CN111825759A - Enzyme-linked immunosorbent assay method for indirectly detecting pirimiphos-methyl - Google Patents
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Abstract
The invention discloses an enzyme-linked immunosorbent assay method for indirectly detecting pirimiphos-methyl, which takes an artificial antigen of bovine serum albumin coupled with 2-diethylamino-6-methyl-4-pyrimidineol as an immunogen and an artificial antigen of chicken ovalbumin coupled with 2-diethylamino-6-methyl-4-pyrimidineol as an envelope antigen. Immunizing test animal with artificial immunogen to make its body produce antibody specifically directed against methyl pyrimidine phosphorus hydrolysateThe product 2-diethylamino-6-methyl-4-pyrimidineol is recognized by opposite sex, and is used for indirectly detecting the content of the pirimiphos-methyl in food after the pesticide raw material is hydrolyzed. The detection limit of the method is 3.2ng/mL, and the half Inhibitory Concentration (IC)50) The detection range is 23.42ng/mL, the linear detection range is 6.09-90.07 ng/mL, and the curve correlation coefficient is 0.9996. The method has the advantages of strong specificity, high sensitivity, simplicity, convenience and rapidness, and wide application prospect in the aspect of detection of the residue of the pirimiphos-methyl in the food.
Description
Technical Field
The invention relates to the technical field of immunodetection, and particularly relates to an enzyme-linked immunosorbent assay method for indirectly detecting pirimiphos-methyl.
Background
Pirimiphos-methyl, also known as O- (2-diethylamino-6-methyl-4-pyrimidinyl) -O, O-dimethyl phosphorothioate, also known as Andership. The molecular weight is 305, yellow liquid, can be hydrolyzed by strong acid and strong base, is unstable to light, can be dissolved in most organic solvents, and has the solubility of about 5mg/L in water. The pirimiphos-methyl is a quick-acting low-toxicity insecticide and acaricide, has the effects of contact killing and fumigation, has good pesticide effect on stored grain beetles, weevils, moths and mites, can be used for controlling pests of field crops and economic crops, and can also be used for controlling pests of fruits, vegetables and families, and is worthy of pointing out that the pirimiphos-methyl has particularly good control effect on stored pests and is widely applied at home and abroad.
The pirimiphos-methyl as a broad-spectrum novel organophosphorus insecticide has the characteristics of low toxicity and high efficiency, and is an ideal pesticide for replacing high-toxicity varieties. The fertilizer is widely applied to agriculture and grain storage production at present, but the excessive residual quantity of the fertilizer poses a potential threat to the health of people to a certain extent. After entering a human body, the pirimiphos-methyl can be combined with cholinesterase in the body to prevent the cholinesterase from catalyzing and hydrolyzing acetylcholine, so that a large amount of acetylcholine is accumulated in the body, cholinergic nerves are continuously motivated, toxic symptoms of excitation and then failure are caused, and coma and even death can be caused seriously.
At present, for the detection of pirimiphos-methyl, instrumental methods such as gas chromatography, gas chromatography-mass spectrometry, high performance liquid chromatography, liquid chromatography-mass spectrometry and the like are mainly adopted for quantitative detection. Although the methods have the advantages of accurate and credible detection results, the methods also have the defects of complicated sample pretreatment, time consumption, high cost, unsuitability for field detection and the like. In comparison, the immunoassay method developed based on antigen-antibody specificity recognition has the advantages of convenient and rapid detection and suitability for rapid detection of large-scale samples, and plays an increasingly important role in the field of rapid detection. The basis of the immunological detection method is to have high-quality and strong-specificity antibodies, so the design of haptens and the synthesis of complete antigens are particularly important.
Small molecule compounds (MW & lt 1000dolton) have reactogenicity and can react with antibodies, but generally have no immunogenicity and cannot be used for directly immunizing animals to generate specific antibodies, and if the small molecules are connected to macromolecules (generally proteins) with immunogenicity to be made into artificial antigens, the organisms can generate immune response and generate antibodies aiming at small molecule structures. For the selection of hapten structure, if the small molecule object to be tested contains functional groups such as-NH 2, -COOH, -OH, -SH and the like, the small molecule object to be tested can be directly activated and then coupled with carrier protein, and then the artificial complete antigen can be synthesized. Although the methylpyrimidine phosphorus has no corresponding active group, the methyl pyrimidine phosphorus can expose hydroxyl after hydrolysis, and can be directly coupled with carrier protein. CN201910890291.9 discloses a preparation method of a pirimiphos-methyl monoclonal antibody, but the hapten prepared by the method has a complex structure and complicated preparation steps.
Disclosure of Invention
The invention aims to provide an enzyme-linked immunosorbent assay method for indirectly detecting pirimiphos-methyl.
The first purpose of the invention is to provide an artificial hapten of pirimiphos-methyl.
The second purpose of the invention is to provide an antibody for detecting pirimiphos-methyl.
The third purpose of the invention is to provide a preparation method of the antibody for detecting pirimiphos-methyl.
The fourth purpose of the invention is to provide an enzyme-linked immunoassay method for indirectly detecting pirimiphos-methyl.
The fifth purpose of the invention is to provide the application of the artificial hapten and/or the antibody in detecting the pirimiphos-methyl.
The above object of the present invention is achieved by the following scheme:
in the invention, firstly, a methylpyrimidine phosphorus hydrolysate (2-diethylamino-6-methyl-4-pyrimidineol, Pr-OH, formula I) is selected as a hapten, and the hapten and a carrier protein are coupled to prepare an artificial antigen (formula II), so that an immunogen (Pr-OH-BSA) and a coating antigen (Pr-OH-OVA) are prepared. The artificial immunogen is used for immunizing test animals, so that the bodies of the animals generate antibodies specifically aiming at the methyl pyrimidine phosphorus hydrolysate, and the antibodies can specifically recognize the methyl pyrimidine phosphorus hydrolysate, namely 2-diethylamino-6-methyl-4-pyrimidineol, and are used for indirectly detecting the content of the methyl pyrimidine phosphorus in food after the pesticide raw material is hydrolyzed. The detection limit of the method is 3.2ng/mL, the semi-inhibitory concentration (IC50) is 23.42ng/mL, the linear detection range is 6.09-90.07 ng/mL, and the curve correlation coefficient is 0.9996. The method has the advantages of strong specificity, high sensitivity, simplicity, convenience and rapidness, and wide application prospect in the aspect of detection of the residue of the pirimiphos-methyl in the food.
Therefore, the invention firstly claims an artificial hapten of pirimiphos-methyl, which is prepared by coupling 2-diethylamino-6-methyl-4-pyrimidineol (formula I) with carrier protein and has a structure shown in a formula II:
preferably, the protein is prepared by coupling carrier protein by an N, N' -carbonyldiimidazole method.
Preferably, the carrier protein is one of Bovine Serum Albumin (BSA), chicken Ovalbumin (OVA), hemocyanin (KLH), Polylysine (PLL), and Human Serum Albumin (HSA).
An antibody for detecting pirimiphos-methyl, which is obtained by immunizing an animal with the artificial hapten.
Preferably, the antibody is a polyclonal antibody.
A preparation method of an antibody for detecting pirimiphos-methyl comprises the step of immunizing an animal with the artificial hapten to obtain the antibody.
After immunizing the animal, the animal produces antibodies specific to the methylpyrimidine phosphate hydrolysate.
An enzyme-linked immunoassay method for indirectly detecting pirimiphos-methyl is characterized in that an artificial antigen of 2-diethylamino-6-methyl-4-pyrimidinol coupled with Bovine Serum Albumin (BSA) is taken as an immunogen, and an artificial antigen of 2-diethylamino-6-methyl-4-pyrimidinol coupled with chicken Ovalbumin (OVA) is taken as an envelope antigen.
Preferably, the method comprises the following steps:
s1, diluting a coating source with a coating buffer solution, coating the coating source in different micropores of an enzyme label plate, incubating, washing the plate, and sealing;
s2, respectively adding a standard solution of 2-diethylamino-6-methyl-4-pyrimidineol or a sample solution to be detected after hydrolysis treatment into corresponding micropores of an ELISA plate;
s3, diluting the antibody solution by a certain multiple, adding the diluted antibody solution into the micropores of the ELISA plate, incubating and washing the plate;
s4, adding the enzyme-labeled secondary antibody solution into all micropores of the enzyme-labeled plate, incubating and washing the plate;
s5, adding a color development liquid into the enzyme label plate for color development;
s6, adding the stop solution into an enzyme label plate, stopping the reaction, and reading the absorbance value of each hole by using an enzyme label instrument;
s7, drawing a standard curve by using a four-parameter fitting module of origin8.5, and calculating corresponding detection parameters of the inhibition curve;
and S8, converting the actual content of the pirimiphos-methyl in the sample to be detected.
More preferably, the color developing solution in step S5 is the substrate solution corresponding to the enzyme in the enzyme-labeled secondary antibody in step S4.
More preferably, the stop solution in step S6 is a concentrated sulfuric acid solution.
More preferably, the stop solution in step S6 is a concentrated sulfuric acid solution with a concentration of 2 mol/L.
The application of the artificial hapten and/or the antibody in detecting the pirimiphos-methyl also belongs to the protection scope of the invention.
Compared with the prior art, the invention has the following beneficial effects:
(1) the hapten provided by the invention has a simple and easily obtained structure, can be directly purchased and directly coupled with carrier protein, avoids fussy hapten design synthesis and identification steps, simultaneously furthest retains the pyrimidine ring characteristic structure of pirimiphos-methyl, and is beneficial to stimulating experimental animals to generate immune response; (2) the polyclonal antibody prepared by the artificial antigen immune experimental animal has high sensitivity and strong specificity, and can meet the detection requirement, so the artificial antigen can be further used for preparing a monoclonal antibody or a genetic engineering antibody with higher quality; (3) the immunoassay detection method established based on the antigen and the antibody has the characteristics of convenient use, low cost, strong specificity and high sensitivity, can be used for simultaneously detecting a large batch of samples, and is suitable for indirectly detecting the residual quantity of the pirimiphos-methyl in various foods.
Drawings
FIG. 1 is a UV scan of hapten I and its immunogen and coatingen.
FIG. 2 is a standard curve for the inhibition of 2-diethylamino-6-methyl-4-pyrimidineol by the antibody prepared from hapten I.
Detailed Description
The present invention is further described in detail below with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
EXAMPLE 1 preparation and characterization of immunogens and coatinggens
The preparation of immunogen and coating antigen is realized by using different carrier proteins. The immunogen carrier protein adopts Bovine Serum Albumin (BSA), the coating original carrier protein adopts chicken Ovalbumin (OVA), and the adopted coupling mode is an N, N-carbonyl diimidazole method. The specific embodiment is as follows:
0.03mmol of the hapten (2-diethylamino-6-methyl-4-pyrimidineol) of the formula I was dissolved in 250. mu.L of DMF, and 8.11mg of N, N-carbonyldiimidazole was added thereto, and the mixture was stirred at 37 ℃ for 2 hours to give solution A. 10mg of carrier protein (BSA or OVA) was weighed and dissolved in 2mL of coating solution, which was solution B. And slowly dropwise adding the solution A to the solution B, and stirring and reacting for 72 hours at 4 ℃. After the reaction, the mixture was put into a dialysis bag previously boiled for 10min, and dialyzed with PBS for 3 days with 3 changes of the dialysate per day. The complete antigen formula II (immunogen Pr-OH-BSA and coating Pr-OH-OVA) obtained is respectively diluted to 1mg/mL, subpackaged and frozen at-20 ℃.
The identification of artificial immunogen and coating antigen adopts ultraviolet scanning method. Preparing a 1mg/mL solution of the carrier protein, the hapten and the artificial antigen thereof by using PBS (phosphate buffer solution) with pH7.4, zeroing by using PBS with 0.01mol/LpH7.4, and performing ultraviolet scanning measurement (200-400 nm) to obtain absorption curves of the carrier protein, the hapten and the artificial antigen thereof.
The results are shown in fig. 1, the ultraviolet scanning curves of the three are not overlapped and the characteristic absorption peaks have corresponding shifts, which indicates that the artificial immunogen and the coating antigen are successfully prepared.
EXAMPLE 2 preparation and characterization of antibodies
1, preparation of polyclonal antiserum
The immunogen Pr-OH-BSA prepared in example 1 is mixed with Freund's adjuvant (complete Freund's adjuvant for the first immunization and incomplete Freund's adjuvant for the subsequent immunization), and after being sufficiently emulsified, the mice are immunized by means of multi-point injection such as back, abdomen, subcutaneous, foot pad, etc. The first immunization was boosted every two weeks three weeks later. Tail blood was collected one week after the third booster immunization and serum titers were determined using indirect competitive ELISA. When the titer does not rise any more, the immunization is strengthened by intraperitoneal injection once, and the eyeball is picked up after one week to draw blood. Standing for 0.5-1 h at room temperature, centrifuging for 10min at 4 ℃ and 12000r/min, taking supernatant, subpackaging in centrifuge tubes, and storing at-20 ℃ for later use.
2, polyclonal antiserum potency assay
The indirect competitive ELISA measures the antibody titer based on the absorbance value of 1.0-1.5. Phosphate buffered saline (PBS, 0.1mol/L, pH7.4) was used as a diluent and reaction buffer for all samples; Pr-OH-OVA is used as a coating antigen; adding 50 mu L of LPBS buffer solution or 1mg/mL of 2-diethylamino-6-methyl-4-pyrimidineol standard solution and 50 mu L of polyclonal antibody with appropriate dilution times into a 96-well enzyme label plate, adding an enzyme-labeled secondary antibody after the competitive reaction is finished, and then measuring the absorbance value through an enzyme label after the color reaction.
The results show that the immunogen prepared in example 1 has an immune polyclonal antiserum titer of 1:32000 and an inhibition rate of 90%.
EXAMPLE 3 establishment of enzyme-linked immunosorbent assay for indirectly determining pirimiphos-methyl
An indirect competition ELISA standard curve was drawn using the antisera prepared in example 2. Phosphate buffered saline (PBS, 0.1mol/L, pH7.4) was used as a diluent and reaction buffer for all samples; the specific examples using Pr-OH-OVA as the coating source are as follows:
(1) coating 1ng/mL coating antigen Pr-OH-OVA on an ELISA plate, coating overnight at 37 ℃, adding 120 mu L of 5% skimmed milk powder solution per hole, sealing at 37 ℃ for 3h, spin-drying, and drying in an oven at 37 ℃ for later use.
(2) Adding a sodium hydroxide solution into the sample treatment solution, adjusting the pH of the solution to 11, and oscillating at 50 ℃ for 2h to obtain a hydrolyzed sample solution;
(3) add 50. mu.L of a series of concentrations (0.128, 0.64, 3.2, 16, 80, 400, 2000ng/mL) of 2-diethylamino-6-methyl-4-pyrimidinol standard solutions or hydrolyzed sample solutions to the prepared coated plate;
(4) adding 50 μ L of antibody diluted 1:4000 times into each well, incubating at 37 deg.C for 30min, washing with washing solution for 5 times, and drying on absorbent paper;
(5) adding 100 mu L of enzyme-labeled secondary antibody solution diluted by 1:5000 times into each hole, incubating for 30min at 37 ℃, washing for 5 times by using washing liquid, and patting dry on absorbent paper;
(6) add 100. mu.L of LH to each well2O2Developing solution of-TMB substrate, incubating at 37 deg.C for 10min
(7) 50 mu L of 2mol/L sulfuric acid solution is added into each hole to stop the reaction, and the absorbance value of each hole is read by a microplate reader under the wavelength of 450 nm.
(8) With B/B0The value is ordinate, the corresponding standard concentration logarithm value is abscissa, curve fitting is carried out by using Origin8.5 software four-parameter logarithm function, and IC calculated by curve50At half inhibitory concentration, with IC10For detection purposes, use IC20～IC80The detection range is shown.
(9) ELISA reaction of several analogous structures of 2-diethylamino-6-methyl-4-pyrimidineol with the antibodies, plotting of standard curves and calculation of IC for each analogue50And calculating the cross-reactivity.
As shown in FIG. 2 and Table 1, the standard curve established with 2-diethylamino-6-methyl-4-pyrimidineol has a typical sigmoid curve, a correlation coefficient of 0.9996, good sensitivity, a detection limit of 3.2ng/mL, high specificity, and no cross-over phenomenon with other similar structures. The method can be used for indirectly detecting the content of the pirimiphos-methyl in the food because the pirimiphos-methyl in the sample can be quickly and efficiently hydrolyzed into the 2-diethylamino-6-methyl-4-pyrimidineol.
TABLE 1 measurement parameters of antisera on 2-diethylamino-6-methyl-4-pyrimidineol
TABLE 2 Cross-reactivity of antisera to 2-diethylamino-6-methyl-4-pyrimidineol and similar structures
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. The artificial hapten of pirimiphos-methyl is characterized by being prepared by coupling 2-diethylamino-6-methyl-4-pyrimidineol with carrier protein and having a structure shown in a formula II:
2. the artificial hapten according to claim 1 wherein the carrier protein is one of bovine serum albumin, chicken egg white albumin, hemocyanin, polylysine, or human serum albumin.
3. An antibody for detecting pirimiphos-methyl, which is obtained by immunizing an animal with the artificial hapten as claimed in claim 1.
4. A method for preparing an antibody for detecting pirimiphos-methyl, comprising immunizing an animal with the artificial hapten of claim 1 to obtain the antibody.
5. An enzyme-linked immunoassay method for indirectly detecting pirimiphos-methyl is characterized in that an artificial antigen of bovine serum albumin coupled with 2-diethylamino-6-methyl-4-pyrimidinol is taken as an immunogen, and an artificial antigen of chicken ovalbumin coupled with 2-diethylamino-6-methyl-4-pyrimidinol is taken as an envelope antigen.
6. The ELISA method of claim 5 comprising the steps of:
s1, diluting a coating source with a coating buffer solution, coating the coating source in different micropores of an enzyme label plate, incubating, washing the plate, and sealing;
s2, respectively adding a standard solution of 2-diethylamino-6-methyl-4-pyrimidineol or a sample solution to be detected after hydrolysis treatment into corresponding micropores of an ELISA plate;
s3, diluting the antibody solution by a certain multiple, adding the diluted antibody solution into the micropores of the ELISA plate, incubating and washing the plate;
s4, adding the enzyme-labeled secondary antibody solution into all micropores of the enzyme-labeled plate, incubating and washing the plate;
s5, adding a color development liquid into the enzyme label plate for color development;
s6, adding the stop solution into an enzyme label plate, stopping the reaction, and reading the absorbance value of each hole by using an enzyme label instrument;
s7, drawing a standard curve by using a four-parameter fitting module of origin8.5, and calculating corresponding detection parameters of the inhibition curve;
and S8, converting the actual content of the pirimiphos-methyl in the sample to be detected.
7. The ELISA method of claim 6 wherein the color reagent of step S5 is the substrate solution corresponding to the enzyme in the enzyme-labeled secondary antibody of step S4.
8. The ELISA method of claim 6 wherein the stop solution of step S6 is a concentrated sulfuric acid solution.
9. The ELISA method of claim 8 wherein the stop solution of step S6 is a concentrated sulfuric acid solution of 2 mol/L.
10. Use of the artificial hapten of claim 1 and/or the antibody of claim 3 for detecting pirimiphos-methyl.
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