CN102455358A - Time-resolved fluoroimmunoassay kit for detecting diazepam and detection method thereof - Google Patents
Time-resolved fluoroimmunoassay kit for detecting diazepam and detection method thereof Download PDFInfo
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- CN102455358A CN102455358A CN201010528958XA CN201010528958A CN102455358A CN 102455358 A CN102455358 A CN 102455358A CN 201010528958X A CN201010528958X A CN 201010528958XA CN 201010528958 A CN201010528958 A CN 201010528958A CN 102455358 A CN102455358 A CN 102455358A
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- 238000001514 detection method Methods 0.000 title claims abstract description 14
- 229960003529 diazepam Drugs 0.000 title abstract description 4
- AAOVKJBEBIDNHE-UHFFFAOYSA-N diazepam Chemical compound N=1CC(=O)N(C)C2=CC=C(Cl)C=C2C=1C1=CC=CC=C1 AAOVKJBEBIDNHE-UHFFFAOYSA-N 0.000 title abstract description 4
- 230000008105 immune reaction Effects 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 18
- 238000013016 damping Methods 0.000 claims description 16
- 239000012530 fluid Substances 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 12
- 238000010790 dilution Methods 0.000 claims description 9
- 239000012895 dilution Substances 0.000 claims description 9
- 241000283707 Capra Species 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000003018 immunoassay Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 229910052693 Europium Inorganic materials 0.000 claims description 4
- 238000004364 calculation method Methods 0.000 claims description 4
- 230000002708 enhancing effect Effects 0.000 claims description 4
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 claims description 4
- 238000005259 measurement Methods 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 238000005728 strengthening Methods 0.000 claims description 2
- 230000000087 stabilizing effect Effects 0.000 claims 1
- 241000251468 Actinopterygii Species 0.000 abstract 1
- 241001465754 Metazoa Species 0.000 abstract 1
- 235000013305 food Nutrition 0.000 abstract 1
- 230000036046 immunoreaction Effects 0.000 abstract 1
- 238000002372 labelling Methods 0.000 abstract 1
- 235000013372 meat Nutrition 0.000 abstract 1
- 230000035945 sensitivity Effects 0.000 abstract 1
- 239000000427 antigen Substances 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 229910052747 lanthanoid Inorganic materials 0.000 description 3
- 150000002602 lanthanoids Chemical class 0.000 description 3
- 210000002700 urine Anatomy 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241001233278 Scalopus aquaticus Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- WVVLURYIQCXPIV-UHFFFAOYSA-N 4,4,4-trifluoro-1-naphthalen-2-ylbutane-1,3-dione Chemical compound C1=CC=CC2=CC(C(=O)CC(=O)C(F)(F)F)=CC=C21 WVVLURYIQCXPIV-UHFFFAOYSA-N 0.000 description 1
- 206010052804 Drug tolerance Diseases 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 230000026781 habituation Effects 0.000 description 1
- 230000010224 hepatic metabolism Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000003326 hypnotic agent Substances 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229960004535 oxazepam Drugs 0.000 description 1
- ADIMAYPTOBDMTL-UHFFFAOYSA-N oxazepam Chemical group C12=CC(Cl)=CC=C2NC(=O)C(O)N=C1C1=CC=CC=C1 ADIMAYPTOBDMTL-UHFFFAOYSA-N 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 235000015277 pork Nutrition 0.000 description 1
- IWZKICVEHNUQTL-UHFFFAOYSA-M potassium hydrogen phthalate Chemical compound [K+].OC(=O)C1=CC=CC=C1C([O-])=O IWZKICVEHNUQTL-UHFFFAOYSA-M 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000003352 sequestering agent Substances 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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Abstract
A kit for detecting Diazepam and a detection method thereof belong to the technical field of time-resolved fluoroimmunoassay (TRFIA), and are used for detecting the DIA content in animal food and feed such as meat, fish and the like. The kit prepared by the invention adopts TRFIA to detect DIA, and the basis of the detection is labeled immune reaction. The plates were coated with DIA-OVA, and the DIA standards or samples were added followed by the DIA antibodies. Free DIA competes with DIA-OVA on the microplate, unbound DIA antibody is washed away, Eu3+ -goat anti-rabbit antibody is added, and unbound Eu3+ -goat anti-rabbit antibody is washed away after the labeling immunoreaction. After adding the enhancement solution, measuring the fluorescence intensity cps by using a time-resolved fluorescence instrument, wherein the fluorescence intensity is inversely proportional to the concentration of DIA in the sample, and determining the content of DIA in the measured sample by contrasting with a standard curve. The DIA detection kit provided by the invention has the advantages of simple structure, convenience in use, low price and high sensitivity.
Description
Technical field
The kit and the detection method thereof of a kind of detection stable (CPZ) belong to time resolved fluoro-immunoassay (TRFIA) technical field, are used for the stable content detection of samples such as urine, tissue, feed.
Background technology
Stable having another name called diazepam (diazepam), is clinical calmness commonly used, hypnotic drug.Take such medicine for a long time and can produce dependence or habituation property.In recent years, conversion ratio and the juice of some Feed Enterprise in order to pursue feed, stable in feed, stable is Oxazepam through liver metabolism, still has biologically active, so continuous application can be accumulated.The national departments concerned hereinafter clearly is decided to be stable class and forbids one of medicine that adds for this reason.
At present, detecting stable method comparatively commonly used has: high performance liquid chromatography (HPLC), thin layer chromatography scanning (TLC), photochemistry XRF (PCFA), enzyme linked immunosorbent assay (ELISA) etc.Wherein immunoassay is because its high specificity, and is highly sensitive, easy and simple to handle, and is particularly suitable for the advantage such as detection of batch samples and more and more paid attention to by people and adopt.
TRFIA is the new immunoassay that last century, early eighties grew up.Its principle of TRFIA is to utilize the sequestrant with bifunctional group structure, and the one of which end combines with lanthanide series, and the Eu3+ labelled antibody is processed in the free amino group connection on the other end and the antibody molecule, and the antigen in it and the testing sample is combined into immune complex.Under the ideal situation; The fluorescence intensity of measuring lanthanide series in the compound just can be confirmed the amount of antigen in the sample, but the fluorescence intensity of in fact this compound quite a little less than, have only to add a kind of enhancing solution (Enhancement Solution) again; Lanthanide series is disintegrated down from compound; And with strengthening β-naphthoyltrifluoroacetone contained in the liquid (β-NTA) form microcapsules again launches very strong fluorescence, up to a million times of reinforced effects under the exciting of light such as ultraviolet.Differentiate luminoscope with the time and measure its fluorescence intensity cps, can confirm the amount of antigen in the sample.
Summary of the invention
The object of the present invention is to provide kit and the detection method thereof of a kind of DIA of detection, be used for detection the stable content of samples such as urine, tissue, feed.To adopt time resolved fluoro-immunoassay method (TRFIA) to detect DIA among the present invention.Technical scheme: the kit of this detection DIA is to encapsulate plate by (1) 96 or 48 holes, (2) damping fluid, and standard is stabilized in (3), the antibody dried frozen aquatic products that (4) are stable, the goat anti-rabbit antibody of (5) europium mark, (6) cleansing solution, (7) strengthen liquid and form.
Assay method of the present invention: the basis of mensuration is the labelled immune reaction.Be coated with the microwell plate of DIA-OVA, add the DIA standard or the sample handled well in micropore separately, add DIA antibody again; Oscillating reactions; Free DIA and the competition of the DIA-OVA on microwell plate DIA antibody, the cleansing solution washing, the DIA antibody that does not have to connect is removed in washing step.Add EU
3+-goat anti-rabbit antibody carries out the labelled immune reaction, and again with the cleansing solution washing, the reaction back does not have the EU of connection
3+-goat anti-rabbit antibody is removed in washing step.After adding the vibration of enhancing liquid, the very strong fluorescence of emission under the exciting of ultraviolet light is differentiated luminoscope with the time and is measured its fluorescence intensity cps, and the concentration in fluorescence intensity and the sample is inversely proportional to, and the reference standard curve can be confirmed the amount of antigen in the sample.
Description of drawings
Fig. 1: DIA-TRFIA reacts synoptic diagram.
Fig. 2: DIA-TRFIA canonical plotting.
Fig. 3: detect stable kit synoptic diagram.(1) 96 or 48 holes encapsulate plate, (2) damping fluid, and standard is stabilized in (3), the antibody dried frozen aquatic products that (4) are stable, the goat anti-rabbit antibody of (5) europium mark, (6) cleansing solution, (7) strengthen liquid.
Specific embodiments
Follow these steps to prepare kit and detect urine sample:
(1) Eu
3+The preparation of-goat anti-rabbit antibody:
Get the 5g/L goat anti-rabbit antibody 1-2m that is dissolved in 50mmol/LPBS pH7.0, through the conversion buffered condition of PD-10 post, eluent is the 50mmol/LNa2CO3-NaHCO3 pH8.5 damping fluid that contains 0.155mol/LNaCl.Collect protein peak, through the quantitative (1.46A of uv absorption analysis
280-0.74A
260), dilute goat anti-rabbit antibody to 2g/L with above-mentioned eluent.The goat anti-rabbit antibody of getting after 500-1000 μ l dilutes adds the Eu that contains 0.2-0.4mg
3+-N
2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl (Eu
3+In-DTTA) the bottle, 30 ℃ of magnetic agitation reactions 20 hours.Reactant liquor is through Sepharose CL-6B post (1 * 40cm) chromatography, A with 80mmol/L Tris-HCl pH7.8 damping fluid balance
280Protein peak is collected in monitoring, and the dilution packing is subsequent use.
(2) encapsulate the preparation of plate solid phase antigen:
DIA-OVA is used 50mmol/L Na
2CO
3-NaHCO
3The pH9.6 damping fluid is diluted to the coating buffer of 5mg/L, and each hole of microwell plate, 96 (or 48) hole adds 100 μ l, and 4 ℃ of placements are spent the night.Discard coating buffer, flushing twice adds .4 ℃ of placement of above-mentioned damping fluid sealing that 150 μ 1 contain 3g/LBSA and spends the night.Discard confining liquid, vacuum is drained, rearmounted-20 ℃ of freezing preservations of lath sealing.
(3) preparation of reagent:
A. standard stable (DIA): (0ng/ml, 0.05ng/ml, 0.1ng/ml, 0.5ng/ml, 1ng/ml 5ng/ml), dilutes from the pure article of DIA and obtains, and dilution is a methyl alcohol: water=7: 3.
B. damping fluid: 8mmol/LNaCl, 0.1%BSA, 50 μ mol/L diethylene triamine pentacetic acid (DTPA)s (DTPA), 0.1ml/LTween-80 and 0.1%NaN
3Tris-HCl pH7.8.
C. cleansing solution: 14.5mmol/L NaCl, 0.2ml/LTween-80 and 0.2%NaN
350mmol/L Tris-HCl pH7.8.
D. strengthen the preparation of liquid: 1 liter of pH3.2 Potassium Hydrogen Phthalate damping fluid contains ketone in 15 μ mol β-naphthalene formyl trifluoro (β-NTA), 50 μ mol TOPOs (TOPO), 1ml triton x-100 (Triton X-100).
(4) reagent that provides of kit:
Reagent in each box enough carries out 96 measurements, and the material in the box is following:
A.1 * 96 orifice plate (8 * 12 hole can be split as single hole) is coated with DIA-OVA.
B.6 * and the DIA titer, the 1.0ml/ bottle, concentration of standard solution is: 0ng/ml, 0.05ng/ml, 0.1ng/ml, 0.5ng/ml, 1ng/ml, 5ng/ml.
C.1 * and DIA antibody dried frozen aquatic products, time spent 0.5ml dissolved in distilled water.
D.1 * EU
3+-goat anti-rabbit antibody dried frozen aquatic products, time spent 0.5ml dissolved in distilled water.
E.1 * enhancing liquid: 15ml.
F.1 * and cleansing solution: 30ml, the time spent is with distilled water dilution in 1: 25.
G.1 * damping fluid: 30ml.
(5) measure points for attention before:
A. before using all reagent are gone up to room temperature (18-30 ℃).
B. immediately all reagent are put back to 2-8 ℃ after using.
If C. the hyperchannel pipettor is used in the big suggestion of sample size.
D. hatch in the process at all constant temperature, avoid irradiate light, use the cap covers micropore.
E. taking-up needs microwell plate and the framework with quantity, no microwell plate is put in the former Fresco Bag and with the drying agent that provides reseal, and is stored in 2-8 ℃.
(6) it is following specifically to detect step:
Get the DIA-OVA lath, add the DIA standard of 50 μ l or the sample handled well in micropore separately, each standard and sample must use new suction nozzle; DIA antibody 50 μ l with damping fluid dilution in 1: 20; 37 ℃ vibrated 1 hour, and cleansing solution is washed four times, with the Eu of damping fluid dilution in 1: 20
3+-goat anti-rabbit antibody 100 μ l, 37 ℃ vibrated 45 minutes, washed six times with cleansing solution, added 200 μ l and strengthened liquid vibration measurement after 5 minutes.DIA content from the typical curve calculation sample is seen table 1 and Fig. 2, and this routine sample concentration is 0.07ng/ml.
Table 1
Follow these steps to prepare kit and detect the pork sample:
(1) the preparation kit is with (1)～(5) of embodiment 1.
(2) it is following specifically to detect step:
Remove the fat of sample and clean and chopping, get an amount of sample and place even matter device homogenate: the sample that takes by weighing after the 1g homogenate places tool plug centrifuge tube, adds the 4mL70% ethanolic solution, places vortex oscillator concussion 1min, places and shakes 15min on the shaking table; After staticly settling, place the centrifugal 10min of hydro-extractor 5000rpm: get supernatant and detect.
Get the DIA-OVA lath, add the DIA standard of 50 μ l or the sample handled well in micropore separately, each standard and sample must use new suction nozzle; DIA antibody 50 μ l with damping fluid dilution in 1: 20; 37 ℃ vibrated 1 hour, and cleansing solution is washed four times, with the Eu of damping fluid dilution in 1: 20
3+-goat anti-rabbit antibody 100 μ l, 37 ℃ vibrated 45 minutes, washed six times with cleansing solution, added 200 μ l and strengthened liquid vibration measurement after 5 minutes.DIA content from the typical curve calculation sample is seen table 2 and Fig. 2, and this routine sample concentration is 0.1ng/ml.
Table 2
Claims (3)
1. time resolved fluoro-immunoassay method kit that detects stable (DIA) is characterized in that strengthening liquid by goat anti-rabbit antibody (6) cleansing solution (7) that (1) 96 or 48 holes encapsulate stable antibody dried frozen aquatic products (5) the europium mark of the stable standard (4) of plate (2) damping fluid (3) forms.
2. a method of stabilizing with the described kit of claim 1 is characterized in that getting the micropore that is coated with DIA-OVA and encapsulates plate, adds the DIA standard or locates good at present sample in micropore separately; Add DIA antibody again, oscillating reactions, cleansing solution washing; The goat anti-rabbit antibody that adds the europium mark carries out the labelled immune reaction, the cleansing solution washing; Add and strengthen liquid vibration back measurement fluorescence intensity cps, the DIA content in the reference standard curve calculation sample.
3. the method that detection according to claim 2 is stable, it is operating as: get the micropore that is coated with DIA-OVA and encapsulate plate, add the DIA standard of 50 μ l or the sample handled well in micropore separately; Add the DIA antibody of 50 μ l with damping fluid (2) dilution; 25 ℃ of vibrations 1 hour, cleansing solution is given a baby a bath on the third day after its birth time, in addition 100 μ l Eu3+-goat anti-rabbit antibodies of damping fluid (2) dilution; 25 ℃ vibrated 0.5 hour; Wash six times with cleansing solution, add the vibration of 200 μ l enhancing liquid and measure fluorescence intensity cps, the DIA content from the typical curve calculation sample after 5 minutes.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010528958XA CN102455358A (en) | 2010-10-28 | 2010-10-28 | Time-resolved fluoroimmunoassay kit for detecting diazepam and detection method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010528958XA CN102455358A (en) | 2010-10-28 | 2010-10-28 | Time-resolved fluoroimmunoassay kit for detecting diazepam and detection method thereof |
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| Publication Number | Publication Date |
|---|---|
| CN102455358A true CN102455358A (en) | 2012-05-16 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102654500A (en) * | 2012-05-17 | 2012-09-05 | 重庆市科学技术研究院 | Detecting reagent kit used for detecting quinoxalinone-2-carboxylic acid and method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7255998B1 (en) * | 1999-09-29 | 2007-08-14 | Japan Science And Technology Corporation | High sensitivity immunoassay method |
| CN101482562A (en) * | 2008-12-30 | 2009-07-15 | 江苏省微生物研究所有限责任公司 | Detection reagent kit and detection method for diethyl stilbestrol |
| CN101713778A (en) * | 2009-12-04 | 2010-05-26 | 江苏省微生物研究所有限责任公司 | Detection kit for ciprofloxacin and detection method thereof |
-
2010
- 2010-10-28 CN CN201010528958XA patent/CN102455358A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7255998B1 (en) * | 1999-09-29 | 2007-08-14 | Japan Science And Technology Corporation | High sensitivity immunoassay method |
| CN101482562A (en) * | 2008-12-30 | 2009-07-15 | 江苏省微生物研究所有限责任公司 | Detection reagent kit and detection method for diethyl stilbestrol |
| CN101713778A (en) * | 2009-12-04 | 2010-05-26 | 江苏省微生物研究所有限责任公司 | Detection kit for ciprofloxacin and detection method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102654500A (en) * | 2012-05-17 | 2012-09-05 | 重庆市科学技术研究院 | Detecting reagent kit used for detecting quinoxalinone-2-carboxylic acid and method |
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Application publication date: 20120516 |