Fipronil hapten, synthetic method thereof, artificial antigen, antibody and application
阅读说明：本技术 一种氟虫腈半抗原及其合成方法、人工抗原、抗体和用途 (Fipronil hapten, synthetic method thereof, artificial antigen, antibody and application ) 是由 冯艳武 卫大群 郭志雄 葛怀娜 韦吉杨 仇雅婷 孙惠洁 于 2021-10-11 设计创作，主要内容包括：本发明公开了一种氟虫腈半抗原及其合成方法、人工抗原、抗体和用途,属于生物化工技术领域。本发明提供了一种氟虫腈半抗原,其合成方法包括以下步骤：S1.将氟虫腈结构类似物与氯甲酸苯酯反应,合成中间体I；S2.将所述中间体I与氨基丙酸叔丁酯反应,合成中间体II；S3.将所述中间体II在酸性条件下脱叔丁基,合成氟虫腈半抗原。本发明合成的人工抗原能够最大程度地保留氟虫腈的基本结构,能够用于制备较高特异性与灵敏度的抗体,将制备的抗体用于酶联免疫试剂盒检测氟虫腈时,具有特异性好、灵敏度和精密度高的优点。(The invention discloses a fipronil hapten, a synthetic method thereof, an artificial antigen, an antibody and application, and belongs to the technical field of biochemical engineering. The invention provides a fipronil hapten, and a synthetic method thereof comprises the following steps: s1, reacting fipronil structural analogues with phenyl chloroformate to synthesize an intermediate I; s2, reacting the intermediate I with tert-butyl aminopropionate to synthesize an intermediate II; and S3, removing tert-butyl from the intermediate II under an acidic condition to synthesize the fipronil hapten. The artificial antigen synthesized by the invention can furthest reserve the basic structure of fipronil, can be used for preparing antibodies with higher specificity and sensitivity, and has the advantages of good specificity, high sensitivity and high precision when the prepared antibodies are used for detecting the fipronil by an enzyme linked immunosorbent assay kit.)
1. A fipronil hapten, which is characterized in that the structural formula of the fipronil hapten is as follows:
。
2. a method for synthesizing a fipronil hapten as claimed in claim 1, characterized in that it comprises the following steps:
s1, reacting fipronil structural analogues with phenyl chloroformate to synthesize an intermediate I, wherein the intermediate I has a structural formula shown as follows:
；
s2, reacting the intermediate I with tert-butyl aminopropionate to synthesize an intermediate II, wherein the intermediate II has a structural formula shown as follows:
；
and S3, removing tert-butyl from the intermediate II under an acidic condition to obtain the fipronil hapten.
3. The method for synthesizing the fipronil hapten according to claim 2, wherein in step S1, the fipronil structural analogue reacts with phenyl chloroformate in acetone at a reaction temperature of 0-40 ℃, and the feeding molar ratio of the fipronil structural analogue to the phenyl chloroformate is 1: 1.0-1.2.
4. The method for synthesizing fipronil hapten according to claim 2, wherein DBU and triethylamine are added in step S2.
5. The method for synthesizing fipronil hapten according to claim 4, wherein in step S2, the feeding molar ratio of the intermediate II, the tert-butyl aminopropionate, DBU and triethylamine is 1:5:0.1:10, the reaction solvent is tetrahydrofuran, and the reaction temperature is 50-80 ℃.
6. The method for synthesizing fipronil hapten according to claim 2, wherein the step S3 is specifically that the intermediate II is dissolved in dichloromethane, and then trifluoroacetic acid is added to react at room temperature to remove tert-butyl group, so as to obtain the fipronil hapten.
7. The method for synthesizing fipronil hapten according to claim 6, wherein 10-20mL of trifluoroacetic acid and 10-20mL of dichloromethane are added per mmol of intermediate II in step S3.
8. An artificial fipronil antigen, which is obtained by coupling the fipronil hapten as described in claim 1 with a carrier protein, or by coupling the fipronil hapten as synthesized by the synthesis method as described in any one of claims 2 to 7 with a carrier protein, and the structural formula of the artificial fipronil antigen is as follows:
whereinIs a carrier protein.
9. An antibody specific to fipronil, which is a monoclonal or polyclonal antibody specifically immunoreactive with fipronil, obtained by immunizing an animal with the fipronil artificial antigen of claim 8.
10. The use of the fipronil hapten is characterized in that the fipronil hapten of claim 1 or the fipronil hapten synthesized by the synthesis method of any one of claims 2 to 7 is used for detecting the residual quantity of fipronil.
Technical Field
The invention relates to a fipronil hapten, a synthetic method thereof, an artificial antigen, an antibody and application, belonging to the technical field of biochemical engineering.
Background
Fipronil, trade name fipronil, is a phenyl pyrazole pesticide with broad insecticidal spectrum, mainly has stomach toxicity to pests, has both contact killing and certain systemic action, and has an action mechanism of hindering chloride metabolism controlled by insect gamma-aminobutyric acid, so that the fipronil has high insecticidal activity to important pests such as aphids, leafhoppers, plant hoppers, lepidoptera larvae, flies, coleoptera and the like, and has no pesticide damage to crops. The agent can be applied to soil or sprayed on leaf surface. The pesticide composition can be applied to soil to effectively prevent and control corn rootworm beetles, wireworms and cutworms. When the pesticide is sprayed on the leaf surfaces, the pesticide has high-level control effect on plutella xylostella, cabbage butterflies, rice thrips and the like, and has long lasting period.
Although fipronil has good effect of preventing and controlling pests, it is extremely unfriendly to the environment, can affect butterflies, dragonflies and the like around crops, and has high toxicity to fish, shrimps, bees and silkworms. And the existing animal experiment research shows that the short-term intake of a large amount of fipronil can cause adverse effects on the nervous system, and the long-term intake of fipronil can damage the liver, the thyroid and the kidney. Therefore, the fipronil is forbidden from 10 months and 1 day in 2009 in China, and can only be used for domestic sanitary pests at present.
Fipronil is a pesticide with large toxic and side effects in ecological environment, and needs to be strictly monitored. The most important factor influencing the government supervision or management effect is a detection means of fipronil, the detection of fipronil at present mainly aims at the detection of pesticide fipronil in vegetables and environment, and the method mainly comprises a high performance liquid chromatography and liquid chromatography mass combination method, and the methods have the advantages of strong specificity and high sensitivity, but are complex to operate, expensive in instruments and not suitable for screening and detecting large-batch samples. The immunoassay method has unique advantages in qualitative and quantitative aspects of antigen and antibody, and has the advantages of simple and rapid operation, low cost and large analysis sample amount.
CN101100456A discloses fipronil artificial hapten and a synthetic method, an antigen, an antibody and application thereof, when the prepared fipronil antigen is prepared and used for ELISA method detection, the minimum detection limit of fipronil can reach 7.6 mu g/L, the average variation coefficient in batch is 4.6%, the cross reaction rate of ethiprole is 6.4%, and the cross reaction rate of flufenoxuron is 0.44%.
CN101100457A discloses a fipronil hapten compound, a synthetic method and application thereof, wherein when the prepared antigen is used for ELISA detection, the lowest detection limit of fipronil can reach 4.8 mu g/L.
CN101100486A discloses fipronil artificial antigen, antibody and application thereof, when the prepared fipronil artificial antigen is used for ELISA method detection, the minimum detection limit of fipronil is 4.8 mug/L, the average variation coefficient in batch is 5.7%, the cross reaction rate of ethiprole is 3.2%, and the cross reaction rate of flufenoxuron is 0.13%.
When the fipronil hapten or fipronil artificial antigen prepared by the method is used for detecting fipronil by an ELISA method, the sensitivity, specificity and precision of the fipronil hapten or the fipronil artificial antigen are still required to be improved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a fipronil hapten and a synthesis method, an artificial antigen, an antibody and application thereof, wherein the synthesized fipronil hapten can reserve the basic structure of fipronil to the maximum extent and can be used for preparing the antibody with higher specificity and sensitivity, and when the prepared antibody is used for detecting the fipronil by an enzyme linked immunosorbent assay kit, the antibody has the advantages of good specificity, high sensitivity and high precision.
In order to achieve the purpose, the invention provides a fipronil hapten, wherein the structural formula of the fipronil hapten is as follows:
。
the invention also provides a synthetic method of the fipronil hapten, which comprises the following steps:
s1, reacting fipronil structural analogues with phenyl chloroformate to synthesize an intermediate I, wherein the intermediate I has a structural formula shown as follows:
；
s2, reacting the intermediate I with tert-butyl aminopropionate to synthesize an intermediate II, wherein the intermediate II has a structural formula shown as follows:
；
and S3, removing tert-butyl from the intermediate II under an acidic condition to obtain the fipronil hapten.
Preferably, in step S1, the fipronil structural analogue reacts with phenyl chloroformate in acetone at 0-40 deg.C, and the molar ratio of the fipronil structural analogue to the phenyl chloroformate is 1: 1.0-1.2.
Preferably, in step S2, DBU and triethylamine are also added.
Preferably, in step S2, the feeding molar ratio of the intermediate II, the tert-butyl aminopropionate, DBU, and triethylamine is 1:5:0.1:10, the reaction solvent is tetrahydrofuran, and the reaction temperature is 50 ℃ to 80 ℃.
Preferably, in step S3, the intermediate II is dissolved in dichloromethane, and then trifluoroacetic acid is added to react at room temperature to remove tert-butyl group, so as to obtain fipronil hapten.
Preferably, in step S3, 10-20mL of trifluoroacetic acid and 10-20mL of dichloromethane are added per mmol of intermediate II.
The invention also provides a fipronil artificial antigen which is obtained by coupling the fipronil hapten and carrier protein or coupling the fipronil hapten synthesized by the synthesis method and the carrier protein, wherein the structural formula of the fipronil artificial antigen is as follows:
whereinIs a carrier protein.
Preferably, the carrier protein is one or more of bovine serum albumin, chicken egg serum protein and hemocyanin.
The invention also provides a synthetic method of the fipronil artificial antigen, which comprises the steps of dissolving fipronil hapten with DMF, adding NHS and EDC hydrochloride, reacting for 16-24h at room temperature to obtain a fipronil hapten active ester solution; weighing carrier protein, dissolving the carrier protein in borate buffer solution, slowly dripping the fipronil hapten active ester solution into the borate buffer solution, reacting at room temperature for 5-24h, and dialyzing to obtain fipronil artificial antigen;
or dissolving the fipronil hapten with DMF, adding n-butylamine and isobutyl chloroformate for reaction to obtain an active mixed anhydride solution of the fipronil hapten; weighing carrier protein, dissolving the carrier protein in borate buffer solution, slowly dripping the active mixed anhydride solution of the fipronil hapten into the borate buffer solution, reacting at room temperature for 5-24h, and dialyzing to obtain the fipronil artificial antigen.
Preferably, the molar ratio of the fipronil hapten to NHS and EDC hydrochloride is 1:1.0-2.0:1.0-2.0, the molar ratio of the fipronil hapten to n-butylamine and isobutyl chloroformate is 1:1.2:1.2, and the reaction time is 1 h.
The invention also provides a fipronil specific antibody, which is a monoclonal or polyclonal antibody which is obtained by immunizing animals with the fipronil artificial antigen and can perform specific immunoreaction with fipronil.
The invention also provides application of the fipronil hapten, and application of the fipronil hapten or the fipronil hapten synthesized by the synthesis method in detecting fipronil residue.
Compared with the prior art, the invention has the following beneficial effects:
(1) the fipronil hapten synthesized by the method can reserve the basic structure of fipronil to the maximum extent, and has higher synthesis purity.
(2) The artificial antigen synthesized by the invention can be used for preparing antibodies with higher specificity and sensitivity, and when the prepared antibodies are used for detecting fipronil by an enzyme linked immunosorbent assay kit, the artificial antigen has the advantages of good specificity, high sensitivity and high precision, and can be used for quickly detecting fipronil.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
The following example describes a method for synthesizing fipronil hapten and fipronil artificial antigen, and the synthetic route of fipronil hapten is shown as follows:
the synthetic route of the fipronil artificial antigen is shown as follows:
example 1
A synthetic method of fipronil hapten comprises the following steps:
s1, reacting fipronil structural analogues with phenyl chloroformate to synthesize an intermediate I, which specifically comprises the following steps:
weighing 4.37g of fipronil structural analogue, dissolving in acetone, cooling in an ice bath, slowly dropwise adding 1.88g of phenyl chloroformate, and raising the temperature to 25 ℃ for reaction overnight after finishing the reaction. The reaction solution was concentrated to obtain [ 5-cyano-2- (2, 6-dichloro-4-trifluoromethyl-phenyl) -4-methylsulfony l-2H-pyrazol-3-yl ] -carbamic acid phenyl ester (intermediate I). The intermediate I can be directly subjected to the next reaction without purification.
S2, reacting the intermediate I with tert-butyl aminopropionate to synthesize an intermediate II, which specifically comprises the following steps:
dissolving 0.50g of intermediate I in 30mL of tetrahydrofuran, sequentially adding 0.01g of DBU (1, 8-diazabicycloundecen-7-ene), 1.21mL of triethylamine and 0.73g of tert-butyl 3-amino propionate, heating the reaction solution to 65 ℃ to react for 12h, cooling to room temperature, concentrating under vacuum to remove tetrahydrofuran, separating 30mL of ethyl acetate and 30mL of water, extracting the water phase with 30mL of ethyl acetate for 3 times, combining the organic phases, washing with 30mL of saturated saline, drying over anhydrous sodium sulfate, concentrating, loading the residue onto a silica gel column for chromatography (volume ratio of ethyl acetate: petroleum ether =1: 2), this gave 3- {3- [ 5-cyano-2- (2, 6-dichloro-4-trifluoromethyl-phenyl) -4-methylsulfoxido-2H-pyrazol-3-yl ] -ureido } -propionic acid tert-butyl ester (intermediate II).
S3, removing tert-butyl from the intermediate II under an acidic condition to synthesize a fipronil hapten, which specifically comprises the following steps:
0.55g of intermediate II is weighed out and dissolved in 20mL of dichloromethane, 20mL of trifluoroacetic acid is added and stirred for 2H at room temperature, the reaction solution is concentrated, and the residue is subjected to silica gel column chromatography (volume ratio methanol: dichloromethane ═ 1: 20) to obtain 3- {3- [ 5-cyano-2- (2, 6-dichloro-4-trifluoromethyl-phenyl) -4-methylsulfonyl-2H-pyrazol-3-yl ] -ureido } -propionic acid (fipronil hapten) as a white solid, wherein the purity of the synthesized fipronil hapten is 99.9%.
A synthetic method of fipronil artificial antigen specifically comprises the following steps:
10mg of fipronil hapten is weighed and dissolved in 200 microliter DMF, 7.7mg of EDC hydrochloride and 2.8mg of NHS are added, and the mixture is stirred for 16h at room temperature to obtain an active ester solution. 50mg of BSA was dissolved in 4mL of borate buffer solution, and the active ester solution was dropped into the BSA solution to react at room temperature for 15 hours. Dialyzing for 3 days, taking out the dialyzate after the dialysis is finished, and freeze-drying to obtain fipronil BSA artificial antigen, and storing at-40 ℃.
Example 2
The procedure is as in example 1, except that:
when the fipronil semi-antigen is synthesized, in step S1, 4.40g of fipronil structural analogue is weighed and dissolved in acetone, the temperature is reduced in an ice bath, 1.73g of phenyl chloroformate is slowly dripped, and after the completion, the temperature is raised to 40 ℃ for reaction overnight.
When the fipronil semi-antigen is synthesized, in step S2, 0.50g of intermediate I is dissolved in 30mL of tetrahydrofuran, 0.01g of DBU, 1.21mL of triethylamine and 0.73g of tert-butyl 3-amino propionate are sequentially added, and the reaction solution is heated to 50 ℃ for reaction for 12 hours.
When synthesizing a fipronil hapten, 0.55g of intermediate II is weighed out and dissolved in 10mL of dichloromethane in step S3, and 10mL of trifluoroacetic acid is added and stirred at room temperature for 2 hours. The purity of the synthesized fipronil hapten is 99.6%.
When the artificial fipronil antigen is synthesized, 10mg of fipronil hapten is weighed and dissolved in 200 mu L of DMF, 7.7mg of EDC hydrochloride and 2.8mg of NHS are added, and the mixture is stirred at room temperature for 20 hours to obtain an active ester solution. 50mg of BSA was dissolved in 4mL of borate buffer solution, and the active ester solution was dropped into the BSA solution to react at room temperature for 24 hours.
Example 3
The procedure is as in example 1, except that:
when the fipronil semi-antigen is synthesized, in step S1, 4.50g of fipronil structural analogue is weighed and dissolved in acetone, the temperature is reduced in an ice bath, 1.61g of phenyl chloroformate is slowly dripped, and the reaction is carried out overnight at 0 ℃.
When the fipronil semi-antigen is synthesized, in step S2, 0.50g of intermediate I is dissolved in 30mL of tetrahydrofuran, 0.01g of DBU, 1.21mL of triethylamine and 0.73g of tert-butyl 3-amino propionate are sequentially added, and the reaction solution is heated to 80 ℃ for reaction for 12 hours.
When synthesizing a fipronil hapten, 0.55g of intermediate II is weighed out and dissolved in 15mL of dichloromethane in step S3, and 15mL of trifluoroacetic acid is added and stirred at room temperature for 2 hours. The purity of the synthesized fipronil hapten is 99.7%.
When the artificial fipronil antigen is synthesized, 10mg of fipronil hapten is weighed and dissolved in 200 mu L of DMF, 7.7mg of EDC hydrochloride and 2.8mg of NHS are added, and the mixture is stirred at room temperature for 24 hours to obtain an active ester solution. 50mg of BSA was dissolved in 4mL of borate buffer solution, and the active ester solution was dropped into the BSA solution to react at room temperature for 5 hours.
Example 4
The procedure is as in example 1, except that:
when the artificial fipronil antigen is synthesized, 10mg of fipronil hapten is weighed and dissolved in 200 mu L of DMF, 7.7mg of EDC hydrochloride and 2.8mg of NHS are added, and the mixture is stirred at room temperature overnight to obtain an active ester solution. 100mg of BSA was weighed and dissolved in 8mL of borate buffer solution, and the active ester solution was dropped into the BSA solution overnight at room temperature. Dialyzing for 3 days, taking out the dialyzate after the dialysis is finished, and freeze-drying to obtain the fipronil BSA artificial antigen, and storing at-40 ℃.
Example 5
The procedure is as in example 1, except that:
when the artificial fipronil antigen is synthesized, 10mg of fipronil hapten is weighed and dissolved in 200 mu L of DMF, 7.7mg of EDC hydrochloride and 2.8mg of NHS are added, and the mixture is stirred at room temperature overnight to obtain an active ester solution. 20mg of BSA was weighed and dissolved in 1mL of borate buffer solution, and the active ester solution was dropped into the BSA solution overnight at room temperature. Dialyzing for 3 days, taking out the dialyzate after the dialysis is finished, and freeze-drying to obtain the fipronil BSA artificial antigen, and storing at-40 ℃.
Example 6
The procedure is as in example 1, except that:
when the artificial fipronil antigen is synthesized, 10mg of hapten is weighed and dissolved in 200 mu of LDMF, ice bath cooling is carried out, then 5.2 mu of L of n-butylamine is added, 2.9 mu of L of isobutyl chloroformate is added, and ice bath reaction is continued for 1 hour to obtain the fipronil hapten active anhydride solution. 10mg of OVA was dissolved in 1mL of borate buffer (pH = 8.7) and the hapten-active anhydride solution was slowly added dropwise to the OVA protein buffer. And after the reaction is finished, reacting for 4 hours at room temperature in a dark place, and dialyzing the reaction solution for three days at a refrigerator at 4 ℃ to obtain the fipronil OVA artificial antigen.
Example 7
The procedure is as in example 1, except that:
when the artificial fipronil antigen is synthesized, 10mg of hapten is weighed and dissolved in 200 mu L of DMF, ice bath cooling is carried out, then 5.2 mu L of n-butylamine is added, 2.9 mu L of isobutyl chloroformate is added, and ice bath reaction is continued for 1h to obtain the fipronil hapten active anhydride solution. 50mg of BSA was dissolved in 4mL of borate buffer (pH = 8.7), and the hapten-active anhydride solution was slowly added dropwise to the BSA protein buffer solution. And after the reaction is finished, reacting for 4 hours at room temperature in a dark place, and dialyzing the reaction solution for three days at a refrigerator at 4 ℃ to obtain the fipronil BSA artificial antigen.
Comparative example 1
An artificial fipronil antigen prepared by the method of example 2 in patent publication No. CN 101100456A.
Comparative example 2
An artificial fipronil antigen prepared by the method of example 4 in patent publication No. CN 101100457A.
Comparative example 3
An artificial fipronil antigen prepared by the method of example 1 in patent publication No. CN 101100486A.
Test example 1 preparation of enzyme-linked immunosorbent assay kit for detecting fipronil
a) Preparation of fipronil monoclonal antibody
Animal immunization: the fipronil artificial antigens obtained in examples 1 to 7 and comparative examples 1 to 3 were injected into Balb/c (8-week) mice at an immunization dose of 100. mu.g/mouse to generate antiserum.
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 fipronil monoclonal antibody.
Freezing and recovering cells: monoclonal hybridoma cell lines were made into cell suspensions of 1X 106 cells/mL using a frozen stock solution and stored in liquid nitrogen for a long period of time. 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: adopting a method of inducing monoclonal antibodies in animals, and collecting ascites after 7 days. Ascites was purified by protein G purification column and stored at-20 ℃.
b) Preparation of enzyme-labeled Secondary antibody
Taking a goat as an immune animal and taking the fipronil monoclonal antibody as an immunogen to immunize the goat without the pathogen to obtain the fipronil anti-antibody. Coupling the fipronil anti-antibody with horseradish peroxidase (HRP) to obtain an enzyme-labeled secondary antibody.
c) Preparation of ELISA plates
Diluting the coating source to 1 μ g/mL with a coating buffer (pH 9.6 carbonate), adding 100 μ L into each well, incubating at 4 deg.C in the dark for 16h, decanting off the liquid in the well, washing with a washing solution for 1 time, standing for 30s, patting to dry, adding 200 μ L of blocking solution into each well, incubating at 37 deg.C in the dark for 2h, decanting off the liquid in the well, patting to dry, and vacuum sealing with an aluminum film for storage.
d) Construction of enzyme linked immunosorbent assay kit for detecting fipronil
An enzyme linked immunosorbent assay kit for detecting fipronil is constructed, and comprises the following components:
1) an ELISA plate coated with fipronil coupled antigen;
2) 6 bottles (concentrated solution solvent methanol) of fipronil standard substance concentrated solution with the concentrations of 0 mug/L, 5 mug/L, 15 mug/L, 45 mug/L, 135 mug/L and 405 mug/L respectively, when in use, the fipronil standard substance concentrated solution is diluted into standard substance solution by 0.02mol phosphate buffer solution (pH value is 7.2) according to the volume ratio of 9:1, and the concentrations after dilution are 0 mug/L, 0.5 mug/L, 1.5 mug/L, 4.5 mug/L, 13.5 mug/L and 40.5 mug/L respectively;
3) horseradish peroxidase-labeled fipronil anti-antibody;
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 solution has a pH value of 7.2, and contains 0.01% of tween-20, 3g/L of sodium azide preservative and 0.01mol/L of phosphate buffer solution, wherein the percentages are weight volume percentages;
7) the compound solution is phosphate buffer solution with pH value of 7.0 and 0.02mol/L, and the percentage is weight volume percentage.
Test example 2 specific detection of fipronil kit
Different concentration standard curves of ethiprole, fipronil sulfoxide and fipronil thioether are respectively configured, wherein the standard curves are 0 mu g/L, 0.5 mu g/L, 1.5 mu g/L, 4.5 mu g/L, 13.5 mu g/L and 40.5 mu g/L, the IC50 values of the ethiprole, the fipronil sulfoxide and the fipronil thioether can be obtained through absorbance values by using fipronil kits prepared in examples 1-7 and comparative examples 1-3, and the calculation mode of the cross reaction rate is as follows:
TABLE 1 Cross-reactivity ratios of ethiprole, fipronil sulfoxide, fipronil sulfide
As can be seen from Table 1, compared with comparative examples 1 to 3, the cross-reaction rates of ethiprole, fipronil sulfoxide and fipronil thioether measured by the fipronil kits prepared in examples 1 to 7 are obviously lower, which indicates that the fipronil detection specificity of the fipronil kits prepared in examples 1 to 7 is higher, and the fipronil artificial antigen prepared in examples 1 to 6 is stronger.
Test example 3 detection of precision of fipronil kit
(1) Detection with a kit
Adding 50 mu L of standard solution/sample into the corresponding micropores, adding 50 mu L/hole of antibody, finally adding 50 mu L/hole of enzyme-labeled secondary antibody, lightly oscillating and uniformly mixing, and covering with a cover plate film for reaction for 45min in a dark environment at 25 ℃. Carefully uncovering the cover plate film, drying the liquid in the holes, fully washing the holes for 4-5 times by using 250 mu L of washing working solution at intervals of 10s, and patting the holes dry by using absorbent paper (bubbles which are not removed after patting the holes dry can be slightly punctured by using an unused gun head). Adding 50 μ L/hole of the substrate solution A, adding 50 μ L/hole of the substrate solution B, gently shaking, mixing, covering with a cover plate, and developing at 25 deg.C in dark environment for 15 min. Adding 50 mu L of stop solution into each hole, slightly oscillating and uniformly mixing, setting the detection wavelength of an enzyme-labeling instrument to be 450nm and the reference wavelength to be 620nm, and determining the OD value of each hole.
(2) Analysis of detection results
The percent absorbance of the standard or sample is equal to the absorbance of the standard or sample divided by the absorbance of the first standard (0 standard) and multiplied by 100% to obtain the percent absorbance 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 fipronil standard substance as an abscissa. 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 corresponding dilution times to obtain the actual concentration of the fipronil in the sample.
10 kits were extracted from each of 3 different kit lots prepared in example 1, and the absorbance values of 10 microwell assay standard solutions (containing 1. mu.g/L of fipronil) were immediately extracted from each microplate, and the coefficient of variation was calculated. The experiment was repeated three times and the results are shown in table 2.
Table 2 concentration and coefficient of variation of standard solution tested by fipronil kit prepared in example 1
The coefficient of variation of the standard solution (containing 1. mu.g/L of fipronil) measured by the fipronil kit prepared in examples 2 to 7 is similar to that of example 1, and as can be seen from Table 2, the coefficient of variation of the standard solution (containing 1. mu.g/L of fipronil) measured by the fipronil kit prepared in example 1 is 2.1 to 2.2%.
10 kits were extracted from each of 3 different kit lots prepared in example 1 and comparative examples 2 to 3, and absorbance values of 10 microwell assay standard solutions (containing 5. mu.g/L of fipronil) were immediately extracted from each microplate, and the coefficient of variation was calculated. The experiment was repeated three times and the results are shown in table 3.
TABLE 3 Standard solution concentration and coefficient of variation for fipronil kit prepared in example 1 and comparative examples 2-3
The coefficient of variation of the standard solution (containing 5 mug/L fipronil) measured by the fipronil kit prepared in the examples 2 to 7 is similar to that of the fipronil kit prepared in the example 1, and as can be seen from Table 3, the coefficient of variation of the fipronil kit prepared in the example 1 in three batches is 0.9%, 0.9% and 0.8%, respectively, which shows that the fipronil kit prepared in the examples 1 to 7 can effectively improve the accuracy of detecting the fipronil content compared with the fipronil kit prepared in the comparative examples 2 to 3.
10 kits were extracted from each of 3 different kit lots prepared in example 1 and comparative example 1, and the absorbance values of 10 microwell assay standard solutions (containing 8. mu.g/L fipronil) were immediately extracted from each microplate, and the coefficient of variation was calculated. The experiment was repeated three times and the results are shown in table 4.
Table 4 concentration and coefficient of variation of standard solution tested by fipronil kit prepared in example 1 and comparative example 1
The coefficient of variation of the standard solution (containing 8 mug/L fipronil) measured by the fipronil kit prepared in the examples 2 to 7 is similar to that of the fipronil kit prepared in the example 1, and as can be seen from Table 4, the coefficient of variation of the fipronil kit prepared in the example 1 in three batches is 0.6%, 0.5% and 0.6%, respectively, which shows that compared with the fipronil kit prepared in the comparative example 1, the fipronil kits prepared in the examples 1 to 7 can effectively improve the accuracy of detecting the fipronil content.
Test example 4 sensitivity detection of fipronil kit
The fipronil kits prepared in examples 1-7 and comparative examples 1-3 were subjected to the minimum detection limit test, and the results are shown in table 5.
TABLE 5 detection results of minimum detection limits of fipronil kits prepared in examples 1 to 7 and comparative examples 1 to 3
As can be seen from Table 5, the fipronil kits prepared in examples 1-7 had lower minimum detection limit of 0.1. mu.g/L as compared with those prepared in comparative examples 1-3, indicating that the fipronil kits prepared in examples 1-7 had higher sensitivity.
While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.