阅读说明：本技术 霜霉威半抗原、人工抗原和抗体及其制备方法和应用 (Propamocarb hapten, artificial antigen and antibody as well as preparation method and application thereof ) 是由 朱亮亮 万宇平 陈姗姗 贾芳芳 王兆芹 王琳琛 鲁晓雄 于 2021-10-18 设计创作，主要内容包括：本发明提供了霜霉威半抗原、人工抗原和抗体及其制备方法,以及将其应用于酶联免疫试剂盒,试剂盒包括：包被有包被原的酶标板、霜霉威标准品溶液、霜霉威抗体、酶结合物浓缩液、酶结合物稀释液、底物显色液、终止液、洗涤液,所述包被原为霜霉威偶联抗原,所述酶结合物为酶标记的霜霉威抗体,所述霜霉威抗体是由免疫原免疫动物得到的。本发明还公开了一种应用上述酶联免疫试剂盒检测霜霉威的方法,它包括：首先进行样品前处理,然后用试剂盒进行检测,最后分析检测结果。本发明提供的酶联免疫试剂盒可用于检测烟叶(采后待烤烟叶、初烤烟叶)样本中霜霉威的含量,其操作简便、费用低廉、灵敏度高、能够现场监控且适合大量样本的筛查。(The invention provides propamocarb hapten, artificial antigen and antibody, a preparation method thereof and application thereof in an enzyme linked immunosorbent assay kit, wherein the kit comprises: the enzyme-linked propamocarb antigen-immobilized enzyme-linked propamocarb antibody is characterized by comprising an enzyme label plate coated with a coating antigen, a propamocarb standard solution, a propamocarb antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate developing solution, a stop solution and a washing solution, wherein the coating antigen is a propamocarb coupled antigen, the enzyme conjugate is an enzyme-labeled propamocarb antibody, and the propamocarb antibody is obtained by immunizing an animal with an immunogen. The invention also discloses a method for detecting propamocarb 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 propamocarb in tobacco leaf (post-harvest tobacco leaf to be cured and flue-cured tobacco leaf) samples, 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.)

1. An enzyme linked immunosorbent assay kit for detecting propamocarb is characterized by comprising: the enzyme-linked immunosorbent assay kit comprises an enzyme label plate coated with a coating antigen, a propamocarb standard solution, a propamocarb antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate developing solution, a stop solution and a washing solution, wherein the coating antigen is a propamocarb coupling antigen, the enzyme conjugate is an enzyme-labeled propamocarb antibody, the propamocarb antibody is obtained by immunizing an animal with an immunogen, the propamocarb coupling antigen is obtained by coupling a propamocarb hapten and a carrier protein, and the propamocarb hapten is obtained by carrying out a series of chemical reactions on propamocarb and 1, 4-cyclohexane diisocyanate and other substances.

2. The kit according to claim 1, wherein the propamocarb hapten has the molecular formula:

3. the kit according to claim 1, characterized in that the immunogen is prepared as follows:

taking 13mg of the cyclohexane isocyanate-propamocarb hapten product, adding 1ml of DMSO for dissolving and clarifying to obtain a hapten solution, and recording the hapten solution as solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 0.1M CB9.56ml for dissolving to obtain solution B, dropwise adding the solution A into the solution B, reacting at room temperature for 4 hours, stopping the reaction, dialyzing and purifying with 0.02M PBS for 3 days, changing the solution 3 times every day, and centrifugally subpackaging to obtain the propamocarb-BSA conjugate, namely the immunogen.

4. A method for detecting the propamocarb content in a sample, comprising the steps of:

(1) detecting with the kit according to any one of claims 1 to 3;

(2) and analyzing the detection result.

Technical Field

The invention relates to a propamocarb hapten, an artificial antigen and an antibody as well as a preparation method and an application technology thereof, in particular to a propamocarb artificial antigen molecular structure and an enzyme linked immunosorbent assay kit for detecting propamocarb, which can qualitatively and quantitatively detect the residual quantity of propamocarb drugs in tobacco leaves (tobacco leaves to be roasted after harvest and tobacco leaves initially roasted).

Background

Propamocarb is a low-toxicity fungicide with a local systemic effect, and belongs to carbamates. Has special effect on oomycetes fungi, can inhibit the formation of germ cell membranes, and reduce the formation of sporangia and the number of zoospores, thereby achieving the purpose of preventing and treating diseases. The fruit tree has excellent effects on downy mildew, epidemic disease and damping-off, is suitable for foliar spraying and soil treatment, and the propamocarb also has the effect of promoting the growth of crops. The propamocarb has low toxicity to higher animals and no irritation to eyes and skin of rabbits. Although propamocarb is a high-efficiency low-toxicity low-residue product, no clear standard exists in China for the residue limit of propamocarb in crops, but the new regulation issued in the United states requires that the residue amount of propamocarb on fruits and vegetables is 2 mg/kg. The residual limits are also specified for propamocarb in japan. This has a certain effect on our export of agricultural products, and therefore, it is essential to detect the drug residue of propamocarb.

The method for detecting propamocarb comprises a gas chromatography, a high performance liquid chromatography-mass spectrometry/mass spectrometry, a liquid chromatography-mass spectrometry/mass spectrometry 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, automatic operation realization and the like. The invention discloses a preparation method of propamocarb artificial antigen, which is applied to an enzyme-linked immunosorbent assay kit, has the advantages of short time, simplicity in 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 propamocarb drug residue in tobacco leaves (after-harvest tobacco leaves to be cured and flue-cured tobacco leaves), 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 propamocarb standard solution, a propamocarb antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate developing solution, a stop solution and a washing solution, wherein the coating antigen is a propamocarb coupling antigen, and the enzyme conjugate is an enzyme-labeled propamocarb antibody.

The propamocarb specific antibody is prepared by taking a propamocarb artificial antigen as an immunogen, and can be a propamocarb monoclonal antibody or a propamocarb monoclonal antibody, wherein the propamocarb 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 propamocarb antibody.

In order to facilitate field monitoring and large-scale sample screening, the kit further comprises a propamocarb standard solution, a substrate developing solution, a stop solution and a washing solution.

The concentration of the propamocarb standard solution in 6 bottles is respectively 0 mug/L, 150 mug/L, 450 mug/L, 1350 mug/L and 4050 mug/L.

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, the coupling antigen is pre-coated on the micropore strip of the ELISA plate, the residual propamocarb in the sample and the coupling antigen pre-coated on the micropore strip of the ELISA plate compete for the enzyme conjugate resisting the propamocarb, the TMB substrate is used for developing color, the absorbance value of the sample is in negative correlation with the content of the residual propamocarb contained in the sample, the absorbance value is compared with a standard curve, and the absorbance value is multiplied by the corresponding dilution multiple, so that the residual amount of the propamocarb in the sample can be obtained.

The invention also provides a method for detecting propamocarb by using 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 propamocarb mainly adopts an ELISA method to qualitatively or quantitatively detect the content of propamocarb in a sample; 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 propamocarb 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 propamocarb hapten

Taking 1.88g of propamocarb, adding 20ml of pyridine for dissolution, adding 2.49g of 1, 4-cyclohexane diisocyanate and 1.3ml of triethylamine for reaction at 80 ℃ for 3h, stopping the reaction, carrying out rotary evaporation to remove pyridine and triethylamine to obtain red oily matter, adding 35ml of diethyl ether/n-hexane (v/v, 1/15) for recrystallization to obtain 1.13g of a cyclohexane isocyanate-propamocarb hapten product, wherein the yield is 31.9%.

2. Preparation of antigens

Immunogen preparation-propamocarb hapten is coupled with Bovine Serum Albumin (BSA) to obtain the immunogen.

Taking 13mg of the cyclohexane isocyanate-propamocarb hapten product, adding 1ml of DMSO for dissolving and clarifying to obtain a hapten solution, and recording the hapten solution as solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 0.1M CB9.56ml for dissolving to obtain solution B, dropwise adding the solution A into the solution B, reacting at room temperature for 4 hours, stopping the reaction, dialyzing and purifying with 0.02M PBS for 3 days, changing the solution 3 times every day, and centrifugally subpackaging to obtain the propamocarb-BSA conjugate, namely the immunogen.

Preparation of coating antigen-coupling propamocarb hapten and Ovalbumin (OVA) to obtain the coating antigen.

Taking 7.8mg of cyclohexane isocyanate-propamocarb hapten product, adding 1ml of DMSO for dissolving and clarifying to obtain a hapten solution, and recording the hapten solution as solution A; dissolving Ovalbumin (OVA)50mg in 0.1M CB9.56ml to obtain solution B, dropwise adding the solution A into the solution B, reacting at room temperature for 4h, stopping reaction, dialyzing and purifying with 0.02M PBS for 3 days, changing the solution 3 times per day, centrifuging and packaging to obtain propamocarb-OVA conjugate, namely the coating antigen.

Propamocarb is used as a raw material, 1, 4-cyclohexane diisocyanate with high activity is heated in the environment of pyridine and triethylamine, and is subjected to acylation reaction, cyclohexane isocyanate is introduced to imine, and coupling protein is used for immunization, so that the specific antibody for propamocarb is prepared.

3. Preparation of propamocarb 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 propamocarb 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

The goat is used as an immune animal, and the propamocarb monoclonal antibody is used as an immunogen to immunize the goat without the pathogen, so that the propamocarb antibody is obtained. The propamocarb antibody is coupled with horseradish 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 propamocarb

An enzyme linked immunosorbent assay kit for detecting propamocarb is constructed, and comprises the following components:

(1) an ELISA plate coated with propamocarb coupling antigen;

(2) 6 bottles of propamocarb standard solution with the concentrations of 0 mug/L, 150 mug/L, 450 mug/L, 1350 mug/L and 4050 mug/L respectively;

(3) a propamocarb antibody labeled with 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 propamocarb in tobacco leaves (after-harvest ready-cured tobacco leaves, flue-cured tobacco leaves)

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 an ordinate and taking the logarithm of the concentration (mu g/L) of the propamocarb standard substance as an abscissa. And substituting the percent absorbance of the sample into the standard curve, reading out the corresponding concentration of the sample from the standard curve, and multiplying the corresponding dilution times by the corresponding concentration to obtain the actual concentration of the propamocarb in the sample.

Example 4 determination of the technical parameters of propamocarb

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 150-4050 mu g/L, IC₅₀(50% inhibitory concentration) floating range is 500-1100 mug/L; the 20 samples are detected, the concentration corresponding to each percent absorbance value is found out from the standard curve, the detection limit is represented by adding 3 times of standard deviation to the average value of the 20 sample concentration, and the result shows that the detection limit of the method for the propamocarb in the tobacco leaves (the tobacco leaves to be roasted after being picked and the tobacco leaves to be primarily roasted) is 300 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 tobacco leaf samples (the tobacco leaves to be baked after being picked and the tobacco leaves primarily baked) are added with propamocarb with the concentrations of 300 mu g/kg and 600 mu g/kg for recovery and determination, 4 samples are parallel to each other, three batches of different reagents are used for determination, and the average recovery rate and precision result of the samples are calculated and are shown in the following table.

TABLE 1 tobacco leaf sample precision and accuracy test

Adding and recovering the tobacco leaf (the tobacco leaf to be roasted after being picked and the tobacco leaf primarily roasted) samples according to propamocarb with two concentrations of 300 mu g/kg and 600 mu g/kg, wherein the average recovery rate is 74.8-118.3%; the relative standard deviation in each batch and between 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), the 50% inhibition concentration and the actual measurement value of propamocarb addition of the kit are 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-reactivity with metalaxyl, fluvalicarb and imidacloprid was determined with the propamocarb kit and the results are shown in table 2.

TABLE 2 specificity test

Name of drug	Rate of cross reaction
		Pericarb	100％
Metalaxyl	＜6％
		Flumetralin	＜1％
Imidacloprid	＜1％