CN105510588A - Enzyme-linked immunoassay (ELISA) kit for detecting glyphosate and detection method thereof - Google Patents
Enzyme-linked immunoassay (ELISA) kit for detecting glyphosate and detection method thereof Download PDFInfo
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- CN105510588A CN105510588A CN201410548726.9A CN201410548726A CN105510588A CN 105510588 A CN105510588 A CN 105510588A CN 201410548726 A CN201410548726 A CN 201410548726A CN 105510588 A CN105510588 A CN 105510588A
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Abstract
The invention discloses an enzyme-linked immunoassay (ELISA) kit for detecting glyphosate and a detection method thereof. The ELISA kit has the advantages of sensitivity, accuracy, rapidness, convenient operation, and strong specificity, and is suitable for detect a large amount of samples. The ELISA kit comprises a glyphosate antigen coated ELISA plate, a glyphosate standard substance, a glyphosate antibody working fluid, a glyphosate enzyme labeled second antibody working 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 for detecting glyphosate is solid phase indirect competition enzyme-linked immunosorbent assay. An extracted sample, an enzyme labeled second antibody working fluid, and an antibody working fluid are added into micro holes of a ELISA plate and incubated for a while, then a substrate liquid A and a substrate liquid B are added into a washing plate, under the effect of enzyme, a color developing agent gives off a blue color, and then a stop solution is added to convert blue into yellow. The depth of the color is in a reverse proportion relationship with the content of glyphosate in a standard substance or a sample. The method can be directly used to measure the residual amount of glyphosate in cereals.
Description
Technical field
The present invention relates to detection of veterinary drugs in food technical field, particularly detect the enzyme linked immunological kit of glyphosate in cereal.
Background technology
Along with the development of agricultural science and technology, a lot of pharmaceutical factory of China can produce efficiently, the herbicide of wide spectrum, low toxicity, and glyphosate is wherein a kind.Glyphosate is that inner sucting conduction type wide spectrum goes out natural disposition glyphosate, mainly through suppressing enolpyruvyl shikimene phosphate synthase in plant, thus suppress shikimene to the conversion of phenylalanine, tyrosine and tryptophane, the synthesis of protein is interfered and causes Plant death.
But in China's Agriculture Production, owing to only have studied the herbicide effect of glyphosate, but do not study poisoning after rescue method, so glyphosate brings certain harm also to the animal husbandry development of China and the mankind.
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 glyphosate.
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 glyphosate.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 glyphosate, comprises ELISA Plate, glyphosate standard items, glyphosate antibody working fluid, glyphosate 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 glyphosate, comprises the following steps: the preparation of the preparation of the preparation of the preparation of ELISA Plate, the preparation of glyphosate standard items, glyphosate antibody working fluid, the preparation of glyphosate 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 glyphosate, concrete steps are that glyphosate 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, glyphosate 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.
Glyphosate standard concentration is respectively 0mg/kg, 0.1mg/kg, 0.3mg/kg, 0.9mg/kg, 2.7mg/kg, 8.1mg/kg.
Described glyphosate antibody working fluid adopts glyphosate artificial antigen immune mouse to obtain monoclonal antibody, is diluted to the preparation of 1:60000 ratio with antibody diluent.
Described glyphosate 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 glyphosate, 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 glyphosate antigen is got, add standard items/sample 50 μ L/ hole in the micropore of correspondence, add glyphosate ELIAS secondary antibody working fluid, 50 μ L/ holes, then glyphosate 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 glyphosate in reference standard curve calculation sample.
Wherein, described testing sample following methods carries out pre-service:
A, grinding representative sample (make the sample of 50% can by 20 object filter screens);
Sample after b, weighing 15g grinding filtration adds 30mL water;
C, to mix in the container of sealing, concussion vortex 1min;
D, room temperature centrifugal more than 4000r/min, 10min;
E., get 10ml supernatant in centrifuge tube, add 10mL methylene chloride, mix in the container of sealing, concussion vortex 1min, room temperature centrifugal more than 4000r/min, 10min;
F, 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 glyphosate and the measuring principle of detection method thereof: antigen-specific sexual competition antibody fixing in the glyphosate in sample and ELISA Plate, add ELIAS secondary antibody, with antibody response, by enzymatic chromogenic reagent, carry out the content of glyphosate in judgement sample according to the depth of colour developing.If the glyphosate 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 glyphosate protein conjugate:
Succinic anhydride method is adopted to obtain being with the glyphosate 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 glyphosate 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 glyphosate envelope antigen:
Glyphosate 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, glyphosate 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.
Glyphosate standard items compound concentration is respectively 0mg/kg, 0.1mg/kg, 0.3mg/kg, 0.9mg/kg, 2.7mg/kg, 8.1mg/kg.
The preparation of glyphosate antibody working fluid: adopt glyphosate artificial antigen immune mouse to obtain monoclonal antibody, be diluted to the preparation of 1:60000 ratio with antibody diluent.
Glyphosate 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 glyphosate:
(1) 96 ELISA Plate × 1 piece, hole;
(2) titer × 6 bottle: (1mL/ bottle) 0mg/kg, 0.1mg/kg, 0.3mg/kg, 0.9mg/kg, 2.7mg/kg, 8.1mg/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 glyphosate residual quantity in cereal sample, implemented by following steps: sample pretreatment, with kit of the present invention carry out detecting, analysis result.
(1) sample pretreatment
A, grinding representative sample (make the sample of 50% can by 20 object filter screens);
Sample after b, weighing 15g grinding filtration adds 30mL water;
C, to mix in the container of sealing, concussion vortex 1min;
D, room temperature centrifugal more than 4000r/min, 10min;
E., get 10ml supernatant in centrifuge tube, add 10mL methylene chloride, mix in the container of sealing, concussion vortex 1min, room temperature centrifugal more than 4000r/min, 10min;
F, get 1mL supernatant, the sample diluting liquid added after 1mL dilution fully vibrates mixing 30s, mixes to be measured.
(2) glyphosate residual quantity in testing sample is detected with kit of the present invention
Get the ELISA Plate being coated with glyphosate 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 glyphosate 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, glyphosate residual quantity 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 glyphosate (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 glyphosate in sample.
Claims (8)
1. detect enzyme linked immunological kit and the detection method thereof of glyphosate, comprise ELISA Plate, glyphosate standard items, glyphosate antibody working fluid, glyphosate 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 glyphosate 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 glyphosate standard items, glyphosate antibody working fluid, the preparation of glyphosate 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 glyphosate according to claim 2 and detection method thereof, it is characterized in that: described ELISA Plate preparation method is for obtain glyphosate envelope antigen by glyphosate 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 glyphosate according to claim 2 and detection method thereof, is characterized in that: the concentration of glyphosate standard items is respectively 0mg/kg, 0.1mg/kg, 0.3mg/kg, 0.9mg/kg, 2.7mg/kg, 8.1mg/kg.
5. the enzyme linked immunological kit of detection glyphosate according to claim 2 and detection method thereof, it is characterized in that: described glyphosate antibody working fluid adopts glyphosate 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 glyphosate according to claim 2, it is characterized in that: described glyphosate 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 glyphosate 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 glyphosate antigen, add standard items/sample respectively according to the order of sequence, glyphosate ELIAS secondary antibody working fluid, the each 50 μ L/ holes of glyphosate 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 glyphosate in reference standard curve calculation sample.
8. method according to claim 7, wherein, described testing sample following methods carries out pre-service:
A, grinding representative sample (make the sample of 50% can by 20 object filter screens);
Sample after b, weighing 15g grinding filtration adds 30mL water;
C, to mix in the container of sealing, concussion vortex 1min;
D, room temperature centrifugal more than 4000r/min, 10min;
E., get 10ml supernatant in centrifuge tube, add 10mL methylene chloride, mix in the container of sealing, concussion vortex 1min, room temperature centrifugal more than 4000r/min, 10min;
F, 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 (2)
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| CN107478828A (en) * | 2017-09-25 | 2017-12-15 | 河南科技大学 | The kit of quick detection glyphosate residual and detection method and the application of glyphosate residual |
| 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 |
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