℃).
(3) Determination of the potency of monoclonal antibodies
The titer of the antibody is 1 (100000-300000) by using an indirect competition ELISA method.
Indirect competitive ELISA method: coating an enzyme label plate with a diclazuril hapten-OVA conjugate, adding a diclazuril standard solution, a diclazuril monoclonal antibody solution and a horseradish peroxidase-labeled goat anti-mouse anti-antibody solution, reacting for 30min at 25 ℃, pouring out liquid in a hole, washing for 3-5 times by using a washing solution, and patting dry by using absorbent paper; adding a substrate color developing solution, reacting for 15min at 25 ℃, and adding a stop solution to stop the reaction; the microplate reader was set to measure the absorbance value per well at a wavelength of 450 nm.
(4) Determination of monoclonal antibody specificity
Antibody specificity refers to the comparison of its ability to bind to a specific antigen with the ability to bind to such antigen analogs, often using cross-reactivity as an evaluation criterion. The smaller the cross-reactivity, the higher the specificity of the antibody.
In the experiment, diclazuril, toltrazuril, monensin, maduramycin and salinomycin are serially diluted, respectively subjected to indirect competitive ELISA with monoclonal antibodies, standard curves are prepared, and IC is obtained by analysis 50 Then calculating the cross as followsReaction rate:
the results show that the cross-reactivity rate of each analog is: 100% of diclazuril, 11.4% of toltrazuril, less than 0.1% of monensin, less than 0.1% of maduramicin and less than 0.1% of salinomycin. The antibody of the invention has no cross reaction to analogs such as toltrazuril, monensin, maduramicin, salinomycin and the like, and only has specific binding to diclazuril.
4. Preparation of goat anti-mouse anti-antibody
The sheep is used as an immune animal, and the pathogen-free sheep is immunized by using the murine antibody as an immunogen to obtain the goat anti-mouse antibody.
5. Preparation of enzyme-labeled anti-antibody
The goat anti-mouse antibody and horseradish peroxidase (HRP) are coupled by a modified sodium periodate method. The traditional sodium periodate method requires that the molar concentration ratio of enzyme to antibody in a reaction system is 4. To solve this problem, we modified the conventional method, namely:
(1) The blocking process of the amino group is omitted, because the amino group capable of generating self amino group connection is practically few;
(2) The molar concentration ratio of the horseradish peroxidase to the antibody is reduced to 2, the improved method is simpler and more convenient than the traditional method, and the loss of enzyme activity is reduced.
6. Preparation of ELISA plates
Diluting the coating antigen (diclazuril hapten-OVA conjugate) to 20 mu g/mL by using a coating buffer solution, adding 100 mu L into each hole, incubating for 2h in the dark at 37 ℃, pouring off liquid in the holes, washing for 2 times by using a washing solution for 30s each time, patting dry, then adding 200 mu L of a sealing solution into each hole, incubating for 2h in the dark at 37 ℃, pouring off liquid in the holes, patting dry, drying, and performing vacuum sealing and storage by using an aluminum film.
Example 2 construction of enzyme-linked immunosorbent assay kit for detecting diclazuril
An enzyme linked immunosorbent assay kit for detecting diclazuril is constructed, and comprises the following components:
(1) An ELISA plate coated with diclazuril coupling antigen;
(2) 5 bottles of diclazuril standard solution with the concentrations of 0 mug/L, 1 mug/L, 3 mug/L, 9 mug/L and 27 mug/L respectively;
(3) Diclazuril monoclonal antibody working solution;
(4) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;
(5) The substrate color development liquid consists of a liquid A and a liquid B, wherein the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;
(6) The stop solution is 2mol/L sulfuric acid;
(7) The washing liquid is phosphate buffer solution with pH value of 7.4, containing 0.5-1.0% of Tween-20, 0.01-0.03% of sodium azide and 0.1-0.3 mol/L;
(8) The complex solution is phosphate buffer solution with pH value of 7.0 and 0.1 mol/L.
Example 3 detection of diclazuril in samples
1. Sample pretreatment
Weighing 1.0g +/-0.05 g of tissue sample into a 50mL centrifuge tube, adding 1mL of 0.1mol/L sodium hydroxide, whirling for 1min by a vortex instrument, adding 7mL of acetonitrile, whirling for 2min by the vortex instrument, and centrifuging for 5min at room temperature (20-25 ℃) by 3000g of acetonitrile; transferring 1mL of the upper organic phase into a 10mL clean dry glass tube, and drying the upper organic phase in a water bath nitrogen flow at the temperature of between 50 and 60 ℃; adding 1mL of redissolution, and whirling for 30s by using a vortex instrument; 50 μ L was taken for analysis.
2. Detection with a kit
Adding 50 mu L/hole of diclazuril standard solution or pretreated sample solution into micropores of an ELISA plate coated with diclazuril coupling antigen, then adding 50 mu L/hole of horseradish peroxidase-labeled goat anti-mouse anti-antibody, then adding 50 mu L/hole of diclazuril monoclonal antibody working solution, lightly oscillating and uniformly mixing, and placing the microplate into a dark environment with the temperature of 25 ℃ for reaction for 30min; pouring out the liquid in the holes, adding 250 mu L of washing liquid into each hole, fully washing for 4-5 times at intervals of 10s each time, and patting dry by using absorbent paper; adding 50 mu L of substrate solution A urea peroxide and 50 mu L of substrate solution B Tetramethylbenzidine (TMB) into each hole, slightly oscillating and uniformly mixing, covering a plate with a cover plate film, then placing the plate in a dark environment at 25 ℃ for reaction for 15min, adding 50 mu L of stop solution 2mol/L sulfuric acid into each hole, slightly oscillating and uniformly mixing, setting the wavelength at 450nm by using an enzyme-linked immunosorbent assay, and measuring the absorbance value (OD value) of each hole.
3. Analysis of detection results
Dividing the average absorbance (B) obtained for each concentration of the standard solution by the absorbance value (B) of the first standard solution (0 standard) 0 ) And then multiplied by 100% to obtain a percent absorbance value. A standard curve is drawn with the log of the diclazuril standard concentration (. Mu.g/L) as the X-axis and the percent absorbance as the Y-axis, as shown in FIG. 2. The percent absorbance values of the sample solutions were calculated in the same way, and the diclazuril content corresponding to each sample was read from the standard curve.
Example 4 determination of technical parameters of Diclizumab ELISA kits
1. Sensitivity and detection limits of the kit
The sensitivity of the kit is determined according to a conventional method, the lowest point of a standard curve of the kit is 1 mu g/L, the range of the standard curve is 1-27 mu g/L, and IC is 50 (50% inhibitory concentration) the floating range is 2.5-4.5 mug/L; and (3) detecting 20 parts of each of the blank pork and chicken samples, finding out the concentration corresponding to each percent absorbance value from the standard curve, and adding 3 times of standard deviation to the average value of the 20 parts of sample concentration to represent the detection limit, so that the detection limit of the method on the tissue sample is 8 mug/kg.
2. Sample precision and accuracy testing
The recovery rate is used as an accuracy evaluation index, and the relative standard deviation (RSD%) of the detection result of a sample with a certain concentration is repeatedly measured as a precision evaluation index. The calculation formula is as follows: recovery (%) = actual measured value/theoretical value × 100%, where theoretical value is the added concentration of the sample; relative standard deviation RSD% = SD/X × 100%, where SD is the standard deviation and X is the average of the measured data.
The blank pork and chicken samples are subjected to addition recovery measurement according to diclazuril with three concentrations of 8 mug/kg, 16 mug/kg and 32 mug/kg, 4 samples are parallelly measured by three different kits, and the results of the average recovery rate and the precision of the samples are calculated as shown in the following table.
TABLE 1 precision and accuracy tests
Adding diclazuril with the concentration of 8, 16 and 32 mu g/kg to the blank pork and chicken samples, wherein the average recovery rate is between 80 and 110 percent; the relative standard deviation in each batch and among the batches is less than 15 percent.
3. Stability test of kit
The storage condition of the kit is 2-8 ℃, and the maximum absorbance value (zero standard), 50% inhibition concentration and the actual measurement value of diclazuril addition of the kit are all within the normal range after 12 months of measurement. Considering that abnormal storage conditions occur in the transportation and use processes, the kit is placed for 7 days under the storage condition of 37 ℃ for accelerated aging experiments, and the result shows that all indexes of the kit completely meet the requirements. And in consideration of the occurrence of the freezing condition of the kit, the kit is frozen for 7 days in a refrigerator at the temperature of-20 ℃, and the determination result also shows that all indexes of the kit are completely normal. From the above results, it was found that the kit can be stored at 2 to 8 ℃ for at least 12 months.
Claims (6)
1. An enzyme linked immunosorbent assay kit for detecting diclazuril, comprising: an ELISA plate coated with diclazuril coupling antigen, a diclazuril monoclonal antibody, an enzyme-labeled anti-antibody, a diclazuril standard solution, a substrate developing solution, a stop solution, a washing solution and a redissolution; the diclazuril monoclonal antibody is characterized by being prepared by taking a diclazuril coupling antigen as an immunogen, wherein the diclazuril coupling antigen is obtained by coupling a diclazuril hapten and a carrier protein, the carrier protein is mouse serum protein, thyroid protein, bovine serum albumin, rabbit serum protein, human serum albumin, ovalbumin, hemocyanin or fibrinogen, and the synthetic method of the diclazuril hapten is as follows: taking 1g of diclazuril, adding 5mL of glacial acetic acid for dissolving and clarifying, adding 20mL of hydrobromic acid aqueous solution, heating at 100 ℃, stirring for reacting for 2 hours, stopping the reaction, adding 100mL of water, adding 1mol/L of NaOH for neutralizing until the pH value is 6, adding 200mL of ethyl acetate multiplied by 3, extracting for three times, combining organic phases, drying by 20g of anhydrous sodium sulfate, evaporating to dryness, loading on a silica gel column, eluting and separating by ethyl acetate/petroleum ether with the volume ratio of 1/1 to obtain 0.91g of diclazuril hapten, wherein the molecular structural formula is as follows:
2. the kit of claim 1, wherein the anti-antibody of the enzyme-labeled anti-antibody is a goat anti-mouse anti-antibody.
3. The kit according to claim 1, wherein the enzyme-labeled anti-antibody labeling enzyme is horseradish peroxidase, the substrate chromogenic solution is composed of a substrate solution A and a substrate solution B, the substrate solution A is hydrogen peroxide or carbamide peroxide, the substrate solution B is o-phenylenediamine or tetramethylbenzidine, and the stop solution is 1-2 mol/L sulfuric acid solution.
4. The kit of claim 1, wherein the washing solution is a phosphate buffer solution with a pH value of 7.4, containing 0.5% -1.0% Tween-20, 0.01-0.03% sodium azide and 0.1-0.3 mol/L; the complex solution is phosphate buffer solution with the pH value of 7.0 and 0.1 mol/L.
5. The kit according to claim 1, wherein the concentration of the diclazuril standard solution is 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, or 27 μ g/L, respectively.
6. A method for detecting the residual amount of diclazuril in a sample, comprising the steps of:
(1) Pretreating a sample;
(2) Detecting with the kit according to any one of claims 1 to 5;
(3) And analyzing the detection result.
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