CN105319353A - Preparation of enzyme linked immunosorbent assay kit used for detecting dihydropyridine drug residues - Google Patents
Preparation of enzyme linked immunosorbent assay kit used for detecting dihydropyridine drug residues Download PDFInfo
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Abstract
The invention discloses preparation of an enzyme linked immunosorbent assay (ELISA) kit used for detecting dihydropyridine drug residues, and a detection method for detecting dihydropyridine drug residues. The enzyme linked immunosorbent assay kit is high in detection sensitivity, accuracy, speed, and specificity; operation is simple; and the detection method is suitable for large-scale sample detection. The enzyme linked immunosorbent assay kit comprises an enzyme labeled plate coated with dihydropyridine drug antigens, a dihydropyridine drug standard substance, a dihydropyridine drug antibody working solution, a dihydropyridine drug enzyme labeled second antibody working solution, a substrate solution A, a substrate solution B, a stopping solution, a concentrated sample diluent, and a concentrated washing liquid. Solid phase indirect competitive enzyme-linked immune response is taken as the principle of the enzyme linked immunosorbent assay kit used for detecting dihydropyridine drugs. According to the detection method, an extracted sample, the dihydropyridine drug enzyme labeled second antibody working solution, and the dihydropyridine drug antibody working solution are added into corresponding enzyme labeled holes for a certain time of incubation; after plate washing, the substrate solution A, and the substrate solution B are added, so that the color of an obtained mixture is changed to be blue under enzyme action; and the stopping solution is added, and the color is changed from blue to yellow. The shade of color developing and the content of dihydropyridine drugs in the standard substance or the sample are inversely related. The direction method can be used for directly detecting dihydropyridine drugs in health care products.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection technical field, particularly detect the enzyme linked immunological kit of Dihydropyridines drugs.
Background technology
Dihydropyridines is the calcium antagonist that a class has obvious hemangiectasis, and dihydropyridine type calcium antagonists is the wider cardiovascular drugs of clinical practice.Long-Time Service with effectively control blood pressure, less causes reflex tachycardia or postural hypotension.To with angina pectoris, high fat of blood, arrhythmia cordis, migrainous hyperpietic, dihydropyridine type calcium antagonists can be used as choice drug.It adapts to the hypertension of various degree, angina pectoris and ARR first-line drug.The price of its definite curative effect, widely effect and relative moderate, has overwhelming superiority compared with the medicine of other types.But be existingly added in health products, corresponding bad reaction can be caused for normal population, health risk.
At present, the assay method of Dihydropyridines drugs mainly contains high performance liquid chromatography, vapor-phase chromatography, thin layer chromatography, tablets by HPLC-MS etc., accurately and reliably, but instrument and equipment is costly for its testing result, and Sample pretreatment process relative complex.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 dihydropyridines.
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 Dihydropyridines drugs.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 Dihydropyridines drugs, comprises ELISA Plate, Dihydropyridines drugs standard items, Dihydropyridines drugs antibody working fluid, the anti-working fluid of Dihydropyridines drugs mark two, 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 Dihydropyridines drugs, comprises the following steps: the preparation of the preparation of the preparation of the preparation of ELISA Plate, the preparation of Dihydropyridines drugs standard items, Dihydropyridines drugs antibody working fluid, the preparation of Dihydropyridines drugs 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 Dihydropyridines drugs, concrete steps are that Dihydropyridines drugs 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, Dihydropyridines drugs envelope antigen is diluted to 1:10000 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 250 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.
Dihydropyridines drugs concentration is respectively 0ng/mL, 10ng/mL, 30ng/mL, 90ng/mL, 270ng/mL, 810ng/mL.
Described Dihydropyridines drugs antibody working fluid adopts Dihydropyridines drugs artificial antigen immune mouse to obtain monoclonal antibody, is diluted to the preparation of 1:40000 ratio with antibody diluent.
Described Dihydropyridines drugs ELIAS secondary antibody working fluid adds diluted by ELIAS secondary antibody and becomes 1:1000 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 Dihydropyridines drugs, 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 Dihydropyridines drugs antigen is got, add standard items/sample 50 μ L/ hole in the micropore of correspondence, add Dihydropyridines drugs ELIAS secondary antibody working fluid, 50 μ L/ holes, then dihydropyridine antibody-like 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 Dihydropyridines drugs in reference standard curve calculation sample.
Wherein, described testing sample following methods carries out pre-service:
Preparation sample diluting liquid (1 × PBS) and sample redissolve liquid (acetonitrile: ethyl acetate=4:1) and grind representative sample (make the sample of 50% can by 20 object filter screens); The sample weighed after 5g grinding filtration adds 10mL sample redissolution liquid, and Sample Dilution is than being 1:2 (w/v); Mix in the container of sealing, concussion vortex 1min; Leave standstill with the centrifugal 5min. of 4000r/min, get upper liquid 1ml, add 1ml sample diluting liquid.Get upper strata with the centrifugal 5min. of 4000r/min and carry out analysis detection.
The present invention detects the enzyme linked immunological kit of Dihydropyridines drugs and the measuring principle of detection method thereof: antigen-specific sexual competition antibody fixing in the Dihydropyridines drugs in sample and ELISA Plate, add ELIAS secondary antibody, with antibody response, by enzymatic chromogenic reagent, carry out the content of Dihydropyridines drugs in judgement sample according to the depth of colour developing.If the Dihydropyridines drugs 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.
Accompanying drawing explanation
Fig. 1 is the typical curve of dihydropyridines.
Embodiment
The preparation of Dihydropyridines drugs protein conjugate:
Succinic anhydride method is adopted to obtain being with the Dihydropyridines drugs 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 Dihydropyridines drugs 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 Dihydropyridines drugs envelope antigen:
Dihydropyridines drugs 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, Dihydropyridines drugs 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 250 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.
Dihydropyridines drugs standard items compound concentration is respectively 0ng/mL, 10ng/mL, 30ng/mL, 90ng/mL, 270ng/mL, 810ng/mL.
The preparation of Dihydropyridines drugs antibody working fluid: adopt Dihydropyridines drugs artificial antigen immune mouse to obtain monoclonal antibody, be diluted to the preparation of 1:40000 ratio with antibody diluent.
Dihydropyridines drugs ELIAS secondary antibody working fluid adds diluted by ELIAS secondary antibody and becomes 1:1000 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 Dihydropyridines drugs:
(1) 96 ELISA Plate × 1 piece, hole;
(2) titer × 6 bottle: (1mL/ bottle) 0ppm, 10ng/mL, 30ng/mL, 90ng/mL, 270ng/mL, 810ng/mL;
(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 Dihydropyridines drugs residual quantity in sample, implemented by following steps: sample pretreatment, with kit of the present invention carry out detecting, analysis result.
(1) sample pretreatment
Preparation sample diluting liquid (1 × PBS) and sample redissolve liquid (acetonitrile: ethyl acetate=4:1) and grind representative sample (make the sample of 50% can by 20 object filter screens); The sample weighed after 5g grinding filtration adds 10mL sample redissolution liquid, and Sample Dilution is than being 1:2 (w/v); Mix in the container of sealing, concussion vortex 1min; Leave standstill with the centrifugal 5min. of 4000r/min, get upper liquid 1ml, add 1ml sample diluting liquid.Get upper strata with the centrifugal 5min. of 4000r/min and carry out analysis detection.
(2) Dihydropyridines drugs residual quantity in testing sample is detected with kit of the present invention
Get the ELISA Plate being coated with Dihydropyridines drugs 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 Dihydropyridines drugs 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 250 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, Dihydropyridines drugs 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 dihydropyridines (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 Dihydropyridines drugs in sample.
Claims (8)
1. detect preparation and the detection method of the ELISA kit of Dihydropyridines drugs, comprise ELISA Plate, Dihydropyridines drugs standard items, Dihydropyridines drugs antibody working fluid, Dihydropyridines drugs ELIAS secondary antibody working fluid, substrate solution A, substrate solution B, stop buffer and concentrated cleaning solution.
2. detect the preparation of ELISA kit and the detection method of Dihydropyridines drugs, comprise the following steps: the preparation of the preparation of ELISA Plate, the making of Dihydropyridines drugs standard items, Dihydropyridines drugs monoclonal antibody and working fluid thereof, the preparation of Dihydropyridines drugs ELIAS secondary antibody working fluid, the preparation of cleansing solution, the preparation of substrate solution A, the preparation of substrate solution B, the preparation of stop buffer.
3. detect preparation and the detection method of the ELISA kit of Dihydropyridines drugs according to claim 2, it is characterized in that: Dihydropyridines drugs haptens and carrier protein couplet obtain by described Dihydropyridines drugs envelope antigen, this carrier protein is bovine serum albumin(BSA) (BSA); With carbonate (CBS) damping fluid of 0.05mol/LpH9.6 as coating buffer, Dihydropyridines drugs antigen diluent is become 1:10000 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 250 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, discard confining liquid for 37 DEG C, the 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.
4. detect preparation and the detection method of the ELISA kit of Dihydropyridines drugs according to claim 2, it is characterized in that: described Dihydropyridines drugs standard concentration is respectively 0ng/ml, 10ng/mL, 30ng/mL, 90ng/mL, 270ng/mL, 810ng/mL.
5. detect preparation and the detection method of the ELISA kit of Dihydropyridines drugs according to claim 2, it is characterized in that: described Dihydropyridines drugs protein conjugate monoclonal antibody adopts Dihydropyridines drugs artificial antigen immunize rabbit gained, and this antibody working fluid antibody diluent is diluted to 1:40000.
6. detect preparation and the detection method of the ELISA kit of Dihydropyridines drugs according to claim 2, it is characterized in that: described Dihydropyridines drugs ELIAS secondary antibody working fluid ELIAS secondary antibody diluted becomes 1:1000 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 concentrated cleaning solution is 10 times of concentrated cleaning solutions, and it comprises 0.5% Tween-20, and the PBST of 0.01mol/L, between pH value range 7.0-7.5.
7. the preparation of the ELISA kit of detection Dihydropyridines drugs according to claim 2 and detection method, carry out indirect competitive enzyme-linked immunosorbent reaction based on 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 working fluid;
(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) get the ELISA Plate being coated with Dihydropyridines drugs antigen, add standard items/sample 50 μ L/ hole in the micropore of correspondence;
(4) add Dihydropyridines drugs ELIAS secondary antibody working fluid, 50 μ L/ holes, then add Dihydropyridines drugs 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;
(5) carefully open cover plate film, liquid in hole is dried, with wash operating solution 250 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;
(6) 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;
(7) 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);
(8) with the absorbance of standard items test and the log concentration value drawing standard curve of standard items, the content of Dihydropyridines drugs in reference standard curve calculation sample.
8. method according to claim 7, wherein, the sample after described process is through the sample of following process:
(1) sample diluting liquid (1 × PBS) and sample redissolution liquid (acetonitrile: ethyl acetate=4:1) is prepared;
(2) representative sample (make the sample of 50% can by 20 object filter screens) is ground;
(3) sample weighed after 5g grinding filtration adds 10mL sample redissolution liquid, and Sample Dilution is than being 1:2 (w/v);
(4) mix in the container of sealing, concussion vortex 1min; With the centrifugal 5min of 4000r/min;
(5) leave standstill, get upper liquid 1ml, add 1ml sample diluting liquid, with the centrifugal 5min of 4000r/min;
(6) get upper strata and carry out analysis detection.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109406643A (en) * | 2017-08-18 | 2019-03-01 | 青岛谱尼测试有限公司 | The measuring method of dihydropyridine in a kind of feed |
| CN109725142A (en) * | 2018-12-27 | 2019-05-07 | 国家食品安全风险评估中心 | The enzyme linked immunological kit of detection fluo quinolone drug residual and application and detection method |
| CN112375744A (en) * | 2020-12-04 | 2021-02-19 | 江南大学 | Dihydropyridine monoclonal antibody hybridoma cell strain and application thereof |
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| EP0175269B1 (en) * | 1984-09-21 | 1991-03-06 | University Of Iowa Research Foundation | Immunogen conjugates and the use thereof in a dihydropyridine assay |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109406643A (en) * | 2017-08-18 | 2019-03-01 | 青岛谱尼测试有限公司 | The measuring method of dihydropyridine in a kind of feed |
| CN109406643B (en) * | 2017-08-18 | 2021-07-13 | 青岛谱尼测试有限公司 | Method for determining dihydropyridine in feed |
| CN109725142A (en) * | 2018-12-27 | 2019-05-07 | 国家食品安全风险评估中心 | The enzyme linked immunological kit of detection fluo quinolone drug residual and application and detection method |
| CN112375744A (en) * | 2020-12-04 | 2021-02-19 | 江南大学 | Dihydropyridine monoclonal antibody hybridoma cell strain and application thereof |
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