CN106610431A - Enzyme-linked immunosorbent assay kit for detecting Benalaxyl and detection method thereof - Google Patents
Enzyme-linked immunosorbent assay kit for detecting Benalaxyl and detection method thereof Download PDFInfo
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- CN106610431A CN106610431A CN201510687767.0A CN201510687767A CN106610431A CN 106610431 A CN106610431 A CN 106610431A CN 201510687767 A CN201510687767 A CN 201510687767A CN 106610431 A CN106610431 A CN 106610431A
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses an enzyme-linked immunosorbent assay kit for detecting Benalaxyl and a detection method thereof. The kit realizes sensitive, accurate and rapid detection, can be simply operated, has strong specificity and is suitable for detection of a large number of samples. The kit comprises an enzyme-labeled plate coated with a Benalaxyl antigen, a Benalaxyl standard product, a Benalaxyl antibody working solution, a Benalaxyl enzyme-labeled secondary antibody working solution, a substrate liquid A, a substrate liquid B, a stop solution, a concentration diluent and a concentration washing solution. The principle of the enzyme-linked immunosorbent assay kit for detection of Benalaxyl is solid phase indirect competition enzyme-linked immunosorbent assay. The detection method comprises adding an extracted sample, the enzyme-labeled secondary antibody working solution and the antibody working solution into corresponding micropores of the enzyme-labeled plate, carrying out incubation for some time, washing the enzyme-labeled plate, adding the substrate liquids A and B into the enzyme-labeled plate, carrying out development under action of the enzyme so that the development agent is blue, and adding the stop solution into the sample so that the blue color becomes yellow color, wherein the depth of the color is inversely proportional with the Benalaxyl content of the standard substance or sample. The detection method can be directly used for detecting residual quantity of Benalaxyl in a detected crop.
Description
Technical field
The present invention relates to detection of veterinary drugs in food technical field, particularly detects the enzyme linked immunological kit of M 9834 in rice.
Background technology
M 9834 is the systemic fungicide for preventing and treating oomycetes diseases.For preventing and treating the downy mildew of the crops such as Fructus Vitis viniferae, Nicotiana tabacum L., melon, Semen sojae atricolor and onion, the epidemic disease on Rhizoma Solani tuber osi, Fructus Lycopersici esculenti, grass poison, ornamental plant.M 9834 can be with alone, also can be mixed with protective agent Mancozeb, folpet etc..Because M 9834 is the kind that easily causes cause of disease mattress to produce drug resistance, mixed, wheel is preferably taken to use or be re-dubbed mixed bactericide.
Enzyme linked immunosorbent assay is a kind of accurate, reliable, quick, special detection method, is suitable for the rapid screening of gross sample, and food safety detection industry is widely used in recent years.It is contemplated that setting up a kind of enzyme linked immunological kit and its detection method of detection M 9834.
The content of the invention
In order to overcome chromatographic deficiency, the present invention to provide a kind of enzyme linked immunological kit and its detection method of detection M 9834.The method is sensitive, accurate, quick, easy to operate, high specificity, it is adaptable to the quick detection of gross sample.
The enzyme linked immunological kit of present invention detection M 9834, including ELISA Plate, M 9834 standard substance, M 9834 antibody working solution, M 9834 ELIAS secondary antibody working solution, substrate solution A, substrate solution B, terminate liquid, concentration and dilution liquid and concentrated cleaning solution.
The preparation of the enzyme linked immunological kit of present invention detection M 9834, comprises the following steps：Preparation, the preparation of M 9834 standard substance, the preparation of M 9834 antibody working solution, the preparation of M 9834 ELIAS secondary antibody working solution, the preparation of substrate solution A, the preparation of substrate solution B, the preparation of terminate liquid, the preparation of concentration and dilution liquid and the preparation of concentrated cleaning solution of ELISA Plate.
It is further characterized by：Described ELISA Plate is prepared via M 9834 antigen coat, comprise the concrete steps that to be coupled M 9834 hapten and the pure albumen of carrier proteins Bovine (BSA) and obtain envelope antigen, with carbonate (CBS) buffer of 0.05 mol/L pH 9.6 as coating buffer, M 9834 envelope antigen is diluted to into 1：40000 ratios, 100 μ L/ holes, 37 DEG C of lucifuges are incubated 2 h, take out ELISA Plate and get rid of liquid in plate, add the μ L/ holes of diluted concentrated cleaning solution 300, board-washing 2 times, 30 s/ time, it is subsequently adding 0.5% bovine serum albumin (BSA) confining liquid, 150 μ L/ holes, 37 DEG C of 1.5 h of placement, get rid of confining liquid and directly pat dry, ELISA Plate after patting dry is placed (25 DEG C) between constant temperature and is dried, and is preserved under the conditions of ELISA Plate is vacuum-sealed in into 4 DEG C after sampling observation is qualified.
M 9834 standard concentration is respectively 0 mg/kg, 0.1mg/kg, 0.3 mg/kg, 0.9 mg/kg, 2.7 mg/kg, 8.1 mg/kg.
The M 9834 antibody working solution is to obtain monoclonal antibody using M 9834 artificial antigen's immune mouse, and with antibody diluent 1 is diluted to：It is prepared by 60000 ratios.
The M 9834 ELIAS secondary antibody working solution adds diluted into 1 by ELIAS secondary antibody：2000 ratios, the substrate solution A is the citrate-phosphate disodium hydrogen buffer solution of the carbamide peroxide containing 0.5 mmol/L, and the substrate solution B is the ethanol solution of tetramethyl biphenyl diamidogen, and the terminate liquid is 2
The sulphuric acid of mol/L, the concentration and dilution liquid is 10 times of concentration and dilution liquid, is the PBS of 0.1 mol/L, between pH value range 7.0-7.5, the concentrated cleaning solution is 10 times of concentrated cleaning solutions, is containing 0.5% tween 20, the PBST of 0.1 mol/L, between pH value range 7.0-7.5.
The enzyme linked immunological kit and its detection method of detection M 9834, based on the indirect competitive enzyme-linked immunosorbent reaction principle of antigen-antibody, the method is comprised the following steps：
(1) pretreatment testing sample, sample treatment that will be to be tested is fluid sample, or uses organic solvent extraction testing sample, and is redissolved in sample diluting liquid；
(2) required reagent is taken out from cold storage environment, is placed in room temperature（20～25 DEG C）30 more than min are balanced, notices that every kind of liquid reagent must shake up using before；
(3) ELISA Plate for being coated with M 9834 antigen is taken, plus the μ L/ holes of standard substance/sample 50 are in corresponding micropore, add M 9834 ELIAS secondary antibody working solution, 50 μ L/ holes, it is subsequently adding M 9834 antibody working solution, 50 μ L/ holes, gently vibration is mixed, and with cover plate membrane cover plate 25 DEG C of light protected environments of rearmounted room temperature 30 min are reacted；
(4) cover plate film is carefully opened, liquid in hole is dried, with the μ L/ holes of wash operating solution 260, fully washed 4 ~ 5 times, per the s of minor tick 10, patted dry (bubble not being eliminated after patting dry can be poked with original pipette tips) with absorbent paper；
(5) the μ L/ holes of substrate solution A 50, the μ L/ holes of substrate solution B 50, gently vibration is added to mix, with 15～20 min of reaction in the rearmounted 25 DEG C of light protected environments of cover plate membrane cover plate；
(6) the μ L/ holes of terminate liquid 50 are added, gently vibration is mixed, setting microplate reader is at 450 nm or the nm of dual wavelength 450/630 is detected, determines per hole absorbance（Please run through data in 5 min）；
(7) with standard concentration (ppb) logarithm as abscissa, standard substance percentage absorbance be vertical coordinate, draw standard curve, reference standard curve calculate sample in M 9834 content.
The enzyme linked immunological kit of present invention detection M 9834 and its measuring principle of detection method：M 9834 in sample and antigen-specific sexual competition antibody fixed in ELISA Plate, add ELIAS secondary antibody, and antibody response, by enzyme catalysiss chromogenic reagent, according to the depth of colour developing come the content of M 9834 in judgement sample.If the M 9834 content in sample is few, colour developing is deep；Conversely, it is shallow then to develop the color.The kit test method of the present invention is easy to operate, detects sensitive, accurate, quick, it is adaptable to the quick detection of batch samples.
Specific embodiment
The preparation of M 9834 protein conjugate：
M 9834 hapten derivant with carboxyl is obtained using succinic anhydrides method, 0.05 mmol and carrier protein BSA is taken afterwards by 10：1 combination ratio is blended in 0.05
In the carbonate buffer solution (CBS) of mol/L pH 9.6, it is subsequently adding 0.15 mmol carbodiimides, the h of room temperature reaction 24 is put in stirring, dialyse two days most in the PBS of 0.2 mol/L pH 7.6, unreacted hapten is removed, the protein conjugate solution for obtaining is saved backup in -20 DEG C.
The preparation of M 9834 antibody：
From the purebred BALA/C mices of healthy adult, take to mix with equivalent complete Freund's adjuvant with the μ g of immunizing antigen 50 of protein molecule preparation and initial immunity is carried out using lumbar injection, equivalent incomplete Freund's adjuvant was added to carry out using lumbar injection with same dose immunizing antigen every 3 weeks afterwards secondary, three immunity, every time tail vein blood determines antiserum titre to certain titre after immunity 6 days, adjuvant is not added with same dose carries out final immunization, spleen is taken after 3 days preparing Spleen cell suspensions and myeloma cell carries out cell fusion, hybridoma cell line required for filtering out carries out cloning, the hybridoma in exponential phase is selected to carry out frozen, for ascites preparation, first lumbar injection 0.5
Ml liquid paraffin is in BALB/C Mus sensitization, pneumoretroperitoneum injection 1 × 10 in 2 weeks6Individual hybridoma, inoculating cell can produce ascites after 7-10 days, ascites is extracted with syringe when ascites is as more as possible, collect repeatedly for several times, 4000
Rpm is centrifuged 15 min, collects supernatant, and purification is carried out to monoclonal antibody using caprylic acid-ammonium purification ascites, and lyophilization obtains lyophilized powder and saves backup after -20 DEG C.
Preparation is coated with the ELISA Plate of M 9834 envelope antigen：
M 9834 hapten and carrier proteins Bovine pure albumen (BSA) are coupled and are obtained by envelope antigen, with carbonate (CBS) buffer of 0.05 mol/L pH 9.6 as coating buffer, by M 9834 antigen diluent into 1:40000 ratios, 100 L/ holes, 37 DEG C of 2 h of placement take out ELISA Plate and get rid of liquid in plate, and with the L/ holes of concentrated cleaning solution 300 after dilution, board-washing 2 times 30 s/ time, is subsequently adding 0.5% bovine serum albumin (BSA) and closes, and 150
L/ holes, 37 DEG C of 1.5 h of placement, discard confining liquid, and ELISA Plate after patting dry is placed (25 DEG C) between constant temperature and dried, inspect by random samples it is qualified after by ELISA Plate vacuum sealing it is rearmounted 4 DEG C at preserve.
M 9834 standard substance compound concentration is respectively 0 mg/kg, 0.1mg/kg, 0.3 mg/kg, 0.9 mg/kg, 2.7 mg/kg, 8.1 mg/kg.
The preparation of M 9834 antibody working solution：Monoclonal antibody is obtained using M 9834 artificial antigen's immune mouse, with antibody diluent 1 is diluted to：It is prepared by 60000 ratios.
M 9834 ELIAS secondary antibody working solution adds diluted into 1 by ELIAS secondary antibody：2000 ratios, substrate solution A is the citrate-phosphate disodium hydrogen buffer solution of the carbamide peroxide containing 0.5 mmol/L, and substrate solution B is the ethanol solution of tetramethyl biphenyl diamidogen, and terminate liquid is 2
The sulphuric acid of mol/L, concentration and dilution liquid is 10 times of concentration and dilution liquid, is the PBS of 0.1 mol/L, and between pH value range 7.0-7.5, concentrated cleaning solution is 10 times of concentrated cleaning solutions, is 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 includes following material for the enzyme linked immunological kit for detecting M 9834：
(1) 96 hole elisa Plates × 1 piece；
(2) titer × 6 bottle：(1mL/ bottles) 0 mg/kg, 0.1mg/kg, 0.3 mg/kg, 0.9 mg/kg, 2.7 mg/kg, 8.1 mg/kg；
(3) mL of antibody working solution 7；
(4) mL of ELIAS secondary antibody working solution 7；
(5) mL of substrate solution A 7；
(6) mL of substrate solution B 7；
(7) mL of terminate liquid 7；
The mL of (8) 10 × concentration and dilution liquid 40；
The mL of (9) 10 × concentrated cleaning solution 40；
When the test kit of the present invention is used to detect M 9834 residual quantity in crop sample, implemented by following steps：Sample pretreatment, detected with test kit of the present invention, analysis result.
(1) with M 9834 residual quantity in test kit of the present invention detection testing sample
The ELISA Plate for being coated with M 9834 antigen is taken, plus the L/ holes of standard substance/sample 50 are in corresponding micropore；ELIAS secondary antibody working solution, 50 L/ holes is added to be subsequently adding M 9834 antibody working solution, 50 L/ holes, gently vibration is mixed, and with cover plate membrane cover plate 25 DEG C of light protected environments of rearmounted room temperature 30 min are reacted；Cover plate film is carefully opened, liquid in hole is dried, with wash operating solution 300
L/ holes, fully washing 4 times, soak 15-30 s, are patted dry with absorbent paper；Add the L/ holes of substrate solution A 50, the L/ holes of substrate solution B 50, gently vibration to mix, with the rearmounted 25 DEG C of light protected environments of cover plate membrane cover plate 15 min are reacted；The L/ holes of terminate liquid 50 are added, gently vibration is mixed, setting microplate reader is in 450
At nm or the nm of dual wavelength 450/630 detections, determine per hole absorbance（Please run through data in 5 min）；The absorbance size of contrast testing sample and standard substance, M 9834 residual quantity in quantitative analyses testing sample.
(2) analysis result
The percentage absorbance of the calculating of percentage absorbance, standard substance or sample is equal to the absorbance of the meansigma methodss (diplopore) divided by first standard (0 standard) of the absorbance of standard substance or sample, then is multiplied by 100%, i.e.,
Percentage absorbance (%)=B/B0×100%
Wherein mean absorbance values of B-standard solution or sample solution, B0—0
The mean absorbance values of ppb standard solution.
As abscissa, standard substance percentage absorbance is vertical coordinate to logarithm with the standard concentration (ppb) of M 9834, draws standard curve, obtains linear equation.The percentage absorbance of sample is substituted in standard curve, the concentration corresponding to sample is read from standard curve, be multiplied by the actual concentrations that its corresponding extension rate is M 9834 in sample.
Claims (7)
1. the enzyme linked immunological kit and its detection method of M 9834, including ELISA Plate, M 9834 standard substance, M 9834 antibody working solution, M 9834 ELIAS secondary antibody working solution, substrate solution A, substrate solution B, terminate liquid, concentration and dilution liquid and concentrated cleaning solution are detected.
2. the enzyme linked immunological kit and its detection method of M 9834 are detected, is comprised the following steps：Preparation, the preparation of M 9834 standard substance, the preparation of M 9834 antibody working solution, the preparation of M 9834 ELIAS secondary antibody working solution, the preparation of substrate solution A, the preparation of substrate solution B, the preparation of terminate liquid, the preparation of concentration and dilution liquid and the preparation of concentrated cleaning solution of ELISA Plate.
3. it is according to claim 2 detection M 9834 enzyme linked immunological kit and its detection method, it is characterised in that：Described ELISA Plate preparation method is to be coupled M 9834 hapten and the pure albumen of carrier proteins Bovine (BSA) to obtain M 9834 envelope antigen, with carbonate (CBS) buffer of 0.05 mol/L pH 9.6 as coating buffer, envelope antigen is diluted to into 1：40000 ratios, 100 μ L/ holes, 37 DEG C of 2 h of incubation, take out ELISA Plate and get rid of liquid in plate, add the μ L/ holes of concentrated cleaning solution 300 after dilution, board-washing 2 times, 30 s/ time, it is subsequently adding 0.5% bovine serum albumin (BSA) closing, 150 μ L/ holes, 37 DEG C of 1.5 h of placement, discard confining liquid and directly pat dry, ELISA Plate after patting dry is placed (25 DEG C) between constant temperature and is dried, and is preserved under the conditions of ELISA Plate is vacuum-sealed in into 4 DEG C after sampling observation is qualified.
4. it is according to claim 2 detection M 9834 enzyme linked immunological kit and its detection method, it is characterised in that：The concentration of M 9834 standard substance be respectively 0 mg/kg, 0.1mg/kg, 0.3
mg/kg、0.9 mg/kg、2.7 mg/kg、8.1 mg/kg。
5. it is according to claim 2 detection M 9834 enzyme linked immunological kit and its detection method, it is characterised in that：Described M 9834 antibody working solution is to obtain monoclonal antibody using M 9834 artificial antigen's immune mouse, and with antibody diluent 1 is diluted to：It is prepared by 60000 ratios.
6. it is according to claim 2 it is a kind of detection M 9834 enzyme linked immunological kit and its detection method, it is characterised in that：Described M 9834 ELIAS secondary antibody working solution adds diluted into 1 by ELIAS secondary antibody：2000 ratios, the substrate solution A is the citrate-phosphate disodium hydrogen buffer solution of the carbamide peroxide containing 0.5 mmol/L, the substrate solution B is the ethanol solution of tetramethyl biphenyl diamidogen, and the terminate liquid is the sulphuric acid of 2 mol/L, and the concentration and dilution liquid is 10 times of concentration and dilution liquid, it is the PBS of 0.1 mol/L, between pH value range 7.0-7.5, the concentrated cleaning solution is 10 times of concentrated cleaning solutions, and it is containing 0.5% tween 20, the PBST of 0.01 mol/L, between pH value range 7.0-7.5.
7. the enzyme linked immunological kit and its detection method of the detection M 9834 described in claim 2, based on the indirect competitive enzyme-linked immunosorbent reaction principle of antigen-antibody, the method is characterized in that：Pretreatment testing sample，Take the ELISA Plate for being coated with M 9834 antigen，Sequentially it is separately added into standard substance/sample、M 9834 ELIAS secondary antibody working solution、The each 50 μ L/ holes of M 9834 antibody working solution are in corresponding micropore，Gently vibration is mixed，30 min are reacted with cover plate membrane cover plate 25 DEG C of light protected environments of rearmounted room temperature，Liquid in hole is dried，4 ~ 5 times are fully washed with wash operating solution，Per the s of minor tick 10，The μ L/ holes of substrate solution A 50 are added after patting dry，The μ L/ holes of substrate solution B 50，Gently vibration is mixed，With 15～20 min of reaction in the rearmounted 25 DEG C of light protected environments of cover plate membrane cover plate，Add the μ L/ holes of terminate liquid 50，Gently vibration is mixed，Setting microplate reader is at 450 nm or the nm of dual wavelength 450/630 is detected，Determine per hole absorbance（Please run through data in 5 min）, with standard concentration (ppb) logarithm as abscissa, standard substance percentage absorbance be vertical coordinate, draw standard curve, reference standard curve calculate sample in M 9834 content.
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| CN201510687767.0A CN106610431A (en) | 2015-10-22 | 2015-10-22 | Enzyme-linked immunosorbent assay kit for detecting Benalaxyl and detection method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113109556A (en) * | 2021-04-19 | 2021-07-13 | 河北农业大学 | Detection method of ganoderic acid A |
| CN113189324A (en) * | 2021-03-03 | 2021-07-30 | 广州市丰华生物工程有限公司 | Blocking reagent and method for blocking immunoreaction carrier by using same |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104655847A (en) * | 2013-11-20 | 2015-05-27 | 南京亿特生物科技有限公司 | Enzyme linked immunosorbent assay kit (ELISA kit) for detecting adprin and detection method thereof |
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2015
- 2015-10-22 CN CN201510687767.0A patent/CN106610431A/en not_active Withdrawn
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104655847A (en) * | 2013-11-20 | 2015-05-27 | 南京亿特生物科技有限公司 | Enzyme linked immunosorbent assay kit (ELISA kit) for detecting adprin and detection method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113189324A (en) * | 2021-03-03 | 2021-07-30 | 广州市丰华生物工程有限公司 | Blocking reagent and method for blocking immunoreaction carrier by using same |
| CN113109556A (en) * | 2021-04-19 | 2021-07-13 | 河北农业大学 | Detection method of ganoderic acid A |
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Application publication date: 20170503 |