CN105301245A - Preparation method of enzyme-linked immunosorbent assay (ELISA) kit for detecting cyproheptadine hydrochloride - Google Patents
Preparation method of enzyme-linked immunosorbent assay (ELISA) kit for detecting cyproheptadine hydrochloride Download PDFInfo
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- CN105301245A CN105301245A CN201410349742.5A CN201410349742A CN105301245A CN 105301245 A CN105301245 A CN 105301245A CN 201410349742 A CN201410349742 A CN 201410349742A CN 105301245 A CN105301245 A CN 105301245A
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Abstract
The invention provides a preparation method of enzyme-linked immunosorbent assay (ELISA) kit for detecting cyproheptadine hydrochloride. The kit is sensitive, accurate and rapid in detection, simple and convenient to operate, high in specificity and suitable for detecting samples in large batch, and comprises an ELISA plate coated with a cyproheptadine hydrochloride antigen, a cyproheptadine hydrochloride standard substance, a cyproheptadine hydrochloride enzyme-labeled antibody working solution, a substrate solution A, a substrate solution B, a stop solution and a concentrated scrubbing solution. According the principle of the kit, an ELISA reaction is performed through direct solid-phase competition; an extracted sample and the enzyme-labeled antibody working solution are added into a corresponding ELISA hole; after a period of incubation, the ELISA plate is scrubbed, and the substrate solution A and the substrate solution B are added; under the enzymatic action, a blue color appears in the ELISA hole; after the stop solution is added, the color changes from blue to yellow, and the color depth has an inversely proportional relationship with the content of cyproheptadine hydrochloride in the standard substance or the sample. The principle can be directly utilized for detecting the content of cyproheptadine hydrochloride in a swine urine sample.
Description
Technical field
The invention belongs to detection technique field, relating to a kind of enzyme linked immunological kit for quantitatively detecting the anarexol residual quantity in pig urine sample and detection method particularly.
Background technology
Anarexol is H1 receptor antagonist, belongs to antihistamine, but is also 5-hydroxytryptamine receptor antagonist simultaneously, legal human drugs, in antagonist, histamine is to the effect of blood vessel, bronchial smooth muscle, thus eliminates allergic symptom, also can be used for the appetite improving patient.Acid hydrochloride salt cyproheptadine is used for aquaculture can promote growth of animal, it has the effect of medmain, can suppress hypothalamic satiety center and stimulate appetite to put on weight, therefore some livestock-raising enterprises it can be used as illegal feed addictive for promoting growth of animals or poultry.Anarexol residual can generation health is in animal body endangered.Clear stipulaties in Dec, 2010 Ministry of Agriculture No. 1519 bulletins " forbidding the material used in feed and drinking water for animals ", prohibit the use such medicine as production promoter for livestock and poultry cultivation, in the same year, the Ministry of Agriculture discloses " the mensuration Liquid Chromatography-Tandem Mass Spectrometry of the fixed and anarexol of feed medium coke ".The method report that at present in the external existing blood to people and urine, acid hydrochloride salt cyproheptadine detects, be mainly high performance liquid chromatography and Liquid Chromatography-Tandem Mass Spectrometry, the analytical approach that Liquid Chromatography-Tandem Mass Spectrometry detects the acid hydrochloride salt cyproheptadine content of illegal interpolation in feed was also once set up in this laboratory, but there is no the examination criteria of acid hydrochloride salt cyproheptadine in animal edible tissues and pig urine at present both at home and abroad.
At present, the method detecting anarexol mainly contains high performance liquid chromatography HPLC, vapor-phase chromatography GC etc.Wherein, high performance liquid chromatography HPLC, vapor-phase chromatography GC are quantitative detection methods, result accurately and reliably, shortcoming is that detection limit is higher, instrument and equipment costly, and the Sample pretreatment method of complexity, limits the widespread use of the method.Enzyme-linked Immunosorbent Assay ELISA method is a kind of accurate, reliable, quick, special detection method, is suitable for the rapid screening of gross sample, in recent years widespread use.The present invention is intended to set up a kind of preparation and the detection method that detect the ELISA kit of acid hydrochloride salt cyproheptadine in pig urine.
Summary of the invention
For problems of the prior art, the invention provides the ELISA kit detecting anarexol, its detection is sensitive, accurate, quick, easy and simple to handle, high specificity, is applicable to the detection of gross sample.The invention provides a kind of preparation of the ELISA kit for detecting anarexol.
Detect the preparation of the ELISA kit of anarexol, comprise ELISA Plate, anarexol standard items, anarexol enzyme labelled antibody working fluid, substrate solution A, substrate solution B, stop buffer and concentrated cleaning solution.
Detect the preparation of the ELISA kit of anarexol, comprise the following steps: the preparation of the preparation of ELISA Plate, the making of anarexol standard items, anarexol enzyme labelled 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.
It is further characterized in that: anarexol haptens and carrier protein couplet obtain by described anarexol 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, anarexol 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 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.
Described anarexol standard concentration is respectively 0ppb, 0.1ppb, 0.3ppb, 0.9ppb, 2.7ppb, 8.1ppb.
The preparation of anarexol enzyme labelled antibody working fluid: adopt horseradish peroxidase coupling anarexol single gram of clonal antibody to obtain, this enzyme labelled antibody working fluid antibody diluent is diluted to 1:20000.
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.
The detection method of the ELISA kit of anarexol, carry out competitive ELISA 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 anarexol antigen, add standard items/sample 50 μ L/ hole in the micropore of correspondence;
(4) add enzyme labelled 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, dried by liquid in hole, with wash operating solution 300 μ L/ hole, fully washing 4 times, soaks 15-30s, pats dry 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 anarexol in reference standard curve calculation sample.
Wherein, the sample after described process is through the sample of following process:
Get 5mL sample in clean glass test tube, add the chromatographically pure normal hexane of 2mL, vibrate after closing the lid 3min, then leaves standstill, and gets supernatant liquor 1mL after layering, dries up in clean glassware room temperature under nitrogen, and the methyl alcohol drying up rear 1mL35% redissolves rear to be measured.
The present invention adopts enzyme linked immunosorbent assay (ELISA) method to detect.For detecting the measuring principle of the ELISA kit of anarexol: antigen-specific sexual competition enzyme labelled antibody fixing in the anarexol in sample and ELISA Plate, by enzymatic chromogenic reagent, carry out the content of anarexol in judgement sample according to the depth of colour developing.If the anarexol 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 detection of batch samples.
Accompanying drawing explanation
Fig. 1 is anarexol canonical plotting.
Embodiment
Detect the ELISA kit of anarexol, it comprises ELISA Plate, anarexol standard items, anarexol enzyme labelled antibody working fluid, substrate solution A, substrate solution B, stop buffer and concentrated cleaning solution.
Specifically describe the preparation detecting the ELISA kit of anarexol in the present invention below,
The preparation of anarexol protein conjugate:
By anarexol haptens and carrier protein BSA by the combination of 12:1 than being blended in the carbonate buffer solution (CBS) of 0.05mol/LpH9.6, then add into carbodiimide, stir l ~ 2h, put room temperature reaction 24h, finally dialyse two days with the PBS of 0.2mol/LpH7.6, remove unreacted haptens, anarexol protein conjugate can be obtained.
The preparation of anarexol antibody:
Select 3 monthly ages healthy White Rabbit, by three immunity, each anarexol protein conjugate immunogene consumption is 50 μ g/0.05mL, and each immunization interval time is 2 weeks.Initial immunity is respectively by immunizing antigen and Fei Shi adjuvant and dangerous adjuvant mixed in equal amounts, and the subcutaneous multi-point injection in strength portion, secondary, three immunity directly inject abdominal cavity.Merge first 3 days every Intraperitoneal injection 25 μ g anarexol protein conjugates and carry out booster immunization, after immunity, 6d ear vein gets blood, adopts direct competive ELISA method to measure and tires.Directly inject at leg muscle with haptens after obtaining more efficient valency, 8d rear neck artery adopts whole blood, is separated antiserum, adopts caprylic acid-ammonium antibody purification, saves backup after making freeze-dried powder in-20 DEG C.
Preparation horseradish peroxidase-labeled anarexol monoclonal antibody: adopt periodates oxidizing process.
Preparation is coated with the ELISA Plate of anarexol envelope antigen:
ELISA Plate bag is by anarexol antigen, and anarexol haptens and carrier protein couplet obtain by 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, anarexol 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 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.
Described anarexol standard items compound concentration is respectively 0ppb, 0.1ppb, 0.3ppb, 0.9ppb, 2.7ppb, 8.1ppb.
The preparation of described anarexol enzyme labelled antibody working fluid: adopt horseradish peroxidase coupling anarexol single gram of clonal antibody to obtain, this enzyme labelled antibody working fluid antibody diluent is diluted to 1:20000.
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.
Based on the reagent of above-mentioned preparation, the present invention comprises following material for the ELISA kit detecting anarexol:
(1) 96 ELISA Plate × 1 piece, hole;
(2) titer × 6 bottle: (1mL/ bottle) 0ppb, 0.1ppb, 0.3ppb, 0.9ppb, 2.7ppb, 8.1ppb;
(3) enzyme labelled antibody working fluid 7mL;
(4) substrate solution A7mL;
(5) substrate solution B7mL;
(6) stop buffer 7mL;
(7) 20 × concentrated cleaning solution 20mL;
Use points for attention during this kit:
(1) room temperature is not got back to room temperature (20 ~ 25 DEG C) lower than 20 DEG C or reagent and sample the OD value of all standards can be caused on the low side;
(2) washing the situation if there is plate hole drying in plate process, then there will be typical curve non-linear, the phenomenon that repeatability is bad.Next step operation should be carried out immediately so wash after plate pats dry;
(3) need to be shaken up before often adding a kind of reagent;
(4) reaction terminating liquid is 2M hydrochloric acid, avoids contacting skin;
(5) kit of date of expiration was not used; Also do not use any reagent in the kit of the term of validity, doping had used the kit of the term of validity can cause the reduction of sensitivity; Do not exchange the reagent used in different lot number kit;
(6) condition of storage: preserve kit in 2 ~ 8 DEG C, not freezing, put no ELISA Plate microwell plate into valve bag and reseal.Standard substance and colourless colour former to photaesthesia, under therefore will avoiding being directly exposed to light;
(7) sign that reagent is rotten: chromogenic agents have any color to show colour former goes bad, should be abandoned it.When absorbance (450/630nm) value of 0 standard is less than 0.5 (A450nm<0.5), represents that reagent may go bad, please don't use;
(8) this kit optimal reaction temperature is 25 DEG C, too high or too low for temperaturely will cause detecting absorbance and sensitivity and changes.
The ELISA kit of anarexol of the present invention is detecting the application in the anarexol residual quantity in pig urine samples:
When kit of the present invention is for detecting the anarexol residual quantity in pig urine sample, implemented by following steps: sample pretreatment, with kit of the present invention carry out detecting, analysis result.
(1) sample pretreatment
Get urine sample to be checked, if when sample is more muddy, can more than 5000r/min, centrifugal 5min or filtration.
(2) anarexol residual quantity in detection pig urine samples is carried out with kit of the present invention
Get the ELISA Plate being coated with anarexol antigen, add standard items/sample 50 μ L/ hole in the micropore of correspondence; Add enzyme labelled 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 300 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, the residual quantity of the anarexol in quantitative test testing sample.
(3) analysis result
With 6 anarexol standard concentration 0ppb, 0.1ppb, 0.3ppb, 0.9ppb, 2.7ppb, 8.1ppb in the kit of above-mentioned preparation, measure absorbance at 450/630nm place.
The calculating of percentage absorptance, the percentage absorptance of standard items or sample equals the absorbance of mean value (diplopore) divided by first standard (0 standard) of the percentage absorbance of standard items or sample, then is multiplied by 100%, that is:
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 standard items percentage absorptance for ordinate, be horizontal ordinate drawing standard curve with the semilog of anarexol standard concentration (ppb), obtain straight-line equation.Typical curve is shown in accompanying drawing 1.Y=－18.550X+99.32，R
2=0.9967。By the B/B of sample
0value substitutes in typical curve, and read the concentration of corresponding sample from typical curve, the extension rate being multiplied by its correspondence is the actual concentrations of anarexol in sample.
Claims (8)
1. detect the preparation of the ELISA kit of anarexol, comprise ELISA Plate, anarexol standard items, anarexol enzyme labelled antibody working fluid, substrate solution A, substrate solution B, stop buffer and concentrated cleaning solution.
2. detect the preparation of ELISA kit of anarexol, comprise the following steps: the preparation of the preparation of ELISA Plate, the making of anarexol standard items, anarexol monoclonal antibody and enzyme labelled 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. the preparation of the ELISA kit of the detection anarexol of anarexol according to claim 2, it is characterized in that: anarexol haptens and carrier protein couplet obtain by described anarexol envelope antigen, this carrier protein is bovine serum albumin(BSA) (BSA); With carbonate (CBS) damping fluid of 0.05mol/LpH9.6 as coating buffer, anarexol 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 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 the detection anarexol of anarexol according to claim 2, is characterized in that: described anarexol standard concentration is respectively 0ppb, 0.1ppb, 0.3ppb, 0.9ppb, 2.7ppb, 8.1ppb.
5. the preparation of the ELISA kit of the detection anarexol of anarexol according to claim 2, it is characterized in that: the preparation of described anarexol enzyme labelled antibody working fluid: adopt horseradish peroxidase coupling anarexol single gram of clonal antibody to obtain, this enzyme labelled antibody working fluid antibody diluent is diluted to 1:20000.
6. the preparation of the ELISA kit of the detection anarexol of anarexol according to claim 2, is characterized in that: 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 anarexol according to claim 2, carry out competitive ELISA reaction based on antigen-antibody, the method comprises the following steps:
(1) pre-service testing sample;
(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 anarexol antigen, add standard items/sample 50 μ L/ hole in the micropore of correspondence;
(4) add enzyme labelled 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, dried by liquid in hole, with wash operating solution 300 μ L/ hole, fully washing 4 times, soaks 15-30s, pats 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 anarexol 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:
Get pig to be checked urine, if when sample is more muddy, can more than 5000r/min, centrifugal 5min or filtration.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104792989A (en) * | 2014-01-16 | 2015-07-22 | 北京维德维康生物技术有限公司 | Cyproheptadine hapten, antigen, and preparation method and applications of cyproheptadine hapten and antigen |
| CN108424880A (en) * | 2018-03-16 | 2018-08-21 | 江南大学 | A kind of hybridoma cell strain and preparation method of secretion cyproheptadine monoclonal antibody |
| CN109813892A (en) * | 2017-11-18 | 2019-05-28 | 丹阳亿太生物科技发展有限公司 | Anarexol rapid time resolved fluorometric immunochromatographiassay assay quantitative detection test paper |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN203148947U (en) * | 2013-02-28 | 2013-08-21 | 上海快灵生物科技有限公司 | Cyproheptadine immunochromatography rapid test paper card |
| CN203178273U (en) * | 2013-04-19 | 2013-09-04 | 北京勤邦生物技术有限公司 | Cyproheptadine ELISA (enzyme-linked immunosorbent assay) test kit |
| CN203178285U (en) * | 2013-04-19 | 2013-09-04 | 北京勤邦生物技术有限公司 | Test paper for rapidly detecting residual cyproheptadine |
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2014
- 2014-07-22 CN CN201410349742.5A patent/CN105301245A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN203148947U (en) * | 2013-02-28 | 2013-08-21 | 上海快灵生物科技有限公司 | Cyproheptadine immunochromatography rapid test paper card |
| CN203178273U (en) * | 2013-04-19 | 2013-09-04 | 北京勤邦生物技术有限公司 | Cyproheptadine ELISA (enzyme-linked immunosorbent assay) test kit |
| CN203178285U (en) * | 2013-04-19 | 2013-09-04 | 北京勤邦生物技术有限公司 | Test paper for rapidly detecting residual cyproheptadine |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104792989A (en) * | 2014-01-16 | 2015-07-22 | 北京维德维康生物技术有限公司 | Cyproheptadine hapten, antigen, and preparation method and applications of cyproheptadine hapten and antigen |
| CN109813892A (en) * | 2017-11-18 | 2019-05-28 | 丹阳亿太生物科技发展有限公司 | Anarexol rapid time resolved fluorometric immunochromatographiassay assay quantitative detection test paper |
| CN108424880A (en) * | 2018-03-16 | 2018-08-21 | 江南大学 | A kind of hybridoma cell strain and preparation method of secretion cyproheptadine monoclonal antibody |
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Application publication date: 20160203 |