CN105301247A - Kit for quickly detecting content of chloroquine in crops - Google Patents
Kit for quickly detecting content of chloroquine in crops Download PDFInfo
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- CN105301247A CN105301247A CN201410352359.5A CN201410352359A CN105301247A CN 105301247 A CN105301247 A CN 105301247A CN 201410352359 A CN201410352359 A CN 201410352359A CN 105301247 A CN105301247 A CN 105301247A
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Abstract
The invention discloses preparation of an ELISA kit for detecting chloroquine and a detecting method of ELISA kit. The ELISA kit is sensitive, accurate and quick in detection, simple and convenient to operate and strong in specificity, and is suitable for batch sample detection. The kit comprises an elisa plate coated with chloroquine antigen, a chloroquine standard product, a chloroquine antibody working solution, a chloroquine enzyme-labeled second antibody working solution, a substrate solution A, a substrate solution B, a stopping solution, a concentrated sample diluting solution and a concentrated cleaning solution. The principle of the chloroquine detecting kit is solid-phase indirect competition ELISA, the extracted sample, enzyme-labeled second antibody working solution and antibody working solution are added into corresponding enzyme-labeled holes, after a period of incubation, the substrate solution A and the substrate solution B are added into a cleaning plate, the holes turn blue under the action of enzymes, then the stopping solution is added, and the color turns from blue into yellow. The shade of coloration is in inversely proportional relationship with the content of chloroquine in the standard product or the sample. The method can be directly used for detecting the content of chloroquine in feed and animal tissues.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection technical field, particularly detect the enzyme linked immunological kit of chloroquine.
Background technology
Chloroquine is comparatively slow human body metabolism, easily causes the reactions such as anorexia, n and V, diarrhoea when content exceedes a certain amount of; Also can occur that pruitus, purpura, depilation, hair bleach, eczema and exfoliative dermatitis, psoriasis; Nose heave, headache, giddy, tinnitus, dizzy, burnout, sleep-disorder, amentia, the visual field are reduced, cornea and retinosis etc.
At present, the assay method of chloroquine mainly contains high performance liquid chromatography, reversed-phased high performace liquid chromatographic, Surface enhanced raman spectroscopy method etc., and 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 agricultural product security 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 chloroquine.
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 chloroquine.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 chloroquine, comprises chloroquine ELISA Plate, chloroquine standard items, chloroquine antibody working fluid, chloroquine 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 chloroquine, comprises the following steps: the preparation of the preparation of the preparation of the preparation of ELISA Plate, the preparation of chloroquine standard items, chloroquine antibody working fluid, the preparation of chloroquine 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.
Chloroquine haptens and carrier protein couplet obtain by chloroquine envelope antigen, and this carrier protein is bovine serum albumin(BSA) (BSA); With carbonate (CBS) damping fluid of 0.05mol/LpH9.6 as coating buffer, chloroquine 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 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, 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.
Chloroquine standard concentration is respectively 0ng/mL, 0.5ng/mL, 1.5ng/mL, 4.5ng/mL, 13.5ng/mL, 40.5ng/mL.
Chloroquine antibody working fluid adopts chloroquine artificial antigen immune mouse to obtain monoclonal antibody, is diluted to the preparation of 1:40000 ratio with antibody diluent.
Chloroquine ELIAS secondary antibody working fluid ELIAS secondary antibody diluted becomes 1:5000 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; 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.
The detection method of the ELISA kit of chloroquine 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 chloroquine antigen, add standard items/sample 50 μ L/ hole in the micropore of correspondence;
(4) add chloroquine ELIAS secondary antibody working fluid, 50 μ L/ holes, then add chloroquine 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 15min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate;
(7) add termination 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 chloroquine in reference standard curve calculation sample.
Testing sample following methods carries out pre-service:
(1) prepared and diluted liquid: water-acetonitrile-methanesulfonic acid solution (7mL methane-sulforic acid joins in 4mL glacial acetic acid, is diluted with water to 100mL)-diethylamine solution (2mL is diluted to 20mL, shakes up) (86:10:2:2);
(2) representative sample or by tissue homogenate is ground;
(3) sample that weighing 160mg handles well adds 80mL methanol solution;
(4) mix in the container of sealing, concussion vortex 30min;
(5) room temperature centrifugal more than 4000r/min, 10min; Get 3ml supernatant;
(6) with the dilution proportion supernatant of 1:9, mix to be measured.
Accompanying drawing explanation
Fig. 1 is the canonical plotting of chloroquine.
Embodiment
The preparation of chloroquine protein conjugate:
Succinic anhydride method is adopted to obtain being with the chloroquine 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 chloroquine 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 chloroquine envelope antigen:
Chloroquine haptens and carrier protein couplet obtain by chloroquine envelope antigen, and this carrier protein is bovine serum albumin(BSA) (BSA); With carbonate (CBS) damping fluid of 0.05mol/LpH9.6 as coating buffer, chloroquine 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 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, 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.
Chloroquine standard items compound concentration is respectively 0ng/mL, 0.5ng/mL, 1.5ng/mL, 4.5ng/mL, 13.5ng/mL, 40.5ng/mL.
Chloroquine antibody working fluid adopts chloroquine artificial antigen immune mouse to obtain monoclonal antibody, is diluted to the preparation of 1:40000 ratio with antibody diluent.
Chloroquine ELIAS secondary antibody working fluid ELIAS secondary antibody diluted becomes 1:5000 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; 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.
Based on the reagent of above-mentioned preparation, the present invention comprises following material for the enzyme linked immunological kit detecting chloroquine:
(1) 96 ELISA Plate × 1 piece, hole;
(2) titer × 6 bottle: (1mL/ bottle) 0ng/mL, 0.5ng/mL, 1.5ng/mL, 4.5ng/mL, 13.5ng/mL, 40.5ng/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 chloroquine residual quantity in sample, implemented by following steps: sample pretreatment, with kit of the present invention carry out detecting, analysis result.
(1) sample pretreatment
The sample weighed after 160mg grinding filtration adds 80mL methanol solution, mixes in the container of sealing, concussion vortex 1min; Room temperature centrifugal more than 4000r/min, 10min; Get 3ml supernatant; With the dilution proportion supernatant of 1:9, mix to be measured.
(2) chloroquine residual quantity in testing sample is detected with kit of the present invention
Get the ELISA Plate being coated with chloroquine 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 chloroquine 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, 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; 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, chloroquine 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 chloroquine (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 chloroquine in sample.
Claims (8)
1. detect preparation and the detection method of the ELISA kit of chloroquine, comprise chloroquine ELISA Plate, chloroquine standard items, chloroquine antibody working fluid, chloroquine ELIAS secondary antibody working fluid, substrate solution A, substrate solution B, stop buffer, concentrating sample dilution and concentrated cleaning solution.
2. detect the preparation of ELISA kit and the detection method of chloroquine, comprise the following steps: the preparation of the preparation of chloroquine ELISA Plate, the making of chloroquine standard items, chloroquine monoclonal antibody and working fluid thereof, the preparation of chloroquine 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, the preparation of concentrating sample dilution.
3. the preparation of the ELISA kit of detection chloroquine according to claim 2 and detection method, it is characterized in that: chloroquine haptens and carrier protein couplet obtain by described chloroquine envelope antigen, this carrier protein is bovine serum albumin(BSA) (BSA); With carbonate (CBS) damping fluid of 0.05mol/LpH9.6 as coating buffer, chloroquine 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 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, 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. the preparation of the ELISA kit of detection chloroquine according to claim 2 and detection method, is characterized in that: described chloroquine standard concentration is respectively 0ng/mL, 0.5ng/mL, 1.5ng/mL, 4.5ng/mL, 13.5ng/mL, 40.5ng/mL.
5. the preparation of the ELISA kit of detection chloroquine according to claim 2 and detection method, it is characterized in that: described chloroquine antibody working fluid adopts chloroquine artificial antigen immune mouse to obtain monoclonal antibody, is diluted to the preparation of 1:40000 ratio with antibody diluent.
6. the preparation of the ELISA kit of detection chloroquine according to claim 2 and detection method, is characterized in that: described chloroquine ELIAS secondary antibody working fluid ELIAS secondary antibody diluted becomes 1:5000 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 chloroquine 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 chloroquine antigen, add standard items/sample 50 μ L/ hole in the micropore of correspondence;
(4) add chloroquine ELIAS secondary antibody working fluid, 50 μ L/ holes, then add chloroquine 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 15min 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 chloroquine 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) prepared and diluted liquid: water-acetonitrile-methanesulfonic acid solution (7mL methane-sulforic acid joins in 4mL glacial acetic acid, is diluted with water to 100mL)-diethylamine solution (2mL is diluted to 20mL, shakes up) (86:10:2:2);
(2) representative sample or by tissue homogenate is ground;
(3) sample that weighing 160mg handles well adds 80mL methanol solution;
(4) mix in the container of sealing, concussion vortex 30min;
(5) room temperature centrifugal more than 4000r/min, 10min; Get 3ml supernatant;
(6) with the dilution proportion supernatant of 1:9, mix to be measured.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410352359.5A CN105301247A (en) | 2014-07-23 | 2014-07-23 | Kit for quickly detecting content of chloroquine in crops |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410352359.5A CN105301247A (en) | 2014-07-23 | 2014-07-23 | Kit for quickly detecting content of chloroquine in crops |
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| CN105301247A true CN105301247A (en) | 2016-02-03 |
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| CN201410352359.5A Pending CN105301247A (en) | 2014-07-23 | 2014-07-23 | Kit for quickly detecting content of chloroquine in crops |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109752526A (en) * | 2017-11-05 | 2019-05-14 | 镇江华维检测技术有限公司 | A kind of enzyme linked immunological kit detecting chloroquine content |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1374867A1 (en) * | 2002-06-28 | 2004-01-02 | Valpharma S.A. | Use of chloroquine, hydroxychloroquine and 4 amino-quinolinic derivatives to obtain a drug for the anti retroviral therapy, active towards hiv sensitive strains and towards hiv strains which are resistant to both nucleosidic and non-nucleosidic reverse transcriptase inhibitors and to proteases inhibitors |
| CN102901782A (en) * | 2012-10-30 | 2013-01-30 | 湖南省烟草公司郴州市公司 | Residue detection method for quinclorac in tobacco leaf and tobacco planting soil |
-
2014
- 2014-07-23 CN CN201410352359.5A patent/CN105301247A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1374867A1 (en) * | 2002-06-28 | 2004-01-02 | Valpharma S.A. | Use of chloroquine, hydroxychloroquine and 4 amino-quinolinic derivatives to obtain a drug for the anti retroviral therapy, active towards hiv sensitive strains and towards hiv strains which are resistant to both nucleosidic and non-nucleosidic reverse transcriptase inhibitors and to proteases inhibitors |
| CN102901782A (en) * | 2012-10-30 | 2013-01-30 | 湖南省烟草公司郴州市公司 | Residue detection method for quinclorac in tobacco leaf and tobacco planting soil |
Non-Patent Citations (3)
| Title |
|---|
| C. ESCAND 等: "Sensitive Radioimmunoassay and Enzyme-Linked lmmunosorbent Assay for the Simultaneous Determination of Chloroquine and Its Metabolites in Biological Fluids", 《JOURNAL OF PHARMACEUTICAL SCIENCES》 * |
| INSAF F KHALIL 等: "Development of ELISA-based methods to measure the anti-malarial drug chloroquine in plasma and in pharmaceutical formulations", 《MALARIA JOURNAL》 * |
| V. ROWELL 等: "A specific ELISA method for determining chloroquine in urine or dried blood spots", 《BULLETIN OF THE WORLD HEALTH ORGANIZATION》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109752526A (en) * | 2017-11-05 | 2019-05-14 | 镇江华维检测技术有限公司 | A kind of enzyme linked immunological kit detecting chloroquine content |
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