CN113281501A - Application of pentachloronitrobenzene artificial antigen in enzyme linked immunosorbent assay kit - Google Patents
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Abstract
The invention provides an enzyme linked immunosorbent assay kit for detecting quintozene, which comprises: the enzyme-linked immunosorbent assay kit comprises an ELISA plate coated with a coating antigen, a pentachloronitrobenzene standard solution, a pentachloronitrobenzene antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate color developing solution, a stop solution and a washing solution, wherein the coating antigen is a pentachloronitrobenzene coupling antigen, the enzyme conjugate is an enzyme-labeled pentachloronitrobenzene antibody, and the pentachloronitrobenzene antibody is obtained by immunizing animals with immunogen. The invention also discloses a method for detecting quintozene 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 quintozene in a penaeus vannamei sample, is simple and convenient to operate, low in cost and high in sensitivity, can be monitored on site, and is suitable for screening of a large number of samples.
Description
Technical Field
The invention relates to an application technology of a pentachloronitrobenzene artificial antigen in an enzyme-linked immunosorbent assay kit, in particular to a molecular structure of the pentachloronitrobenzene artificial antigen and the enzyme-linked immunosorbent assay kit for detecting the pentachloronitrobenzene, which can qualitatively and quantitatively detect the residual quantity of a pentachloronitrobenzene drug in penaeus vannamei.
Background
The quintozene belongs to an organic chlorine protective bactericide, is insoluble in water, soluble in an organic solvent, stable in chemical property, difficult to volatilize, oxidize and decompose, and difficult to be influenced by sunlight, acid and alkali, but can be exploded and decomposed under the condition of high-temperature drying, so that the drug effect is reduced. Low toxicity to human, livestock and fish, and long residual effect period in soil. The problem of the use safety of the quintozene has attracted extensive attention in countries around the world. Measures for forbidding or limiting the use range have been provided in both the European Union and the United states, but more work in China is directed to research on analysis methods for detecting the pentachloronitrobenzene in different samples. In order to ensure the yield increase and harvest of grains and protect the safety of ecological environment, although the use of the pentachloronitrobenzene is not forbidden under the current conditions of China, the environmental problem caused by the pentachloronitrobenzene is emphasized greatly, and corresponding supervision and management measures are gradually adopted.
The method for detecting the quintozene comprises a gas chromatography method, a gas chromatography-mass spectrometry method and the like, and compared with the method, the immunochemical detection method has the advantages of rapidness, specificity, sensitivity, accuracy, batch monitoring, simple sample treatment, realization of automatic operation and the like for detecting the medicine. The invention discloses a preparation method of an artificial antigen of quintozene, which is applied to an enzyme linked immunosorbent assay kit, has the advantages of short time, simple operation and lower cost, and is suitable for detecting samples in bulk by basic units.
Disclosure of Invention
The invention aims to provide an enzyme linked immunosorbent assay kit capable of detecting the residual quantity of a pentachloronitrobenzene drug in penaeus vannamei, 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: the enzyme-linked immunosorbent assay kit comprises an ELISA plate coated with a coating antigen, a pentachloronitrobenzene standard solution, a pentachloronitrobenzene antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate color developing solution, a stop solution and a washing solution, wherein the coating antigen is a pentachloronitrobenzene coupling antigen, and the enzyme conjugate is an enzyme-labeled pentachloronitrobenzene antibody.
The specific antibody of the pentachloronitrobenzene is prepared by taking a pentachloronitrobenzene artificial antigen as an immunogen, and the specific antibody of the pentachloronitrobenzene can be a pentachloronitrobenzene monoclonal antibody or a pentachloronitrobenzene polyclonal antibody, wherein the pentachloronitrobenzene monoclonal antibody is preferred.
The labeled enzyme of the enzyme conjugate is horseradish peroxidase or alkaline phosphatase extracted from bacteria, wherein horseradish peroxidase is preferred; the enzyme conjugate is obtained by coupling enzyme and a pentachloronitrobenzene antibody.
In order to facilitate field monitoring and large-scale sample screening, the kit further comprises a quintozene standard solution, a substrate developing solution, a stopping solution and a washing solution.
The concentration of the quintozene standard solution is 0 mug/L, 0.1 mug/L, 0.3 mug/L, 0.9 mug/L, 2.7 mug/L and 8.1 mug/L respectively in 6 bottles.
When the labeled enzyme is horseradish peroxidase, the substrate color development solution consists of a substrate solution A and a substrate solution B, wherein the A is hydrogen peroxide or carbamide peroxide, the B is o-phenylenediamine or tetramethylbenzidine, and the stop solution is 1-2 mol/L sulfuric acid solution or hydrochloric acid buffer solution; when the marker enzyme is bacteria extracted alkaline phosphatase, the substrate color developing solution is a para-nitrophosphate buffer solution, and the stop solution is 1-2 mol/L sodium hydroxide solution.
The washing liquid preferably has a pH value of 7.4, and contains 0.5-1.0% of Tween-20, 0.01-0.03% of sodium azide preservative and 0.1-0.3 mol/L of phosphate buffer solution, wherein the percentages are weight volume percentages.
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 carbonate buffer solution with the pH value of 7.1-7.5, contains 1-3% of casein and 0.1-0.3 mol/L of phosphate buffer solution, and the percentage is weight volume percentage.
The preparation process of the ELISA plate comprises the following steps: diluting the coating source into 20 mu g/mL by using a coating buffer solution, adding 100 mu l into each hole, incubating for 2h in a dark place at 25 ℃ or overnight at 4 ℃, pouring off liquid in the holes, washing for 2 times by using a washing solution for 30s each time, patting to dry, then adding 150-200 mu l of a sealing solution into each hole, incubating for 1-2 h in a dark place at 25 ℃, pouring off liquid in the holes, patting to dry, drying, and performing vacuum sealing and storage by using an aluminum film.
The detection principle of the invention is as follows:
the kit adopts an ELISA method, conjugate antigens are pre-coated on an enzyme label plate microporous strip, the residual pentachloronitrobenzene in a sample and the pre-coated conjugate antigens on the enzyme label plate microporous strip compete for an enzyme conjugate resisting the pentachloronitrobenzene, a TMB substrate is used for developing color, the absorbance value of the sample is in negative correlation with the content of the residual pentachloronitrobenzene, and the absorbance value is compared with a standard curve and multiplied by the corresponding dilution multiple to obtain the residual amount of the pentachloronitrobenzene in the sample.
The invention also provides a method for detecting quintozene by applying the enzyme linked immunosorbent assay kit, which comprises the following steps:
(1) detecting by using the kit;
(2) and analyzing the detection result.
The enzyme linked immunosorbent assay kit for detecting the pentachloronitrobenzene qualitatively or quantitatively detects the content of the pentachloronitrobenzene in a sample mainly by an ELISA method; the pretreatment requirement on the sample is low, the pretreatment process of the sample is simple, and a large amount of samples can be detected quickly; 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 screening of large-batch samples.
Drawings
FIG. 1: molecular structural formula of pentachloronitrobenzene hapten
FIG. 2: synthetic route to pentachloronitrobenzene hapten
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. Preparation of pentachloronitrobenzene hapten
Taking 3.16g of 1.3-dichloro-5-iodo-2-nitrobenzene, adding 100ml of absolute ethanol for dissolving, dropwise adding 15ml of aqueous solution containing 2.4g of carboxymethyl hydroxylamine, adding 1.08g of sodium methoxide, heating and refluxing for 4 hours for reaction, stopping the reaction, carrying out rotary evaporation, removing ethanol, adding 100ml of water, adjusting the pH value to 6 by using 1mol/L hydrochloric acid, adding 100ml multiplied by 3 of ethyl acetate, extracting for three times, combining organic phases, evaporating to dryness, loading on a silica gel column, and eluting and separating by using petroleum ether/ethyl acetate (v/v,1/1) to obtain 2.3g of carboxyl o-dichloronitrobenzene hapten product, wherein the yield is 82.4%.
2. Preparation of antigens
Immunogen preparation-the coupling of the pentachloronitrobenzene hapten and Bovine Serum Albumin (BSA) to obtain the immunogen.
Dissolving 11mg of carboxyl o-dichloronitrobenzene in 1ml of DMSO (dimethyl sulfoxide), clarifying, adding 6.7mg of NHS and 9.7mg of EDC, fully dissolving and uniformly mixing, and reacting at room temperature for 2 hours to obtain a hapten activating solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 0.1M CB 9.56 ml for dissolving to obtain solution B, dropwise adding the solution A into the solution B, reacting for 4 hours at room temperature, stopping the reaction, dialyzing and purifying for 3 days by using 0.02M PBS, changing the solution 3 times every day, and centrifugally subpackaging to obtain the carboxyl o-dichloronitrobenzene-BSA conjugate, namely the immunogen.
Preparation of coating antigen-coupling of pentachloronitrobenzene hapten and Ovalbumin (OVA) to obtain the coating antigen.
Dissolving 9mg of carboxyl o-dichloronitrobenzene in 1ml of DMSO (dimethyl sulfoxide), clarifying, adding 5.4mg of NHS and 8.2mg of EDC, fully dissolving and uniformly mixing, and reacting at room temperature for 2 hours to obtain a hapten activating solution A; dissolving Ovalbumin (OVA)50mg in 0.1M CB 9.56 ml to obtain solution B, dropwise adding the solution A into the solution B, reacting at room temperature for 4h, stopping the reaction, dialyzing and purifying with 0.02M PBS for 3 days, changing the solution 3 times every day, centrifuging and packaging to obtain the carboxyl o-dichloronitrobenzene-OVA conjugate, namely the coating antigen.
The specific antibody against the pentachloronitrobenzene is prepared by taking 1, 3-dichloro-5-iodo-2-nitrobenzene as a raw material, carrying out nucleophilic substitution reaction with iodine on a benzene ring under the catalysis of sodium methoxide and then synthesizing a specific structural fragment of the pentachloronitrobenzene through nucleophilic substitution reaction with the iodine on the benzene ring, and immunizing with coupling protein.
3. Preparation of pentachloronitrobenzene monoclonal antibody
Animal immunization: injecting the immunogen obtained in the above steps into Balb/c mice, wherein the immunization dose is 150 mug/mouse, so that antiserum is generated.
Cell fusion and cloning: after the serum determination result of the mouse is higher, spleen cells are taken and fused with SP2/0 myeloma cells according to the ratio of 8:1 (quantitative ratio), indirect competitive ELISA is adopted to determine cell supernatant, and positive holes are screened. Cloning the positive hole by using a limiting dilution method until obtaining a hybridoma cell strain secreting the pentachloronitrobenzene monoclonal antibody.
Freezing and recovering cells: preparation of monoclonal hybridoma cell line into 1 × 10 frozen stock solution6Cell suspension per mL, preserved for long period in liquid nitrogen. Taking out the frozen tube during recovery, immediately putting the tube into a water bath at 37 ℃ for fast melting, centrifuging to remove frozen liquid, and transferring the tube into a culture bottle for culture.
Production and purification of monoclonal antibodies: injecting sterilized paraffin oil 0.5 mL/mouse into the abdominal cavity of Balb/c mouse, and injecting stable monoclonal hybridoma cell strain 5X 10 into the abdominal cavity after 7 days5Ascites were collected 7 days later. Purifying ascites by octanoic acid-saturated ammonium sulfate method, and storing at-20 deg.C.
4. Preparation of enzyme conjugates
Taking a goat as an immune animal and taking a pentachloronitrobenzene monoclonal antibody as an immunogen to immunize a goat without a pathogen to obtain the pentachloronitrobenzene antibody. The pentachloronitrobenzene antibody is coupled with Horse Radish Peroxidase (HRP) to obtain an enzyme conjugate.
5. Preparation of ELISA plates
Diluting the coating source to 20 mu g/mL by using a coating buffer solution, adding 100 mu l of the coating source into each hole, incubating for 2h at 25 ℃ in a dark place, pouring off liquid in the holes, washing for 2 times by using a washing solution for 30s each time, patting to dry, then adding 200 mu l of a sealing solution into each hole, incubating for 2h at 25 ℃ in a dark place, pouring off liquid in the holes, patting to dry, and performing vacuum sealing and storage by using an aluminum film after drying.
Example 2 construction of enzyme-linked immunosorbent assay kit for detecting quintozene
An enzyme linked immunosorbent assay kit for detecting the quintozene is constructed, and comprises the following components:
(1) an ELISA plate coated with a pentachloronitrobenzene coupling antigen;
(2) 6 bottles of quintozene standard solution with the concentrations of 0 mug/L, 0.1 mug/L, 0.3 mug/L, 0.9 mug/L, 2.7 mug/L and 8.1 mug/L respectively;
(3) a pentachloronitrobenzene antibody marked by horseradish peroxidase;
(4) 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;
(5) the stop solution is 2mol/L sulfuric acid;
(6) the washing liquid has a pH value of 7.4, and contains 0.5-1.0% of tween-20, 0.01-0.03% of sodium azide preservative and 0.1-0.3 mol/L of phosphate buffer solution, wherein the percentages are weight volume percentages;
example 3 detection of Penaeus vannamei Penaeus
1. Detection with a kit
And numbering the corresponding micropores of the samples and the standard products in sequence, making 2 holes in parallel for each sample and standard product, and recording the positions of the standard holes and the sample holes. Adding 50 mul of standard substance/sample into corresponding micropores, then adding 50 mul/pore of the working solution of the enzyme conjugate, and reacting for 30min at 25 ℃ in a dark environment. And (5) spin-drying the liquid in the holes, washing for 4-5 times, and patting dry. Adding 50 mul/hole of the substrate solution A, adding 50 mul/hole of the substrate solution B, mixing uniformly, and reacting for 15min at 25 ℃ in a dark environment. Adding 50 mul/well of stop solution, mixing, setting an enzyme-labeling instrument at 450nm, and measuring the OD value of each well.
2. Analysis of detection results
The percent absorbance of the standard or sample is equal to the average of the absorbance values of the standard or sample (double well) divided by the average of the absorbance values of the first standard (0 standard) and multiplied by 100% to obtain the percent absorbance value of the standard or sample. And drawing a standard curve graph by taking the percent absorbance of the standard substance as a vertical coordinate and taking the logarithm of the concentration (mu g/L) of the quintozene standard substance as a horizontal coordinate. And substituting the percent absorbance of the sample into the standard curve, reading out the concentration corresponding to the sample from the standard curve, and multiplying the concentration by the corresponding dilution times to obtain the actual concentration of the quintozene in the sample.
Example 4 determination of technical parameters of Pentachloronitrobenzene
1. Sensitivity and detection limit of kit
The sensitivity of the kit is determined according to a conventional method, the range of a standard curve is 0.1-8.1 mu g/L, and IC is50The floating range of (50% inhibition concentration) is 0.35-0.78 mu g/L; and (3) detecting 20 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 sample concentration to represent the detection limit, wherein the result shows that the detection limit of the method on the quintozene in the penaeus vannamei boone is 1 mu g/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 and used as a precision evaluation index. The calculation formula is as follows: recovery (%) — actual measurement/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 method comprises the steps of adding, recovering and measuring penaeus vannamei boone samples according to 1 mu g/kg and 2 mu g/kg of pentachloronitrobenzene, performing measurement by using three batches of different reagents in parallel with 4 samples, and calculating the average recovery rate and precision results of the samples as shown in the following table.
TABLE 1 Penaeus vannamei sample precision and accuracy test
The penaeus vannamei boone sample is subjected to addition recovery measurement according to 1 mu g/kg and 2 mu g/kg of pentachloronitrobenzene, and the average recovery rate is 81.7-93.4%; the relative standard deviation in each batch and between batches is less than 10 percent.
3. Stability test of kit
The storage condition of the kit is 2-8 ℃, and the maximum absorbance value (zero standard), the 50% inhibition concentration and the actual measurement value of the quintozene 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 results show 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 can be obtained that the kit can be stored at 2 to 8 ℃ for at least 12 months.
4. Kit specificity test
The cross reaction rate with pentachlorophenol, hexachlorobenzene, tetrachlorophthalide and chlorothalonil is determined by using a pentachloronitrobenzene kit, and the result is shown in
Table 2.
TABLE 2 specificity test
| Name of drug | Rate of cross reaction |
| Pentachloronitrobenzene | 100％ |
| Pentachlorophenol | 2.5％ |
| Hexachlorobenzene | 6.8％ |
| Tetrachlorophthalide | 29.1％ |
| Chlorothalonil | 16.4％ |
Claims (4)
1. An enzyme linked immunosorbent assay kit for detecting quintozene is characterized by comprising: the enzyme-linked immunosorbent assay kit comprises an enzyme label plate coated with a coating antigen, a quintozene standard solution, a quintozene antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate color development solution, a stop solution and a washing solution, wherein the coating antigen is a quintozene conjugate antigen, the enzyme conjugate is an enzyme-labeled quintozene antibody, the quintozene antibody is obtained by immunizing animals with immunogen, the quintozene conjugate antigen is obtained by coupling quintozene hapten and carrier protein, and the quintozene hapten is obtained by carrying out a series of chemical reactions on substances such as 1, 3-dichloro-5-iodo-2-nitrobenzene and carboxymethyl hydroxylamine.
2. The kit of claim 1, wherein the molecular structural formula of the pentachloronitrobenzene hapten is:
3. the kit according to claim 1, characterized in that the immunogen is prepared as follows:
dissolving 11mg of carboxyl o-dichloronitrobenzene in 1ml of DMSO (dimethyl sulfoxide), clarifying, adding 6.7mg of NHS and 9.7mg of EDC, fully dissolving and uniformly mixing, and reacting at room temperature for 2 hours to obtain a hapten activating solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 0.1M CB 9.56 ml for dissolving to obtain solution B, dropwise adding the solution A into the solution B, reacting for 4 hours at room temperature, stopping the reaction, dialyzing and purifying for 3 days by using 0.02M PBS, changing the solution 3 times every day, and centrifugally subpackaging to obtain the carboxyl o-dichloronitrobenzene-BSA conjugate, namely the immunogen.
4. A method for detecting the content of pentachloronitrobenzene in a sample comprises the following steps:
(1) detecting with the kit according to any one of claims 1 to 3;
(2) and analyzing the detection result.
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2021
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