CN101315374A - ELISA detection reagent kit suitable for diazepam relict analysis - Google Patents
ELISA detection reagent kit suitable for diazepam relict analysis Download PDFInfo
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- CN101315374A CN101315374A CNA2008100221598A CN200810022159A CN101315374A CN 101315374 A CN101315374 A CN 101315374A CN A2008100221598 A CNA2008100221598 A CN A2008100221598A CN 200810022159 A CN200810022159 A CN 200810022159A CN 101315374 A CN101315374 A CN 101315374A
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Abstract
The invention relates to an enzyme-linked immune detection kit suitable for the analysis of diazepam residues and belongs to the technology field of the enzyme-linked immune adsorption analysis kit. The kit comprises an enzyme label plate coated with the envelope antigen of the diazepam, a sponge support, a diazepam standard, a diazepam polyclonal antibody, an enzyme sign second antibody, a concentrating and washing liquid, a colored solution and a reaction stopping solution; the envelope antigen of the diazepam is the coupling compound of 3-half succinate diazepam and an egg-white protein; and the enzyme sign second antibody is a horseradish peroxidase labeled goat anti-rabbit antibody. The kit adopts the diazepam polyclonal antibody, and can accurately and sensitively detect the diazepam residues or other structurally similar benzodiazepine residues in urine or tissue; the process of pretreating samples is simple; the time consumption is low; a large quantity of samples can be tested simultaneously; furthermore, the cost for sample detection is lower than that of the traditional instrument detection method; the kit has a long retention time, no radioactive pollution, and practical significance for realizing on-site monitoring of the diazepam residues of the large quantity of samples.
Description
Technical field
The present invention relates to a kind of enzyme-linked immunologic detecting kit that is applicable to diazepam (DZP) retention analysis, belong to Enzyme Linked Immunoadsorbent Assay (ELISA) kit technical field.
Background technology
Diazepam (Diazepam) is the Benzodiazepines sedative hypnotics, and chemical name is: 1-methyl-5-phenyl-7-chloro-1,3-dihydro-2H-1,4-Benzodiazepine-2-ketone.Once as animal feed additive,, had drowsiness few the moving of the animal of making, the effect of grow fast and the meat that changes as growth promoter.Also be used in butcher with animal quarantine before, or with animal shift butcher before, play sedation, to alleviate the animal tense situation, reduce animal injury and mortality ratio.This type of medicine is common drowsiness, as seen unable, headache, dizzy, nauseating, constipation, bad reactions such as accidental fash, hepatic lesion, bone marrow suppression.If with being intended to add this type of medicine in the feed, the medicine of accumulating enters human body by food chain, will cause huge harm.In the forbidden drugs list is listed this type of medicine by many for this reason countries.At present, the detection of relevant diazepam both at home and abroad mainly contains high performance liquid chromatography (HPLC), vapor-phase chromatography (GC), thin-layered chromatography (TLC), gas-matter coupling method (GC-MS), liquid-matter coupling method (LC-MS) etc., but these methods not only need expensive instrument and equipment, to the requirement of sample also than higher, need further purification processes just can carry out, this can not reach modern and detect quick, convenient, requirement accurately.In recent years, abroad carried out the research to the Benzodiazepines immune analysis method, but domestic in this respect research starts late, most testing product also needs to rely on external import.In order to strengthen the supervision of domestic meat products and to ensure people health, be necessary to launch research to the Benzodiazepines immune analysis method, be necessary to set up a kind of enzyme-linked immunosorbent assay method of diazepam fast and effectively.
Summary of the invention
(1) technical matters that will solve
The object of the present invention is to provide a kind of have high sensitivity, high specific, pin-point accuracy, pinpoint accuracy, the simple enzyme-linked immuno sorbent assay kit of method of operating, be used for the residual batch of diazepam, fast detecting.
(2) technical scheme
For achieving the above object, the invention provides a kind of enzyme-linked immunologic detecting kit of diazepam relict analysis, this kit comprises by the ELISA Plate of diazepam envelope antigen, sponge bracket, diazepam standard items, diazepam polyclonal antibody, ELIAS secondary antibody, concentrated cleaning solution, colour developing liquid and reaction terminating liquid.
Wherein, bag is by in the preparation process of ELISA Plate of diazepam envelope antigen, used coating antigen is the haptens 3-hemisuccinic acid ester diazepam of diazepam and the coupled complex of ovalbumin (OVA), used coating buffer is 0.05 M pH, 9.6 sodium carbonate buffers, and used confining liquid is the above-mentioned coating buffer that contains 2%OVA.
Wherein, the diazepam polyclonal antibody is the coupled complex conventional method immunity preparation with 3-hemisuccinic acid ester diazepam and bovine serum albumin(BSA) (BSA).
Wherein, ELIAS secondary antibody is the horseradish peroxidase-labeled goat anti-rabbit antibody.
Wherein, the prescription of concentrated cleaning solution (PBST) is to add 8g sodium chloride in the 20mL distilled water, the 0.2g potassium dihydrogen phosphate, and the 3g sodium hydrogen phosphate, 0.4g potassium chloride, the 0.5mL Tween-20, the concentration of concentrated cleaning solution is 50 times when normally using.
Wherein, colour developing liquid comprises A liquid and B liquid, and the A formula of liquid is to add 0.933g citric acid, 3.68g Na in every 100mL water
2HPO
412H
2O and 18 μ L30%H
2O
2The B formula of liquid is dissolved in 100mL ethylene glycol for the 60mg tetramethyl benzidine, during use in A: B=5: 1 ratio is used.
The check and analysis principle of kit of the present invention is: each Kong Jun on the ELISA Plate is coated with the diazepam antigen of same amount, after adding diazepam sample to be measured and diazepam polyclonal antibody, solid-phase coating antigen and diazepam to be measured are vied each other and polyclonal antibody reacts, because the solid phase antigen in each hole and the polyclonal antibody uniform content of adding cause, so when the diazepam concentration of testing sample is high, the polyclonal antibody that then is bonded on the solid phase antigen is few, the ELIAS secondary antibody that adds is few with the antibodies amount that is fixed, add substrate solution (liquid A liquid promptly develops the color) and colour developing liquid (being B liquid) with cleansing solution washing back, chromogenic reaction is shallow, the light absorption value that detects with microplate reader is low, shows the inhibiting rate height; Otherwise, when the diazepam concentration of testing sample is hanged down, the light absorption value height of then being surveyed, inhibiting rate is low.According to detecting the typical curve of being done, can extrapolate the concentration of the diazepam of testing sample with known diazepam concentration.
(3) beneficial effect
Diazepam residue detection kit provided by the invention has adopted the diazepam polyclonal antibody, the Benzodiazepines that can accurately detect diazepam and other structural similarities in urine, the tissue delicately is residual, the pre-treatment process of sample is simple, consuming time few, can detect a large amount of samples simultaneously, the sample detection cost is far below traditional instrument detecting method, and the reagent holding time of the present invention is long, "dead" pollution.The present invention has important practical significance to the residual on-site supervision technology of diazepam that solves batch samples.
Description of drawings
The standard of Fig. 1 diazepam suppresses curve.
Embodiment
Below be that embodiment is used for further specifying of the present invention, but be not used for limiting invention which is intended to be protected.
Operation of embodiment 1 kit and result calculate:
Testing sample is after pre-treatment, and is standby with the PBST constant volume.Take vacuum packaging bag apart and take out ELISA Plate, at room temperature balance 5 minutes is standby.Preparation 0ng/mL, 0.7ng/mL, 1.4ng/mL, 2.8ng/mL, 5.6ng/mL, 11.2ng/mL, 56ng/mL, the diazepam titer of 112ng/mL, the testing sample that adds 50 μ L standard specimens or handle well is in each hole, standard specimen and sample are done 4 repetitions, add the antibody of 50 μ L dilution, hatch 30 minutes for 37 ℃; Pour out the liquid in the hole, the PBST good with dilution washes 5 times, and ELISA Plate is upside down in thieving paper arsis thousand; Add by the good enzyme mark goat-anti rabbit two anti-100 μ L of dilution in 1: 3000, hatched 30 minutes for 37 ℃; Pour out the liquid in the hole, the PBST good with dilution washes plate 5 times, pats dry; Get A liquid and B liquid by 5: 1 mixings, every hole adds 100pL, in the dark develops the color 10～15 minutes, and every hole adds the stop buffer cessation reaction of 50 μ L, and measuring each hole on the microplate reader is the light absorption value at 450nm place at wavelength.
The light absorption value that will contain 0ng/mL standard items hole is decided to be B
0, the light absorption value in all the other holes is decided to be B; With B/B
0Value is ordinate, and the log value of respective standard product concentration is a horizontal ordinate, draws the diazepam standard and suppresses curve.The concentration of diazepam in the corresponding testing sample can be obtained according to the regression equation of curve, also diazepam IC can be obtained
50(B/Bo=50%) and minimum detectable level LOD (concentration of 90% inhibiting rate correspondence).IC
50Be 50.12ng/mL, LOD is 5.12ng/mL, and sensing range (concentration of 20%～80% inhibiting rate correspondence) is 9.12～251.2ng/mL, linear equation: y=-0.4068x+1.1899, R
2=0.9921.
The establishment of embodiment 2 diazepam relict analysis enzyme-linked immuno sorbent assay kits:
In this example, kit comprises as the lower part:
(1) bag is by the enzyme mark of diazepam antigen;
(2) sponge bracket;
(3) 1mg/mL diazepam standard items (Sigma company);
(4) diazepam polyclonal antibody;
(5) horseradish peroxidase mark goat anti-rabbit antibody (health becomes bio-engineering corporation);
(6) the thickening and washing formula of liquid is: 8g sodium chloride, 0.2g potassium dihydrogen phosphate, 3g sodium hydrogen phosphate, 0.4g potassium chloride, 0.5mL Tween-20 and 20mL distilled water;
(7) colour developing liquid A formula of liquid: 0.933g citric acid, 3.68g Na
2HPO
412H
2O, 18 μ L30%H
2O
2With the 100mL ultrapure water
(8) colour developing liquid B formula of liquid: the 60mg tetramethyl benzidine is dissolved in 100mL ethylene glycol
The experiment of embodiment 3 storage lives:
Kit is positioned over 4 ℃ of preservations, get 0,10,20,30,60,90,120,150 and the kit of 180d respectively, with 5.25 μ g/mL antigen working concentrations and 1.2 μ g/mL antibody working concentrations is working concentration, carries out standard model and detects to measure its detection effect.The storage life measurement result sees Table 1, shows IC
50Change not quite, kit can be preserved more than 6 months under 4 ℃.
Table 1 kit is preserved experiment
The experiment of embodiment 4 kit specificitys:
The analogue of selecting diazepam is as determinand, records concentration (IC in the inhibition of various materials
50), use the cross reactivity of following formula calculating antibody again to these materials; Cross reacting rate is littler, and then antibody is stronger to the specificity of diazepam, on the contrary the poor specificity of antibody then.
Cross reaction (CR%)=IC
50(diazepam)/IC
50(for the examination thing) * 100%.
Measuring the results are shown in Table 2, adopts indirect elisa method, and the diazepam polyclonal antibody has specificity preferably to other similar benzodiazepine of molecular structure, and cross reacting rate is all less.
Table 2 cross reaction
Embodiment 5 adds the recovery experiment:
(1) extraction and cleaning of sample: get the 1mL urine sample in centrifuge tube, add 4mL, 0.1MNaOH solution vibration 2-5min, add the normal hexane of 5mL, behind the whirlpool 5min, 15 ℃ of centrifugal 5min of 3000rpm get upper organic phase, nitrogen dries up, and adds the standard items dilution of 1mL, supplies to analyze as sample.
(2) add the standard items liquid storage in people's urine of blank, making its concentration is 5.0ng/mL, 15.0ng/mL, 50.0ng/mL, 100.0ng/mL.Each concentration prepares five parts in sample respectively, measures its content, and measured concentration and interpolation concentration are compared.
In blank urine sample, add the diazepam of variable concentrations, extract, concentrate post analysis by above-mentioned pre-treating method.The results are shown in Table 3.As can be seen, the recovery in the urine sample is between 68.8%～92.5%.This analytical approach repeatability is good, and relative standard deviation is lower than 4.24%.
Table 3 adds the mensuration of the recovery
Claims (4)
1. an enzyme-linked immunologic detecting kit that is applicable to diazepam relict analysis is characterized in that comprising by the ELISA Plate of diazepam envelope antigen, sponge bracket, diazepam standard items, diazepam polyclonal antibody, ELIAS secondary antibody, concentrated cleaning solution, colour developing liquid and reaction terminating liquid;
Described diazepam envelope antigen is the coupled complex of 3-hemisuccinic acid ester diazepam and ovalbumin;
Described ELIAS secondary antibody is the horseradish peroxidase-labeled goat anti-rabbit antibody.
2. enzyme-linked immunologic detecting kit according to claim 1, the prescription that it is characterized in that concentrated cleaning solution are to add 8g sodium chloride, 0.2g potassium dihydrogen phosphate, 3g sodium hydrogen phosphate, 0.4g potassium chloride, 0.5mL Tween-20 in the 20mL distilled water.
3. enzyme-linked immunologic detecting kit according to claim 1, the liquid that it is characterized in that developing the color comprises A liquid and B liquid, the A formula of liquid is to add 0.933g citric acid, 3.68g Na in every 100mL water
2HPO
412H
2O and 18 μ L 30%H
2O
2The B formula of liquid is dissolved in 100mL ethylene glycol for the 60mg tetramethyl benzidine.
4. enzyme-linked immunologic detecting kit according to claim 1 is characterized in that reaction terminating liquid is the sulfuric acid liquid of 2mol/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100221598A CN101315374A (en) | 2008-06-30 | 2008-06-30 | ELISA detection reagent kit suitable for diazepam relict analysis |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100221598A CN101315374A (en) | 2008-06-30 | 2008-06-30 | ELISA detection reagent kit suitable for diazepam relict analysis |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101936986A (en) * | 2010-08-03 | 2011-01-05 | 中国农业大学 | Method for detecting diazepam and chemoluminescence immunoassay kit special for same |
| CN105319370A (en) * | 2014-07-25 | 2016-02-10 | 江苏维赛科技生物发展有限公司 | Preparation of kit used for detecting benzodiazepine drug residues |
| CN106645764A (en) * | 2016-12-26 | 2017-05-10 | 北京勤邦生物技术有限公司 | Enzyme-linked immunosorbent assay kit for detecting diazepam and application thereof |
-
2008
- 2008-06-30 CN CNA2008100221598A patent/CN101315374A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101936986A (en) * | 2010-08-03 | 2011-01-05 | 中国农业大学 | Method for detecting diazepam and chemoluminescence immunoassay kit special for same |
| CN101936986B (en) * | 2010-08-03 | 2013-03-06 | 中国农业大学 | Method for detecting diazepam and chemoluminescence immunoassay kit special for same |
| CN105319370A (en) * | 2014-07-25 | 2016-02-10 | 江苏维赛科技生物发展有限公司 | Preparation of kit used for detecting benzodiazepine drug residues |
| CN106645764A (en) * | 2016-12-26 | 2017-05-10 | 北京勤邦生物技术有限公司 | Enzyme-linked immunosorbent assay kit for detecting diazepam and application thereof |
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Application publication date: 20081203 |