CN1176376C - Fipronil immune detecting method - Google Patents
Fipronil immune detecting method Download PDFInfo
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- CN1176376C CN1176376C CNB021485348A CN02148534A CN1176376C CN 1176376 C CN1176376 C CN 1176376C CN B021485348 A CNB021485348 A CN B021485348A CN 02148534 A CN02148534 A CN 02148534A CN 1176376 C CN1176376 C CN 1176376C
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Abstract
The present invention relates to a method for detecting fipronil immunity, which is characterized in that a coating antigen dilute solution is processed through incubation; a confining solution is added for incubation; a preprocessed sample is mixed with an enzyme labelled antibody with equal volume for incubation; an enzyme labelled board is taken out, a substrate solution is added for incubation; a sulfuric acid solution is added for stopping the reaction; the OD value is measured under the wave length of 450MM, and the fipronil content in the sample is set through detection and determination. The present invention has the advantages that the method can be used for environment monitoring, food, agricultural chemical toxicology or pharmacology, or can be used in research fields; because the method can be directly used for detecting samples, the present invention has the advantages of simple operation, short detection process and strong fluidity; the present invention is especially suitable for fast detecting the pollution conditions in the field of aquatic products, vegetables and food by base departments and market monitoring departments.
Description
Technical field:
The present invention relates to a kind of fluorine worm nitrile immunologic detection method.
Background technology:
Fluorine worm nitrile is a kind of novel benzene Insecticidal pyrazolines, in China good prospects for application is arranged, but studies show that it is a kind of chronic neurotoxicity, when the per os dosage of 300ppm, can cause the Mouse thyroid cancer, be decided to be C class carcinogen, especially its metabolism in environment is slow, there are two kinds of metabolins higher more than 10 times to mammiferous toxicity than parent agricultural chemicals, and in biosome fat inrichment is arranged, especially this agricultural chemicals is to aquatic shellfish severe toxicity, to the honeybee strong toxicity, be the bigger agricultural chemicals of a kind of ecologic environment toxic side effect, necessary to the supervision after this agricultural chemicals reinforcement application.
He Yibing discloses a kind of gas chromatography in 2000 in " pesticide science management " on the 3rd phase, instrument and equipment requires than higher, also need before the test sample is done necessary extraction and purification, not only required time is long, sample extraction method complexity, and the parent agricultural chemicals is different with the metabolin testing conditions, needs many residue detection, and is very inconvenient.
Zhu Guonian etc. 2000 have reported degradation pathway research in the similar fluorine worm nitrile simulation rice field ecology the in " Pesticide Science journal " the 2nd phase, also need in its analytical approach sample is extracted and purifies, the circumstances in which people get things ready for a trip of going forward side by side spectrum detects, and adopts this method that fluorine worm nitrile and metabolin are respectively the concentration limit of surveying water, soil and rice plant: 0.028-0.37mg/L, 0.001-0.008mg/kg and 0.001-0.009mg/kg.
External also have the scholar that the detection technique of fluorine worm nitrile is also launched research, discloses some detection methods about gas chromatography or chromatogram-mass spectrometry, and concentration limit is 0.005mg/kg.Average recovery rate is 85.4-95.8%, and the coefficient of variation is 2.3-12.0%, and testing result generally needs two days, the testing process complexity.
Summary of the invention:
The objective of the invention is to: the practical problems at existing in the detection to the immunity of fluorine worm nitrile at present provides a kind of new fluorine worm nitrile immunologic detection method.
The object of the present invention is achieved like this: the fine immunologic detection method of a kind of fluorine worm is characterized in that:
A, every hole adds the 100ul coating buffer in ELISA Plate, and 4 ℃ are spent the night or 37 ℃ of incubations 2 hours;
B, take out ELISA Plate, discard coating buffer,, dry, and in every hole, add the 200ul confining liquid, 37 ℃ of incubations 1 hour with the phosphate buffer physiological saline washing of Tween-20 three times;
C, taking-up ELISA Plate, phosphate buffer physiological saline with Tween-20 washs three times, dry, one to six leu preface with A, B, the C of ELISA Plate in capable is mixed pretreated sample with equal-volume enzyme labelled antibody mixed liquor, every hole 100ul, the every hole of the 7th row with A, B, the C of ELISA Plate in capable adds the phosphate buffer physiological saline of 100ul, and the every hole of the 8th row with A, B, the C of ELISA Plate in capable adds the antibody of 50ul and the phosphate buffer physiological saline of 50ul, 37 ℃ of incubations 2 hours;
D, take out ELISA Plate,, dry, add the substrate solution 100ul that now joins in every hole, 37 ℃ of incubations 15 minutes with the phosphate buffer physiological saline washing of Tween-20 three times;
The 2M sulfuric acid solution cessation reaction that adds 50ul in e, the every hole;
After f, the cessation reaction, blot ELISA Plate bottom with thieving paper immediately, ELISA Plate is placed on the microplate reader, under the 450nm wavelength, the 8th each hole of row with A, B, the C of ELISA Plate in capable is blank zeroing, measures the OD value in each hole;
G, set the mean value zeroing in A, B, the C of ELISA Plate the 8th each hole of row in capable, the mean value in the 7th each hole of row with A, B, the C of ELISA Plate in capable is B
0, measure mean value of A, B, the C of ELISA Plate one to six each row of row in capable simultaneously, determine the fluorine worm nitrile content in the sample.The particular location of the sample in each hole of ELISA Plate, standard items (standard specimen), contrast, blank well is referring to table one.
Table one:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | Contrast | Blank | ||||
| B | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | Contrast | Blank | ||||
| C | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | Contrast | Blank | ||||
| D | Standard specimen 1 | Standard specimen 2 | Standard specimen 3 | Standard specimen 4 | Standard specimen 5 | Standard specimen 6 | ||||||
| E | Standard specimen 1 | Standard specimen 2 | Standard specimen 3 | Standard specimen 4 | Standard specimen 5 | Standard specimen 6 | ||||||
| F | Standard specimen 1 | Standard specimen 2 | Standard specimen 3 | Standard specimen 4 | Standard specimen 5 | Standard specimen 6 | ||||||
| G | ||||||||||||
| H |
The invention has the advantages that: this method can be in application or research field uses such as environmental monitoring, food security, agricultural chemicals toxicity or pharmacology, because this method can directly detect sample, have simple to operate, test process is short, mobile strong, be fit to basic unit and market monitorings department the pollution condition in aquatic products, the vegetable food field is carried out fast detecting.
Embodiment:
The present invention includes two types of solid phase antigen indirect ELISA and direct ELISA.The measuring principle of indirect ELISA is: sharp strength thing haptens one albumen composition is combined with solid phase carrier (polystyrene microtiter plates): eccysis is adsorption antigen not, add fluorine worm nitrile to be measured (standard specimen or sample) and antibody mixed liquor, behind the competition incubation, form antigen-antibody complex at surface of solid phase carriers; The antibody that eccysis does not combine with solid phase antigen, add zymolyte after adding enzyme connection staphylococcal protein A then, under the catalytic action of enzyme, substrate generation degradation reaction, produce coloured product, detect the degradation amount (how many OD value height after with colour developing are represented) of zymolyte by microplate reader, enzyme amount and added fluorine worm nitrile content are negative correlation, according to the corresponding OD value of standard specimen concentration and gained, and behind mathematical conversion, can get a typical curve.For unknown sample, need only the OD value behind the assaying reaction under similarity condition, just can learn the content of fluorine worm nitrile with interpolation method according to typical curve.Directly ELSA be with the direct mark of enzyme to antibody, make solid phase antigen and free antigen directly and the enzyme labelled antibody competitive reaction, make minute shorten greatly.
Embodiment 1: the preparation method of envelope antigen.
Earlier the itrile group on the fluorine worm nitrile pyrazoles ring is transformed into carboxyl and utilizes active ester method that the amino of carboxyl and ovalbumin is carried out coupling, obtain fluorine worm nitrile-ovalbumin conjugate, obtain coating buffer.
Embodiment 2: solid phase indirect detection step.
1, bag quilt: every hole adds the 100ul coating buffer in ELISA Plate, and 4 ℃ are spent the night or 37 ℃ of incubations 2 hours;
2, sealing: take out ELISA Plate, discard coating buffer,, dry, and in every hole, add the 200ul confining liquid, 37 ℃ of incubations 1 hour with the phosphate buffer physiological saline washing of Tween-20 three times;
3, application of sample: take out ELISA Plate, phosphate buffer physiological saline with Tween-20 washs three times, dry, sample is removed suspending sundries after, mix with equal-volume antibody mixed liquor, press and inject in the ELISA Plate shown in the table one, every hole 100ul, simultaneously, the every hole of the 7th row with A, B, the C of ELISA Plate in capable adds the phosphate buffer physiological saline of 100ul, the every hole of the 8th row with A, B, the C of ELISA Plate in capable adds the antibody of 50ul and the phosphate buffer physiological saline of 50ul, 37 ℃ of incubations 2 hours;
4, enzyme-added staphylococcal protein A: take out ELISA Plate, the phosphate buffer physiological saline washing with Tween-20 dries, and adds the enzyme-added staphylococcal protein A dilution of 100ul in every hole, 37 ℃ of incubations 45 minutes;
5, add substrate solution: take out ELISA Plate, use the phosphate buffer physiological saline of Tween-20 to wash three times, dry.In every hole, add the substrate solution now join, 37 ℃ of incubations 15 minutes.
6, cessation reaction: add 50u12M sulfuric acid solution cessation reaction in every hole.
7, reading adds and blots paper with thieving paper immediately behind the sulfuric acid and blot the ELISA Plate bottom, and ELISA Plate is placed on the microplate reader, and under the 450nm wavelength, the every hole of the 8th row with A, B, the C of ELISA Plate in capable is blank zeroing, measures the OD value in each hole.
8, the mean value of setting the OD value in A, B, the C of the control wells ELISA Plate every hole of the 7th row in capable is B
0, A, the B of one to six row, the OD value mean value in C hole are B
n(n=1,2,3,4,5,6), the OD value mean value of the repeating hole of same sample are Byp (samples that the yp representative is different).Every group of data all are as the criterion with the mean value of three repetitions.Common logarithm log[c with fluorine worm nitrile standard specimen concentration] be horizontal ordinate (X), corresponding inhibiting rate I=100%[(B
0-Byp)/B
0] be ordinate (Y), can get a y=a+bx straight line.Byp through being converted into Yyp, in the substitution straight line, can be got Xyp then,, can learn fluorine worm nitrile content Cyp in the sample Xyp negate logarithm.
During enforcement, described antibody can adopt monoclonal antibody also can adopt polyclonal antibody, and described sample can adopt vegetables and fruits class or liquid matter.In the present embodiment, described antibody is monoclonal antibody, and described sample is a liquid matter.
Embodiment 3: solid phase directly detects step.
1, bag quilt: every hole adds 100ul envelope antigen dilution in ELISA Plate, and 4 ℃ are spent the night or 37 ℃ of incubations 2 hours;
2, sealing: take out ELISA Plate, discard coating buffer,, dry, and in every hole, add the 200ul confining liquid, 37 ℃ of incubations 1 hour with the phosphate buffer physiological saline washing of Tween-20 three times;
3, application of sample: take out ELISA Plate, phosphate buffer physiological saline with Tween-20 washs three times, dry, sample is extracted with 95: 5 with acetone and water, extract is purified and concentrates, sample after the processing mixes with equal-volume antibody mixed liquor, press and inject in the ELISA Plate shown in the table one, every hole 100ul, simultaneously, the every hole of the 7th row with A, B, the C of ELISA Plate in capable adds the phosphate buffer physiological saline of 100ul, and the every hole of the 8th row during the A of ELISA Plate, B, C are capable adds the antibody of 50ul and the phosphate buffer physiological saline of 50ul, 37 ℃ of incubations 2 hours.
4, add substrate solution: take out ELISA Plate, use the phosphate buffer physiological saline of Tween-20 to wash three times, dry.In every hole, add the substrate solution now join, 37 ℃ of incubations 15 minutes.
5, cessation reaction: the 2M sulfuric acid solution cessation reaction that adds 50ul in every hole.
6, reading adds and blots paper with thieving paper immediately behind the sulfuric acid and blot the ELISA Plate bottom, and ELISA Plate is placed on the microplate reader, and under the 450nm wavelength, the every hole of the 8th row with A, B, the C of ELISA Plate in capable is blank zeroing, measures the OD value in each hole.
7, the mean value of setting the OD value in A, B, the C of the control wells ELISA Plate every hole of the 7th row in capable is B
0, A, the B of one to six row, the OD value mean value in C hole are B
n(n=1,2,3,4,5,6), the OD value mean value of the repeating hole of same sample are Byp (samples that the yp representative is different).Every group of data all are as the criterion with the mean value of three repetitions.Common logarithm log[c with fluorine worm nitrile standard specimen concentration] be horizontal ordinate (X), corresponding inhibiting rate 1=100%[(B
0-Byp)/B
0] be ordinate (Y), can get a Y=a+bx straight line.Byp through being converted into Yyp, in the substitution straight line, can be got Xyp then,, can learn fluorine worm nitrile content Cyp in the sample Xyp negate logarithm.
During enforcement, described antibody can adopt polyclonal antibody or monoclonal antibody, and described sample can adopt vegetables and fruits class or liquid matter.In the present embodiment, described antibody is polyclonal antibody, and described sample is the vegetables and fruits class.
Claims (7)
1, the fine immunologic detection method of a kind of fluorine worm is characterized in that:
A, every hole adds the 100ul coating buffer in ELISA Plate, and 4 ℃ are spent the night or 37 ℃ of incubations 2 hours;
B, take out ELISA Plate, discard coating buffer,, dry, and in every hole, add the 200ul confining liquid, 37 ℃ of incubations 1 hour with the phosphate buffer physiological saline washing of Tween-20 three times;
C, taking-up ELISA Plate, phosphate buffer physiological saline with Tween-20 washs three times, dry, the sample that one to six leu preface with A, B, the C of ELISA Plate in capable will be removed suspending sundries mixes with equal-volume enzyme labelled antibody mixed liquor, every hole 100ul, the every hole of the 7th row with A, B, the C of ELISA Plate in capable adds the phosphate buffer physiological saline of 100ul, the every hole of the 8th row with A, B, the C of ELISA Plate in capable adds the antibody of 50ul and the phosphate buffer physiological saline of 50ul, 37 ℃ of incubations 2 hours;
D, take out ELISA Plate,, dry, add the substrate solution 100ul that now joins in every hole, 37 ℃ of incubations 15 minutes with the phosphate buffer physiological saline washing of Tween-20 three times;
The 2M sulfuric acid solution cessation reaction that adds 50ul in e, the every hole;
After f, the cessation reaction, blot ELISA Plate bottom with thieving paper immediately, ELISA Plate is placed on the microplate reader, under the 450nm wavelength, the 8th each hole of row with A, B, the C of ELISA Plate in capable is blank zeroing, measures the OD value in each hole;
G, set the mean value zeroing in A, B, the C of ELISA Plate the 8th each hole of row in capable, the mean value in the 7th each hole of row with A, B, the C of ELISA Plate in capable is B
0, measure mean value of A, B, the C of ELISA Plate one to six each row of row in capable simultaneously, determine the fluorine worm nitrile content in the sample.
2, the fine immunologic detection method of fluorine worm according to claim 1, it is characterized in that: remove the sample of suspending sundries and the mixed liquor of equal-volume antibody in adding among the step C, 37 ℃ of incubations are after 2 hours, take out ELISA Plate, the washing of phosphate buffer physiological saline, drying with Tween-20, add 100ul enzyme connection staphylococcal protein A dilution in every hole, 37 ℃ of incubations carry out the postorder test after 45 minutes again.
3, fluorine worm nitrile immunologic detection method according to claim 1 and 2 is characterized in that: described coating buffer is to adopt the itrile group on the fluorine worm nitrile pyrazoles ring to be transformed into carboxyl, utilizes the amino of active ester method and ovalbumin to carry out diluting after the coupling again and obtains.
4, the fine immunologic detection method of fluorine worm according to claim 1 and 2, it is characterized in that: described sample is the vegetables and fruits class, and quantitatively extract with 95: 5 with acetone and water the back, and extract is purified and concentrates, sample after the processing mixes with equal-volume antibody, detects.
5, the fine immunologic detection method of fluorine worm according to claim 1 and 2, it is characterized in that: described sample is a liquid matter, then removes suspending sundries, sample after the processing and equal-volume antibody mixed liquor detect.
6, fluorine worm nitrile immunologic detection method according to claim 4, it is characterized in that: described antibody is polyclonal antibody or monoclonal antibody.
7, fluorine worm nitrile immunologic detection method according to claim 5, it is characterized in that: described antibody is polyclonal antibody or monoclonal antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021485348A CN1176376C (en) | 2002-12-13 | 2002-12-13 | Fipronil immune detecting method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021485348A CN1176376C (en) | 2002-12-13 | 2002-12-13 | Fipronil immune detecting method |
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| CN1414390A CN1414390A (en) | 2003-04-30 |
| CN1176376C true CN1176376C (en) | 2004-11-17 |
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| CNB021485348A Expired - Fee Related CN1176376C (en) | 2002-12-13 | 2002-12-13 | Fipronil immune detecting method |
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007187650A (en) * | 2005-12-16 | 2007-07-26 | Horiba Ltd | Fipronyl measuring kit using immunoassay |
| CN100403030C (en) * | 2006-05-10 | 2008-07-16 | 北京望尔生物技术有限公司 | ELISA kit for detecting Sudan red medicines and detection method thereof |
| CN106018818A (en) * | 2016-07-01 | 2016-10-12 | 安邦（厦门）生物科技有限公司 | Time-resolved immunofluorescent quantitative detection method and kit used by time-resolved immunofluorescent quantitative detection method |
| CN107037149B (en) * | 2017-04-08 | 2019-09-20 | 中国热带农业科学院农产品加工研究所 | Fipronil and its metabolite residue amount method for measuring in a kind of egg |
| CN109813894A (en) * | 2017-11-18 | 2019-05-28 | 镇江亿特生物科技发展有限公司 | The chemiluminescence immune detection reagent kit of ethiprole in a kind of detection rice |
| CN108226489A (en) * | 2018-01-03 | 2018-06-29 | 河南省农业科学院 | A kind of colloid gold label test strip for quickly detecting micro ethiprole and preparation method thereof |
| CN108226490A (en) * | 2018-01-03 | 2018-06-29 | 河南省农业科学院 | A kind of quantum dot test strips for quickly detecting micro ethiprole and preparation method thereof |
| CN117497081B (en) * | 2023-12-29 | 2024-04-05 | 北京市农林科学院 | Logic gate construction based on porphyrin COF fipronil and application thereof |
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Correction item: Inventor Correct: Liu Xianjin|Yu Xiangyang|Yan Chunrong|Dong Jian False: Liu Xianjin|Yan Chunrong|Dong Jian|Yu Xiangyang Number: 46 Page: 508 Volume: 20 |
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Correction item: Inventor Correct: Liu Xianjin|Yu Xiangyang|Yan Chunrong|Dong Jian False: Liu Xianjin|Yan Chunrong|Dong Jian|Yu Xiangyang Number: 46 Page: The title page Volume: 20 |
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