CN105510589A - Enzyme linked immunosorbent assay (ELISA) kit for detecting carbendazim and detection method thereof - Google Patents
Enzyme linked immunosorbent assay (ELISA) kit for detecting carbendazim and detection method thereof Download PDFInfo
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Abstract
The invention discloses an enzyme linked immunosorbent assay (ELISA) kit for detecting carbendazim and a detection method thereof. The ELISA kit and detection method thereof have the advantages of sensitivity, accuracy, rapidness, convenient operation, and strong specificity, and can be used to detect a large amount of samples. The kit comprises an ELISA plate with an embedded carbendazim antigen, a carbendazim standard, a carbendazim antibody work fluid, a carbendazim enzyme marked second antibody work fluid, a substrate liquid (A), a substrate liquid (B), a stopping solution, a concentrated diluent, and a concentrated washing liquid. The principle of the ELISA kit is solid indirect competitive ELISA reactions. An extracted sample, an enzyme marked second antibody work fluid, and an antibody work fluid are added into the corresponding micro holes of an ELISA plate, after an incubation period, adding a substrate liquid (A) and a substrate liquid (B) into a washing plate, under the action of enzyme, a color developing agent turns into a blue color, then a stopping solution is added, and the color changes from blue to yellow. The depth of the color and the content of carbendazim in a standard or a sample are in an inverse proportion relationship. The provided method can be directly used to detect the residual amount of carbendazim in vegetable.
Description
Technical field
The present invention relates to detection of veterinary drugs in food technical field, particularly detect the enzyme linked immunological kit of carbendazim in vegetables.
Background technology
Carbendazim (Carbendazin), chemical name 2-(methoxyformamido) benzimidazole [2-(methoxy-carbanmoyl)-benzimidaxole], be a kind of high-efficiency low-toxicity germifuge, can be used for preventing and treating plurality of plant diseases.Carbendazim is a kind of agricultural chemicals conventional in vegetable growing process, and China's victual export volume is very large, and residues of pesticides are very important restriction indexs.Therefore regularly carbendazim is remained to the necessary means detecting the monitoring becoming many countries.European Union specifies that the maximum residue limit(MRL) of carbendazim in vegetables (MRL) is 0.1mg/kg.
Enzyme linked immunosorbent assay is a kind of accurate, reliable, quick, special detection method, is suitable for the rapid screening of gross sample, has been widely used in food safety detection industry in recent years.The present invention is intended to set up a kind of enzyme linked immunological kit and the detection method thereof that detect carbendazim.
Summary of the invention
In order to overcome chromatographic deficiency, the invention provides a kind of enzyme linked immunological kit and the detection method thereof that detect carbendazim.The method is sensitive, accurate, quick, easy and simple to handle, high specificity, is applicable to the quick detection of gross sample.
The present invention detects the enzyme linked immunological kit of carbendazim, comprises ELISA Plate, carbendazim standard product, carbendazim antibody working fluid, carbendazim ELIAS secondary antibody working fluid, substrate solution A, substrate solution B, stop buffer, concentration and dilution liquid and concentrated cleaning solution.
The present invention detects the preparation of the enzyme linked immunological kit of carbendazim, comprises the following steps: the preparation of the preparation of the preparation of the preparation of ELISA Plate, the preparation of carbendazim standard product, carbendazim antibody working fluid, the preparation of carbendazim ELIAS secondary antibody working fluid, substrate solution A, the preparation of substrate solution B, stop buffer, the preparation of concentration and dilution liquid and the preparation of concentrated cleaning solution.
It is further characterized in that: described ELISA Plate is via the antigen coated preparation of carbendazim, concrete steps are that carbendazim haptens and the pure albumen of carrier proteins Bovine (BSA) coupling are obtained envelope antigen, with carbonate (CBS) damping fluid of 0.05mol/LpH9.6 as coating buffer, carbendazim envelope antigen is diluted to 1:40000 ratio, 100 μ L/ holes, 37 DEG C of lucifuges hatch 2h, take out ELISA Plate and get rid of liquid in plate, add diluted concentrated cleaning solution 300 μ L/ hole, wash plate 2 times, 30s/ time, then 0.5% bovine serum albumin(BSA) (BSA) confining liquid is added, 150 μ L/ holes, place 1.5h for 37 DEG C, get rid of confining liquid directly to pat dry, ELISA Plate after patting dry is placed (25 DEG C) between constant temperature and is dried, inspect by random samples qualified after ELISA Plate is vacuum-sealed in 4 DEG C of conditions under preserve.
Carbendazim standard product concentration is respectively 0mg/kg, 0.05mg/kg, 0.15mg/kg, 0.45mg/kg, 1.35mg/kg, 4.05mg/kg.
Described carbendazim antibody working fluid adopts carbendazim artificial antigen immune mouse to obtain monoclonal antibody, is diluted to the preparation of 1:60000 ratio with antibody diluent.
Described carbendazim ELIAS secondary antibody working fluid adds diluted by ELIAS secondary antibody and becomes 1:2000 ratio, described substrate solution A is the citrate-phosphate disodium hydrogen buffer solution of the carbamide peroxide containing 0.5mmol/L, described substrate solution B is the ethanolic solution of tetramethyl biphenyl diamines, described stop buffer is the sulfuric acid of 2mol/L, described concentration and dilution liquid is 10 times of concentration and dilution liquid, for the PBS of 0.1mol/L, between pH value range 7.0-7.5, described concentrated cleaning solution is 10 times of concentrated cleaning solutions, for containing 0.5% Tween-20, the PBST of 0.1mol/L, between pH value range 7.0-7.5.
Detect enzyme linked immunological kit and the detection method thereof of carbendazim, based on the indirect competitive enzyme-linked immunosorbent reaction principle of antigen-antibody, the method comprises the following steps:
(1) pre-service testing sample is fluid sample by sample preparation to be tested, or uses Solvent Extract methods testing sample, and is redissolved in sample diluting liquid;
(2) required reagent is taken out from cold storage environment, be placed in room temperature (20 ~ 25 DEG C) balance more than 30min, notice that often kind of liquid reagent must shake up before using;
(3) ELISA Plate being coated with carbendazim antigen is got, add standard items/sample 50 μ L/ hole in the micropore of correspondence, add carbendazim ELIAS secondary antibody working fluid, 50 μ L/ holes, then carbendazim antibody working fluid is added, 50 μ L/ holes, mixing of vibrating gently, reacts 30min with in the rearmounted room temperature of cover plate membrane cover plate 25 DEG C of light protected environment;
(4) carefully open cover plate film, liquid in hole is dried, with wash operating solution 260 μ L/ hole, fully washs 4 ~ 5 times, every minor tick 10s, pat dry (bubble be not eliminated after patting dry can be poked with original rifle head) with thieving paper;
(5) add substrate solution A50 μ L/ hole, substrate solution B50 μ L/ hole, mixing of vibrating gently, react 15 ~ 20min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate;
(6) add stop buffer 50 μ L/ hole, mixing of vibrating gently, setting microplate reader detects in 450nm place or dual wavelength 450/630nm, measures every hole absorbance (please running through data in 5min);
(7) with the logarithm of standard concentration (ppb) for horizontal ordinate, standard items percentage absorbance is ordinate, drawing standard curve, the content of carbendazim in reference standard curve calculation sample.
Wherein, described testing sample following methods carries out pre-service:
A, get 20g sample, be placed in 250mL triangular flask through tissue mashing homogenate;
B, add 40mL acetone, ultrasound wave extracts 30min, adds 16g anhydrous sodium sulfate, concussion vortex 1min;
C, room temperature centrifugal more than 4000r/min, 10min;
D., get 5ml supernatant in centrifuge tube, add 5mL methylene chloride, mix in the container of sealing, concussion vortex 1min, room temperature centrifugal more than 4000r/min, 10min;
E, get 1mL supernatant, the sample diluting liquid added after 1mL dilution fully vibrates mixing 30s, mixes to be measured.
The present invention detects the enzyme linked immunological kit of carbendazim and the measuring principle of detection method thereof: antigen-specific sexual competition antibody fixing in the carbendazim in sample and ELISA Plate, add ELIAS secondary antibody, with antibody response, by enzymatic chromogenic reagent, carry out the content of carbendazim in judgement sample according to the depth of colour developing.If the carbendazim content in sample is few, colour developing is dark; Otherwise, then develop the color shallow.Kit test method of the present invention is easy and simple to handle, detects sensitive, accurate, quick, is applicable to the quick detection of batch samples.
Embodiment
The preparation of carbendazim protein conjugate:
Succinic anhydride method is adopted to obtain being with the carbendazim hapten derivant of carboxyl, get 0.05mmol and the carrier protein BSA combination by 10:1 afterwards than being blended in the carbonate buffer solution (CBS) of 0.05mol/LpH9.6, then 0.15mmol carbodiimide is added, room temperature reaction 24h is put in stirring, finally dialyse two days in the PBS damping fluid of 0.2mol/LpH7.6, remove unreacted haptens, the protein conjugate solution obtained is saved backup in-20 DEG C.
The preparation of carbendazim antibody:
Select the purebred BALA/C mouse of healthy adult, get the immunizing antigen 50 μ g prepared with protein molecule to mix with equivalent complete Freund's adjuvant and adopt lumbar injection to carry out initial immunity, adding equivalent incomplete Freund's adjuvant every 3 weeks with same dose immunizing antigen afterwards adopts lumbar injection to carry out secondary, three immunity, after after each immune 6 days, tail vein blood measures antiserum titre to certain titre, do not add adjuvant with same dose and carry out final immunization, get spleen after 3 days and prepare Spleen cell suspensions and myeloma cell carries out Fusion of Cells, filter out required hybridoma cell line and carry out cloning, the hybridoma being in exponential phase is selected to carry out frozen, prepare for ascites, first lumbar injection 0.5ml whiteruss is in BALB/C mouse sensitization, pneumoretroperitoneum injection 1 × 10 in 2 weeks
6individual hybridoma, ascites can be produced after inoculating cell 7-10 days, ascites is extracted with syringe when ascites is many as far as possible, repeatedly collect for several times, the centrifugal 15min of 4000rpm, collect supernatant, adopt caprylic acid-ammonium purifying ascites to carry out purifying to monoclonal antibody, freeze drying saves backup in-20 DEG C after obtaining freeze-dried powder.
Preparation is coated with the ELISA Plate of carbendazim envelope antigen:
Carbendazim haptens and the pure albumen of carrier proteins Bovine (BSA) coupling obtain by envelope antigen, with carbonate (CBS) damping fluid of 0.05mol/LpH9.6 as coating buffer, carbendazim antigen diluent is become 1:40000 ratio, 100 μ L/ holes, place 2h for 37 DEG C, take out ELISA Plate and get rid of liquid in plate, with the concentrated cleaning solution 300 μ L/ hole after dilution, wash plate 2 times, 30s/ time, then add 0.5% bovine serum albumin(BSA) (BSA) to close, 150 μ L/ holes, place 1.5h for 37 DEG C, discard confining liquid, ELISA Plate after patting dry is placed (25 DEG C) between constant temperature and is dried, inspect by random samples qualified after by rearmounted for ELISA Plate vacuum seal 4 DEG C preserve.
Carbendazim standard product compound concentration is respectively 0mg/kg, 0.05mg/kg, 0.15mg/kg, 0.45mg/kg, 1.35mg/kg, 4.05mg/kg.
The preparation of carbendazim antibody working fluid: adopt carbendazim artificial antigen immune mouse to obtain monoclonal antibody, be diluted to the preparation of 1:60000 ratio with antibody diluent.
Carbendazim ELIAS secondary antibody working fluid adds diluted by ELIAS secondary antibody and becomes 1:2000 ratio, substrate solution A is the citrate-phosphate disodium hydrogen buffer solution of the carbamide peroxide containing 0.5mmol/L, substrate solution B is the ethanolic solution of tetramethyl biphenyl diamines, stop buffer is the sulfuric acid of 2mol/L, concentration and dilution liquid is 10 times of concentration and dilution liquid, for the PBS of 0.1mol/L, between pH value range 7.0-7.5, concentrated cleaning solution is 10 times of concentrated cleaning solutions, for containing 0.5% Tween-20, the PBST of 0.01mol/L, between pH value range 7.0-7.5.
Based on the reagent of above-mentioned preparation, the present invention comprises following material for the enzyme linked immunological kit detecting carbendazim:
(1) 96 ELISA Plate × 1 piece, hole;
(2) titer × 6 bottle: (1mL/ bottle) 0mg/kg, 0.05mg/kg, 0.15mg/kg, 0.45mg/kg, 1.35mg/kg, 4.05mg/kg;
(3) antibody working fluid 7mL;
(4) ELIAS secondary antibody working fluid 7mL;
(5) substrate solution A7mL;
(6) substrate solution B7mL;
(7) stop buffer 7mL;
(8) 10 × concentration and dilution liquid 40mL;
(9) 10 × concentrated cleaning solution 40mL;
When kit of the present invention is for detecting Determination of carbendazim residue in chicken sample, implemented by following steps: sample pretreatment, with kit of the present invention carry out detecting, analysis result.
(1) sample pretreatment
A, get 20g sample, be placed in 250mL triangular flask through tissue mashing homogenate;
B, add 40mL acetone, ultrasound wave extracts 30min, adds 16g anhydrous sodium sulfate, concussion vortex 1min;
C, room temperature centrifugal more than 4000r/min, 10min;
D., get 5ml supernatant in centrifuge tube, add 5mL methylene chloride, mix in the container of sealing, concussion vortex 1min, room temperature centrifugal more than 4000r/min, 10min;
E, get 1mL supernatant, the sample diluting liquid added after 1mL dilution fully vibrates mixing 30s, mixes to be measured.
(2) Determination of carbendazim residue in testing sample is detected with kit of the present invention
Get the ELISA Plate being coated with carbendazim antigen, add standard items/sample 50 μ L/ hole in the micropore of correspondence; Add ELIAS secondary antibody working fluid, 50 μ L/ holes, then add carbendazim antibody working fluid, 50 μ L/ holes, mixing of vibrating gently, react 30min with in the rearmounted room temperature of cover plate membrane cover plate 25 DEG C of light protected environment; Carefully open cover plate film, dried by liquid in hole, with wash operating solution 300 μ L/ hole, fully washing 4 times, soaks 15-30s, pats dry with thieving paper; Add substrate solution A50 μ L/ hole, substrate solution B50 μ L/ hole, mixing of vibrating gently, react 15min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate; Add stop buffer 50 μ L/ hole, mixing of vibrating gently, setting microplate reader detects in 450nm place or dual wavelength 450/630nm, measures every hole absorbance (please running through data in 5min); The absorbance size of contrast testing sample and standard items, Determination of carbendazim residue in quantitative test testing sample.
(3) analysis result
The calculating of percentage absorbance, the percentage absorbance of standard items or sample equals the absorbance of mean value (diplopore) divided by first standard (0 standard) of the absorbance of standard items or sample, then is multiplied by 100%, namely
Percentage absorbance (%)=B/B
0× 100%
The wherein mean absorbance values of B-standard solution or sample solution, B
0the mean absorbance values of-0ppb standard solution.
With the logarithm of the standard concentration of carbendazim (ppb) for horizontal ordinate, standard items percentage absorbance is ordinate, and drawing standard curve, obtains straight-line equation.Substituted in typical curve by the percentage absorbance of sample, read the concentration corresponding to sample from typical curve, the extension rate being multiplied by its correspondence is the actual concentrations of carbendazim in sample.
Claims (8)
1. detect enzyme linked immunological kit and the detection method thereof of carbendazim, comprise ELISA Plate, carbendazim standard product, carbendazim antibody working fluid, carbendazim ELIAS secondary antibody working fluid, substrate solution A, substrate solution B, stop buffer, concentration and dilution liquid and concentrated cleaning solution.
2. detect the enzyme linked immunological kit of carbendazim and detection method thereof, comprise the following steps: the preparation of the preparation of the preparation of the preparation of ELISA Plate, the preparation of carbendazim standard product, carbendazim antibody working fluid, the preparation of carbendazim ELIAS secondary antibody working fluid, substrate solution A, the preparation of substrate solution B, stop buffer, the preparation of concentration and dilution liquid and the preparation of concentrated cleaning solution.
3. the enzyme linked immunological kit of detection carbendazim according to claim 2 and detection method thereof, it is characterized in that: described ELISA Plate preparation method is for obtain carbendazim envelope antigen by carbendazim haptens and the pure albumen of carrier proteins Bovine (BSA) coupling, with carbonate (CBS) damping fluid of 0.05mol/LpH9.6 as coating buffer, envelope antigen is diluted to 1:40000 ratio, 100 μ L/ holes, hatch 2h for 37 DEG C, take out ELISA Plate and get rid of liquid in plate, add the concentrated cleaning solution after dilution 300 μ L/ hole, wash plate 2 times, 30s/ time, then add 0.5% bovine serum albumin(BSA) (BSA) to close, 150 μ L/ holes, place 1.5h for 37 DEG C, discard confining liquid directly to pat dry, ELISA Plate after patting dry is placed (25 DEG C) between constant temperature and is dried, inspect by random samples qualified after ELISA Plate is vacuum-sealed in 4 DEG C of conditions under preserve.
4. the enzyme linked immunological kit of detection carbendazim according to claim 2 and detection method thereof, is characterized in that: the concentration of carbendazim standard product is respectively 0mg/kg, 0.05mg/kg, 0.15mg/kg, 0.45mg/kg, 1.35mg/kg, 4.05mg/kg.
5. the enzyme linked immunological kit of detection carbendazim according to claim 2 and detection method thereof, it is characterized in that: described carbendazim antibody working fluid adopts carbendazim artificial antigen immune mouse to obtain monoclonal antibody, is diluted to the preparation of 1:60000 ratio with antibody diluent.
6. a kind of enzyme linked immunological kit and detection method thereof detecting carbendazim according to claim 2, it is characterized in that: described carbendazim ELIAS secondary antibody working fluid adds diluted by ELIAS secondary antibody and becomes 1:2000 ratio, described substrate solution A is the citrate-phosphate disodium hydrogen buffer solution of the carbamide peroxide containing 0.5mmol/L, described substrate solution B is the ethanolic solution of tetramethyl biphenyl diamines, described stop buffer is the sulfuric acid of 2mol/L, described concentration and dilution liquid is 10 times of concentration and dilution liquid, it is the PBS of 0.1mol/L, between pH value range 7.0-7.5, described concentrated cleaning solution is 10 times of concentrated cleaning solutions, it is for containing 0.5% Tween-20, the PBST of 0.01mol/L, between pH value range 7.0-7.5.
7. the enzyme linked immunological kit of detection carbendazim according to claim 2 and detection method thereof, based on the indirect competitive enzyme-linked immunosorbent reaction principle of antigen-antibody, the method is characterized in that: pre-service testing sample, get the ELISA Plate being coated with carbendazim antigen, add standard items/sample respectively according to the order of sequence, carbendazim ELIAS secondary antibody working fluid, the each 50 μ L/ holes of carbendazim antibody working fluid are in the micropore of correspondence, to vibrate gently mixing, 30min is reacted with in the rearmounted room temperature of cover plate membrane cover plate 25 DEG C of light protected environment, liquid in hole is dried, 4 ~ 5 times are fully washed with wash operating solution, every minor tick 10s, substrate solution A50 μ L/ hole is added after patting dry, substrate solution B50 μ L/ hole, to vibrate gently mixing, 15 ~ 20min is reacted with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate, add stop buffer 50 μ L/ hole, to vibrate gently mixing, setting microplate reader detects in 450nm place or dual wavelength 450/630nm, measure every hole absorbance (please running through data in 5min), with the logarithm of standard concentration (ppb) for horizontal ordinate, standard items percentage absorbance is ordinate, drawing standard curve, the content of carbendazim in reference standard curve calculation sample.
8. method according to claim 7, wherein, described testing sample following methods carries out pre-service: a, get 20g sample, is placed in 250mL triangular flask through tissue mashing homogenate;
B, add 40mL acetone, ultrasound wave extracts 30min, adds 16g anhydrous sodium sulfate, concussion vortex 1min;
C, room temperature centrifugal more than 4000r/min, 10min;
D., get 5ml supernatant in centrifuge tube, add 5mL methylene chloride, mix in the container of sealing, concussion vortex 1min, room temperature centrifugal more than 4000r/min, 10min;
E, get 1mL supernatant, the sample diluting liquid added after 1mL dilution fully vibrates mixing 30s, mixes to be measured.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106124766A (en) * | 2016-07-05 | 2016-11-16 | 天津师范大学 | Use the method for carbendazim content in carbendazim detection of specific antibody edible fungi |
| CN108226476A (en) * | 2016-12-15 | 2018-06-29 | 镇江亿特生物科技发展有限公司 | A kind of enzyme linked immunological kit and its detection method for detecting Madumycin |
| CN108226475A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | A kind of enzyme linked immunological kit and its detection method for detecting sarafloxacin |
| CN108614109A (en) * | 2016-12-12 | 2018-10-02 | 丹阳亿太生物科技发展有限公司 | A kind of enzyme linked immunological kit and its detection method of detection sodium sulfocyanate |
| CN109725143A (en) * | 2018-12-27 | 2019-05-07 | 国家食品安全风险评估中心 | Aflatoxin enzyme linked immunological kit and detection method |
| CN109813892A (en) * | 2017-11-18 | 2019-05-28 | 丹阳亿太生物科技发展有限公司 | Anarexol rapid time resolved fluorometric immunochromatographiassay assay quantitative detection test paper |
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| CN103808921A (en) * | 2012-11-06 | 2014-05-21 | 江苏维赛科技生物发展有限公司 | Enzyme-linked immunosorbent assay kit for detecting residual zilpaterol and use method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106124766A (en) * | 2016-07-05 | 2016-11-16 | 天津师范大学 | Use the method for carbendazim content in carbendazim detection of specific antibody edible fungi |
| CN108614109A (en) * | 2016-12-12 | 2018-10-02 | 丹阳亿太生物科技发展有限公司 | A kind of enzyme linked immunological kit and its detection method of detection sodium sulfocyanate |
| CN108226476A (en) * | 2016-12-15 | 2018-06-29 | 镇江亿特生物科技发展有限公司 | A kind of enzyme linked immunological kit and its detection method for detecting Madumycin |
| CN108226475A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | A kind of enzyme linked immunological kit and its detection method for detecting sarafloxacin |
| CN109813892A (en) * | 2017-11-18 | 2019-05-28 | 丹阳亿太生物科技发展有限公司 | Anarexol rapid time resolved fluorometric immunochromatographiassay assay quantitative detection test paper |
| CN109725143A (en) * | 2018-12-27 | 2019-05-07 | 国家食品安全风险评估中心 | Aflatoxin enzyme linked immunological kit and detection method |
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