CN103226146A - Clenbuterol, ractopamine and salbutamol three-in-one euzyme linked immunosorbent assay, special kit and kit detection method - Google Patents
Clenbuterol, ractopamine and salbutamol three-in-one euzyme linked immunosorbent assay, special kit and kit detection method Download PDFInfo
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Abstract
The invention discloses a clenbuterol, ractopamine and salbutamol disposable method for euzyme linked immunosorbent assay detection, which is characterized in that a monoclonal antibody is precoated on an enzyme label plate, clenbuterol, ractopamine and salbutamol in a sample to be detected and enzyme-labelled antigen compete for binding with the coated monoclonal antibody, a developer is used for developing, the content of clenbuterol, ractopamine and salbutamol in the sample to be detected is in inverse proportion with percent absorbency of the sample to be detected, compared with a standard curve, the content of clenbuterol, ractopamine and salbutamol in the sample to be detected can be obtained, and a special detection kit used for the above mentioned detection method comprises the enzyme label plate, a standard substance solution, a concentrated enzyme marker, an enzyme marker diluents, the developer, a concentrated washing lotion and a stopping solution. The invention uses a direct competition enzyme linked immunosorbent assay technology, three kinds of clenbuterol hydrochloride which are clenobuterol hydrochloride, ractopamine and salbutamol are performed with disposable analysis detection, the usage is simple, the specificity is high, and the detection scope is 0.1ng/mL-2mumg/mL.
Description
Technical field
The present invention relates to beta-agonist detection technique field, is the euzymelinked immunosorbent assay (ELISA) of a kind of Clenbuterol, Ractopamine, the disposable detection of salbutamol and the detection method of dedicated test kit and kit thereof specifically.
Background technology
Along with improving constantly of people's living standard, people begin the healthy of oneself also more and more paid close attention to, in daily life, people's meat product of can often cooking, strengthen self immunity and extra-nutrition, but when cooking meat product, people tend to worry a problem---" whether containing beta-agonist (clenbuterol hydrochloride) in the meat product ", beta-agonist is a kind of medicated premix that can promote the lean meat growth, and for example clenobuterol hydrochloride is acid hydrochloride salt Clenbuterol, Ractopamine, salbutamol etc.It can make pig improve the speed of growth, increases lean meat percentage, and can make the ruddy light of bristles color, sells the lover; After butchering, yellowish pink scarlet, fat deposit as thin as a wafer, skin is pasting lean meat often, lean meat is plentiful, has side effects but the people known from experience, and gently then causes cardiac arrhythmia, seriously a bit will cause heart disease.
Detect the beta-agonist method of mainly containing at present methods such as chromatographic technique and immuno analytical method are arranged.
Chromatography comprises high performance liquid chromatography and gas chromatography mass spectrometry chromatography.The advantage of high performance liquid chromatography (HPLC) method is to detect the precision height, and false positive rate is low, but exist the sample preparation time long, testing process is loaded down with trivial details, instrument is expensive, be difficult to shortcoming such as operation, is subjected to certain restriction in actual applications.And gas chromatography mass spectrometry method (GC-MS) needs the hydroxyl on the molecule, amino isopolarity group derivatization before detecting, and is difficult to use as conventional method, can not carry out the scene and detect.
Immuno analytical method comprises radioimmunoassay technology, gold-marking immunity analysis and EIA enzyme immunoassay technology technology etc.The radioimmunoassay technology have high specificity, highly sensitive, accurate, quick, easy and simple to handle, be easy to advantages such as standardization, but its used instrument costliness, and the radioactive isotope that uses exists radiation and pollution, and its discarded object is difficult for handling, and has limited the widespread use of this kind method; The gold-marking immunity analytical technology can be screened a large amount of samples, but its false positive rate is higher, the detection sensitivity shortcoming, and stability is not enough, and result accurately can't be provided.
Summary of the invention
The purpose of this invention is to provide the euzymelinked immunosorbent assay (ELISA) of a kind of Clenbuterol, Ractopamine, the disposable detection of salbutamol and the detection method of dedicated test kit and kit thereof, to overcome present prior art above shortcomings, highly sensitive, good stability, accurately and reliably, the step that simplifies the operation greatly and reaction time, and reduce cost, be fit to the examination of a large amount of samples.
The objective of the invention is to be achieved through the following technical solutions:
A kind of Clenbuterol, Ractopamine, the enzyme-linked immune detection method of the disposable detection of salbutamol, it is characterized in that: monoclonal antibody on pre-bag quilt on the ELISA Plate, Clenbuterol in the detected sample, salbutamol, Ractopamine, also have enzyme-labelled antigen to compete together in conjunction with after the monoclonal antibody of wrapping quilt, use chromogenic reagent, Clenbuterol in the detected sample, Ractopamine, the percentage absorbance of salbutamol content and detected sample is inverse ratio, relatively can draw Clenbuterol in the detected sample with typical curve, salbutamol, Ractopamine content, described typical curve is with Clenbuterol, Ractopamine, salbutamol standard items concentration percentage absorbance is an ordinate, with Clenbuterol, Ractopamine, the logarithm value of salbutamol standard items concentration is that the horizontal ordinate drafting forms, the monoclonal anti physical efficiency of described pre-bag quilt is simultaneously to Clenbuterol, Ractopamine, salbutamol generation cross reaction, described percentage absorbance is calculated as follows:
Wherein B is the mean light absorbency value of standard solution or sample solution; B0 is the mean light absorbency value of first standard solution.
Described enzyme-labelled antigen is by horseradish peroxidase HRP mark Clenbuterol, salbutamol, Ractopamine and obtaining, described detected sample is a pig urine to be checked, the detected sample of pork liver or blood serum, if the limpid urine sample of pig urine detected sample, just directly measure, as the muddy palpus filtration of urine sample or centrifugal until limpid, wouldn't use the urine sample detected sample to answer freezing preservation, avoid multigelation to take, described enzyme-labelled antigen is by horseradish peroxidase HRP mark Clenbuterol, salbutamol, Ractopamine and obtaining, described blood serum detected sample disposal route is: get serum, add enzyme labeling thing diluted, concussion mixes and gets final product.
A kind of three-in-one enzyme-linked immunologic detecting kit of Clenbuterol, Ractopamine, salbutamol that is exclusively used in aforementioned detection method is characterized in that described kit comprises: 1) the described pre-bag of claim 1 is by the ELISA Plate of last monoclonal antibody; 2) the described Clenbuterol of claim 1, Ractopamine, the three-in-one standard solution of salbutamol; 3) concentrate the enzyme labeling thing; 4) enzyme labeling thing dilution; 5) colour developing liquid; 6) concentrate washing lotion; 7) stop buffer; Described enzyme labeling thing is direct oxidation horseradish peroxidase HRP under low pH condition, and horseradish peroxidase HRP is through NaIO
4The hydroformylation enzyme that forms after the oxidation links to each other with the amino of the antigen molecule of Clenbuterol, salbutamol, three kinds of materials of Ractopamine, forms this Fu Shi alkali, further uses NaBH
4Reduction generates stable material.
The monoclonal antibody of described pre-bag quilt is merged through bone marrow cell by synthetic good comlete antigen immune mouse, cultivates, and filters out the hybrid cell strain of secretory antibody, and the hybrid cell strain is carried out cloning and obtained.
The Clenbuterol of described detection kit, Ractopamine, salbutamol cross reacting rate are respectively: 100%, 80%, 80%, and the recovery that described detection kit detects urine and serum all is 60～140%.
The sensitivity that described detection kit detects urine and serum is respectively 0.1ppb, 0.3ppb.
Be limited to 0.1ppb under the lowest detection of described detection kit standard items, be limited to 2ppb on the highest detection, the detected sample that exceeds this concentration needs to detect after an amount of dilution again.
Described detection kit has following characteristic:
1) sets up typical curve: the typical curve of making direct competitive ELISA (enzyme linked immunosorbent assay (ELISA)) of ELISA Plate in the kit and standard solution, logarithm with drug concentration is a horizontal ordinate, inhibiting rate is an ordinate, mapping obtains the typical curve of ELISA kit, and the ELISA Chinese meaning is enzyme linked immunosorbent assay (ELISA);
2) criticize interior and differences between batches: get with a collection of product, do parallel typical curve, calculate the variation within batch coefficient of each drug concentration, the product drug concentration is the parallel laboratory test that 1 μ g/L does corresponding number between getting batch, calculate the batch variation coefficient of variation, be direct competitive ELISA, testing result to same batch is analyzed, batch interior average coefficient of variation that obtains is 4.88%, testing result to different batches is analyzed, obtain batch between average coefficient of variation be 6.96%, show that this research method repeatability is better;
3) get blank urine sample, the standard solution to wherein adding the variable concentrations gradient detects with kit, calculate its recovery, measure the urine of variable concentrations, record its recovery all between 80%-120%, illustrate that this kit is more accurate to the result that detected sample detects;
4) cross reacting rate of various analogues satisfies 100% Clenbuterol cross reacting rate, the cross reacting rate of 80% Ractopamine, the cross reacting rate of 80% salbutamol, and the high specificity that this kit detects detected sample is described.
The detection method of the enzyme-linked immunologic detecting kit that a kind of Clenbuterol, Ractopamine, salbutamol are three-in-one, wherein ELISA Plate is made into the ELISA Plate capillary strip, may further comprise the steps:
1) before the use described kit is placed room temperature 20-25 ℃ more than the balance 30min, note to shake up before every kind of liquid reagent uses, taking-up needs the ELISA Plate capillary strip of quantity to insert in the framework, and no ELISA Plate capillary strip is put into sealing bag, be stored in 2-8 ℃ not freezing;
2) numbering: the micropore on the corresponding ELISA Plate capillary strip of detected sample and standard items is numbered according to the order of sequence, and it is parallel that each detected sample and standard items all need be done 2 holes;
3) add standard solution or test liquid 50 μ l/ holes in the micropore on the ELISA Plate capillary strip, add enzyme labeling thing 100 μ l/ holes then, behind the mixing, with cover plate film shrouding, the room temperature lucifuge leaves standstill reaction 30min;
4) pour out liquid in the hole, ELISA Plate is upside down on the thieving paper pats, remove liquid in the hole, add dilution back, 250 μ l/ holes washing lotion, will concentrate washing lotion, pour out liquid in the hole behind the 15s with 10 times of distilled water dilutings, pat dry with thieving paper, so repetitive operation is washed plate 3 times altogether;
5) colour developing: every hole adds developer 100 μ l, the mixing that vibrates gently, room temperature lucifuge colour developing 20min;
6) measure: every hole adds stop buffer 100 μ l, and the mixing that vibrates is gently set microplate reader in the 450nm place, measures every hole absorbance.
The detection method of described kit, its testing result is judged:
1) qualitatively judge: mean light absorbency value and standard value with detected sample relatively can draw the contained Clenbuterol of detected sample, Ractopamine, salbutamol concentration range, and concentration is the ng/ml of unit;
2, quantitatively judge: with standard items percentage absorptance is ordinate, the logarithm value of standard items concentration is a horizontal ordinate, the drawing standard curve, in the percentage absorptance substitution typical curve with detected sample, read the pairing concentration of detected sample from typical curve, multiply by the actual concentrations that its corresponding extension rate is Clenbuterol in the detected sample, Ractopamine, salbutamol.
Beneficial effect of the present invention is:
1, adopt the direct competitive Enzyme-multiplied immune technique, high specificity, the susceptibility height, it is simple to operate that testing result can judge that accurately and reliably, and cost is low by qualitative, quantitative fast, small investment is convenient to the screening to great amount of samples;
2, can do disposable check and analysis to the clenobuterol hydrochloride in urine, the blood serum, Ractopamine, salbutamol simultaneously, have more advantage than other single variety kits;
3, this detection kit is highly sensitive, reaches 0.1ppb, and each crossing-over rate that detects composition reaches more than 80%, and the range of linearity is 0.1ng/mL～2ng/mL;
4, analyze detecting data by typical curve, can improve the precision and the accuracy of this kit.
In a word, the present invention uses the direct competitive Enzyme-multiplied immune technique, be that clenobuterol hydrochloride, Ractopamine, salbutamol are done disposable analyzing and testing to three kinds of clenbuterol hydrochlorides simultaneously, easy to use, the specificity height, highly sensitive, good stability, simplify operation steps and reaction time greatly, and reduced cost, be fit to very much the examination of a large amount of samples.
Description of drawings
Fig. 1 the present invention is exclusively used in the typical curve of the ELISA kit of the three-in-one enzyme-linked immunologic detecting kit of Clenbuterol, Ractopamine, the salbutamol of aforementioned detection method.
Embodiment
Clenbuterol of the present invention, Ractopamine, the enzyme-linked immune detection method of the disposable detection of salbutamol, it is monoclonal antibody on pre-bag quilt on the ELISA Plate, Clenbuterol in the detected sample, salbutamol, Ractopamine, also have enzyme-labelled antigen to compete together in conjunction with after the monoclonal antibody of wrapping quilt, use chromogenic reagent, Clenbuterol in the detected sample, Ractopamine, the percentage absorbance of salbutamol content and detected sample is inverse ratio, relatively can draw Clenbuterol in the detected sample with typical curve, salbutamol, Ractopamine content, described typical curve is with Clenbuterol, Ractopamine, salbutamol standard items concentration percentage absorbance is an ordinate, with Clenbuterol, Ractopamine, the logarithm value of salbutamol standard items concentration is that the horizontal ordinate drafting forms, the monoclonal anti physical efficiency of described pre-bag quilt is simultaneously to Clenbuterol, Ractopamine, salbutamol generation cross reaction, described percentage absorbance is calculated as follows:
Wherein B is the mean light absorbency value of standard solution or sample solution; B0 is the mean light absorbency value of first standard solution.
Enzyme-labelled antigen is to obtain by horseradish peroxidase HRP mark Clenbuterol, salbutamol, Ractopamine.Wherein to the processing of detected sample:, must filter or centrifugal 10min (15 ℃ 4000r/min) until limpid, wouldn't be used sample to answer freezing preservation, but avoid multigelation to take as urine sample is muddy if the limpid urine sample of urine sample sample is just directly measured.The blood serum sample treatment is: get 0.25mL serum, add 0.5mL enzyme labeling thing dilution with dilution in 1: 2, after concussion mixes, get 50 μ L and join and carry out check and analysis in the ELISA Plate hole.
The present invention is exclusively used in the three-in-one enzyme-linked immunologic detecting kit of Clenbuterol, Ractopamine, salbutamol of aforementioned detection method, comprising: 1) aforesaid pre-bag is by the ELISA Plate of last monoclonal antibody; 2) aforesaid Clenbuterol, Ractopamine, the three-in-one standard solution of salbutamol, Clenbuterol promptly is a clenobuterol hydrochloride; 3) concentrate the enzyme labeling thing; 4) enzyme labeling thing dilution; 5) colour developing liquid; 6) concentrate washing lotion; 7) stop buffer.Wherein the enzyme labeling thing is direct oxidation horseradish peroxidase HRP under low pH condition, and horseradish peroxidase HRP is through NaIO
4The hydroformylation enzyme that forms after the oxidation links to each other with the amino of the antigen molecule of Clenbuterol, salbutamol, three kinds of materials of Ractopamine, forms this Fu Shi alkali, further uses NaBH
4The stable material that reduction generates.
The monoclonal antibody of wherein wrapping quilt is in advance merged through bone marrow cell by synthetic good comlete antigen immune balb/c mice, cultivates, and filters out the hybrid cell strain of secretory antibody, and the hybrid cell strain is carried out three time cloningizations and obtained.The Clenbuterol of detection kit, Ractopamine, salbutamol cross reacting rate are respectively 100%, 80%, 80%.The recovery that detection kit detects urine and serum all is 60～140%.The sensitivity that detection kit detects urine and serum is respectively 0.1ppb, 0.3ppb.Be limited to 0.1ppb under the lowest detection of detection kit standard items, the light absorption value of the light absorption value of this concentration and negative standard solution (0ppb) has evident difference.Be limited to 2ppb on the highest detection, the inspection product that exceed this concentration need detecting after an amount of dilution.
Detection kit of the present invention adopts the direct competitive Enzyme-multiplied immune technique, can do disposable check and analysis to clenobuterol hydrochloride, Ractopamine, salbutamol (beta-agonist) simultaneously, and the detecting operation time was less than 60 minutes.
Described detection kit has following characteristic:
1) set up typical curve: doing the typical curve of direct competitive ELISA with ELISA Plate in the kit and standard solution, is horizontal ordinate with the logarithm of drug concentration, and inhibiting rate is an ordinate, and mapping obtains the typical curve of ELISA kit;
2) criticize interior and differences between batches: get with a collection of product, do parallel typical curve, calculate the variation within batch coefficient of each drug concentration, the product drug concentration is the parallel laboratory test that 1 μ g/L does corresponding number between getting batch, calculate the batch variation coefficient of variation, be direct competitive ELISA, testing result to same batch is analyzed, batch interior average coefficient of variation that obtains is 4.88%, testing result to different batches is analyzed, obtain batch between average coefficient of variation be 6.96%, show that this research method repeatability is better;
3) get blank urine sample, the standard solution to wherein adding the variable concentrations gradient detects with kit, calculate its recovery, measure the urine of variable concentrations, record its recovery all between 80%-120%, illustrate that this kit is more accurate to the result that detected sample detects;
4) cross reacting rate of various analogues satisfies 100% Clenbuterol cross reacting rate, the cross reacting rate of 80% Ractopamine, the cross reacting rate of 80% salbutamol, and the high specificity that this kit detects detected sample is described.
The detection method of the enzyme-linked immunologic detecting kit that Clenbuterol of the present invention, Ractopamine, salbutamol are three-in-one, wherein ELISA Plate is made into the ELISA Plate capillary strip, may further comprise the steps and following step in each value be that strictness is followed:
1) before the use described kit is placed room temperature 20-25 ℃ more than the balance 30min, note to shake up before every kind of liquid reagent uses, taking-up needs the ELISA Plate capillary strip of quantity to insert in the framework, and no ELISA Plate capillary strip is put into sealing bag, be stored in 2-8 ℃ not freezing;
2) numbering: the micropore on the corresponding ELISA Plate capillary strip of detected sample and standard items is numbered according to the order of sequence, and it is parallel that each detected sample and standard items all need be done 2 holes;
3) add standard solution or test liquid 50 μ l/ holes in the micropore on the ELISA Plate capillary strip, add enzyme labeling thing 100 μ l/ holes then, behind the mixing, with cover plate film shrouding, the room temperature lucifuge leaves standstill reaction 30min;
4) pour out liquid in the hole, ELISA Plate is upside down on the thieving paper pats, remove liquid in the hole, add dilution back, 250 μ l/ holes washing lotion, will concentrate washing lotion, pour out liquid in the hole behind the 15s with 10 times of distilled water dilutings, pat dry with thieving paper, so repetitive operation is washed plate 3 times altogether;
5) colour developing: every hole adds developer 100 μ l, the mixing that vibrates gently, room temperature lucifuge colour developing 20min;
6) measure: every hole adds stop buffer 100 μ l, and the mixing that vibrates is gently set microplate reader in the 450nm place, measures every hole absorbance.
The detection method of aforementioned agents box, its testing result is judged:
1) qualitatively judge:
Mean light absorbency value and standard value with sample relatively can draw the contained clenobuterol hydrochloride of sample, Ractopamine, salbutamol (beta-agonist) concentration range (ng/ml).The absorbance of assumes samples 1 is 0.313, and the absorbance of sample 2 is 1.032, and the titer absorbance is respectively: 0ppb is 1.892; 0.1ppb be 1.501; 0.25ppb be 1.175; 0.5ppb be 0.751; 1ppb is 0.421; 2ppb is 0.198.Then the concentration range of sample 1 is 1ppb-2ppb; The concentration range of sample 2 is 0.25ppb-0.5ppb.(multiply by corresponding extension rate again)
2) quantitatively judge:
Each the concentration standard solution that is obtained and the mean value (B) of sample absorbance multiply by 100% again divided by the absorbance (B0) of first standard (0 standard), i.e. the percentage absorbance.Computing formula is seen before and is stated percentage absorbance calculating formula.
With standard items percentage absorbance is ordinate, is horizontal ordinate with the logarithm of clenobuterol hydrochloride, Ractopamine, salbutamol (beta-agonist) standard items concentration (ng/ml), the drawing standard curve map.In the percentage absorbance substitution typical curve with sample, read the pairing concentration of sample, multiply by its corresponding extension rate and be Clenbuterol in the sample, Ractopamine, salbutamol (beta-agonist) actual concentrations from typical curve.
Typical curve is set up: the typical curve of being direct competitive ELISA of lath in the kit and reagent.Logarithm with drug concentration is a horizontal ordinate, and inhibiting rate is that the percentage absorbance is an ordinate, mapping.The typical curve of ELISA kit as shown in Figure 1, R
2=0.994, the range of linearity is 0.1～2 μ g/L.
Criticize interior and differences between batches: get with a collection of product, do 6 parallel typical curves, calculate the variation within batch coefficient of each drug concentration.Getting 5 batches of drug concentrations in the product is 5 parallel laboratory tests of doing of 1 μ g/L, and calculates the batch variation coefficient of variation.
Be direct competitive ELISA, testing result to same batch is analyzed, in following batch and batch between shown in the testing result table, batch interior average coefficient of variation that obtains is 4.88%, testing result to different batches is analyzed, obtain batch between average coefficient of variation be 6.96%, show that this research method repeatability is better.
Batch in and batch between the testing result table:
Precision and accuracy: get blank urine sample,, detect, calculate its recovery with kit to the standard solution that wherein adds the variable concentrations gradient.
Measure the urine of variable concentrations, record its recovery all between 80%-120%, illustrate that this kit is more accurate to the result of sample detection.
Specificity: the cross reacting rate of various analogues is as shown in the table.
If utilize kit specialty analysis software to calculate, accurate, the express-analysis of a large amount of samples of being more convenient for.
Illustrated in greater detail detection kit of the present invention more below:
The detection kit component:
Elisa plate: 8 holes/bar, 12/plate;
Clenbuterol, salbutamol, Ractopamine (beta-agonist) standard solution (1ml/ bottle): 0ppb, 0.1ppb, 0.25ppb, 0.5ppb, 1ppb, 2ppb;
150 times of concentrated enzyme labeling thing 0.15ml; Enzyme labeling thing dilution 50ml; Developer 12ml; Stop buffer 13ml; 10 * concentrated washing lotion 50ml;
The detection kit technical indicator:
Kit sensitivity: 0.1ppb
Pattern detection lower limit: urine sample 0.1ppb; Blood serum 0.3ppb.
Cross reacting rate: Clenbuterol (Clenbuterol) 100%; Salbutamol (Salbutamal) 80%; Ractopamine (Ractopamine) 80%.
The recovery: urine sample 100% ± 40%; Blood serum 100% ± 40%.
Required instrument: micropore microplate reader, nitrogen blow-dry device, printer, homogenizer, oscillator, hydro-extractor, constant temperature oven, balance (sensibility reciprocal 0.01g), graduated pipette, micropipettor (single track 20 μ l-200 μ l, 100 μ l-1000 μ l, multiple tracks 250 μ l).
Required reagent: distilled water.
The kit application notice:
1) with before all temperature of reagent are gone up to room temperature 20-25 ℃ (rising again at least 40 minutes).
2) immediately all reagent are put back to 2-8 ℃ of refrigerator after using up.
3) preparation of cleaning fluid: with distilled water with 10 * concentrate washing lotion with 1: 9 dilution proportion (be 1mL10 * concentrate washing lotion+9mL distilled water).Whether fully annotate: use the back of must determining to rise again, and check dissolving.
4) preparation of enzyme labelled antibody: with distilled water 150 times of concentrated enzyme labeling things are annotated with 1: 149 dilution proportion (being that 0.1mL150 doubly concentrates enzyme labeling thing+14.9mL distilled water): please use as early as possible after the dilution, if be stored in-20 ℃, storage life is 7 days.
5) repeatability in elisa assay depends on the consistance of washing plate to a great extent, is main points in the ELISA mensuration program according to the plate sequential operation of recommending of washing carefully.
6) all constant temperature are hatched in the process, avoid irradiate light, seal microwell plate with the cover plate film.
7) room temperature is lower than 20 ℃ or reagent and sample and does not get back to room temperature (20-25 ℃) and can cause the OD value of all standards on the low side.
8) in washing the plate process if the plate hole dry situation, can be accompanied by then that typical curve to occur non-linear, the phenomenon that repeatability is bad.So should carry out next step operation immediately after washing plate and patting dry.
9) mixing is wanted evenly, otherwise can the bad phenomenon of duplicating property.
10) reaction terminating liquid is a 2M sulfuric acid, avoids contacting skin.
11) do not use the kit of date of expiration, diluted or mixed up the variation that use can cause sensitivity, OD value.Do not exchange reagent in the box that uses different lot numbers.
12) no microwell plate is put valve bag into and is resealed; Therefore standard substance and colourless colour former will be avoided being directly exposed under the light to photaesthesia.
13) colour developing liquid is rotten if there is any color to show, should abandon it.The absorbance of 0 standard was less than 0.5 o'clock, and expression reagent may go bad.
Notice before the sample process: 1) must use disposable tip in the experiment, when drawing different reagent, will change suction nozzle; 2) must check before the experiment whether various experiment utensils are clean, can clean the experiment utensil in case of necessity, to avoid polluting the interference experiment result.
The present invention is not limited to above-mentioned preferred forms; anyone can draw other various forms of products under enlightenment of the present invention; no matter but on its shape or structure, do any variation; every have identical with a application or akin technical scheme, all drops within protection scope of the present invention.
Claims (10)
1. Clenbuterol, Ractopamine, the enzyme-linked immune detection method of the disposable detection of salbutamol, it is characterized in that: monoclonal antibody on pre-bag quilt on the ELISA Plate, Clenbuterol in the detected sample, salbutamol, Ractopamine, also have enzyme-labelled antigen to compete together in conjunction with after the monoclonal antibody of wrapping quilt, use chromogenic reagent, Clenbuterol in the detected sample, Ractopamine, the percentage absorbance of salbutamol content and detected sample is inverse ratio, relatively can draw Clenbuterol in the detected sample with typical curve, salbutamol, Ractopamine content, described typical curve is with Clenbuterol, Ractopamine, salbutamol standard items concentration percentage absorbance is an ordinate, with Clenbuterol, Ractopamine, the logarithm value of salbutamol standard items concentration is that the horizontal ordinate drafting forms, the monoclonal anti physical efficiency of described pre-bag quilt is simultaneously to Clenbuterol, Ractopamine, salbutamol generation cross reaction, described percentage absorbance is calculated as follows:
Wherein B is the mean light absorbency value of standard solution or sample solution; B0 is the mean light absorbency value of first standard solution.
2. the enzyme-linked immune detection method of Clenbuterol according to claim 1, Ractopamine, the disposable detection of salbutamol, it is characterized in that: described detected sample is the detected sample of pig urine to be checked, pork liver and/or blood serum, if the limpid urine sample of pig urine detected sample, just directly measure, as the muddy palpus filtration of urine sample or centrifugal until limpid, wouldn't use the urine sample detected sample to answer freezing preservation, avoid multigelation to take
Described enzyme-labelled antigen is to obtain by horseradish peroxidase HRP mark Clenbuterol, salbutamol, Ractopamine,
Described blood serum detected sample disposal route is: get serum, add enzyme labeling thing diluted, concussion mixes and gets final product.
3. three-in-one enzyme-linked immunologic detecting kit of Clenbuterol, Ractopamine, salbutamol that is exclusively used in the described detection method of claim 1 is characterized in that described kit comprises:
1) the described pre-bag of claim 1 is by the ELISA Plate of last monoclonal antibody;
2) the described Clenbuterol of claim 1, Ractopamine, the three-in-one standard solution of salbutamol;
3) concentrate the enzyme labeling thing;
4) enzyme labeling thing dilution;
5) colour developing liquid;
6) concentrate washing lotion;
7) stop buffer;
Described enzyme labeling thing is direct oxidation horseradish peroxidase HRP under low pH condition, and horseradish peroxidase HRP is through NaIO
4The hydroformylation enzyme that forms after the oxidation links to each other with the amino of the antigen molecule of Clenbuterol, salbutamol, three kinds of materials of Ractopamine, forms this Fu Shi alkali, further uses NaBH
4The stable material that reduction generates.
4. Clenbuterol according to claim 3, Ractopamine, the three-in-one enzyme-linked immunologic detecting kit of salbutamol, it is characterized in that: the monoclonal antibody of described pre-bag quilt is by synthetic good comlete antigen immune mouse, merge through bone marrow cell, cultivate, filter out the hybrid cell strain of secretory antibody, the hybrid cell strain is carried out cloning and is obtained.
5. Clenbuterol according to claim 4, Ractopamine, the three-in-one enzyme-linked immunologic detecting kit of salbutamol, it is characterized in that: the Clenbuterol of described detection kit, Ractopamine, salbutamol cross reacting rate are respectively: 100%, 80%, 80%, and the recovery that described detection kit detects urine and serum all is 60～140%.
6. Clenbuterol according to claim 4, Ractopamine, the three-in-one enzyme-linked immunologic detecting kit of salbutamol is characterized in that: the sensitivity that described detection kit detects urine and serum is respectively 0.1ppb, 0.3ppb.
7. Clenbuterol according to claim 4, Ractopamine, the three-in-one enzyme-linked immunologic detecting kit of salbutamol, it is characterized in that: be limited to 0.1ppb under the lowest detection of described detection kit standard items, be limited to 2ppb on the highest detection, the detected sample that exceeds this concentration needs to detect after an amount of dilution again.
8. Clenbuterol according to claim 4, Ractopamine, the three-in-one enzyme-linked immunologic detecting kit of salbutamol is characterized in that described detection kit has following characteristic:
1) sets up typical curve: the typical curve of making the direct competitive enzyme linked immunosorbent assay (ELISA) of ELISA Plate in the kit and standard solution, logarithm value with drug concentration is a horizontal ordinate, the percentage absorbance is an ordinate, and mapping obtains the typical curve of ELISA kit;
2) criticize interior and differences between batches: get with a collection of product, do parallel typical curve, calculate the variation within batch coefficient of each drug concentration, the product drug concentration is the parallel laboratory test that 1 μ g/L does corresponding number between getting batch, calculate the batch variation coefficient of variation, be direct competitive ELISA, testing result to same batch is analyzed, batch interior average coefficient of variation that obtains is 4.88%, testing result to different batches is analyzed, obtain batch between average coefficient of variation be 6.96%, show that this research method repeatability is better;
3) get blank urine sample, the standard solution to wherein adding the variable concentrations gradient detects with kit, calculate its recovery, measure the urine of variable concentrations, record its recovery all between 80%-120%, illustrate that this kit is more accurate to the result that detected sample detects;
4) cross reacting rate of various analogues satisfies 100% Clenbuterol cross reacting rate, the cross reacting rate of 80% Ractopamine, the cross reacting rate of 80% salbutamol, and the high specificity that this kit detects detected sample is described.
9. the detection method of the three-in-one enzyme-linked immunologic detecting kit of a Clenbuterol, Ractopamine, salbutamol, wherein ELISA Plate is made into the ELISA Plate capillary strip, may further comprise the steps:
1) before the use described kit is placed room temperature 20-25 ℃ more than the balance 30min, note to shake up before every kind of liquid reagent uses, taking-up needs the ELISA Plate capillary strip of quantity to insert in the framework, and no ELISA Plate capillary strip is put into sealing bag, be stored in 2-8 ℃ not freezing;
2) numbering: the micropore on the corresponding ELISA Plate capillary strip of detected sample and standard items is numbered according to the order of sequence, and it is parallel that each detected sample and standard items all need be done 2 holes;
3) add standard solution or test liquid 50 μ l/ holes in the micropore on the ELISA Plate capillary strip, add enzyme labeling thing 100 μ l/ holes then, behind the mixing, with cover plate film shrouding, the room temperature lucifuge leaves standstill reaction 30min;
4) pour out liquid in the hole, ELISA Plate is upside down on the thieving paper pats, remove liquid in the hole, add dilution back, 250 μ l/ holes washing lotion, will concentrate washing lotion, pour out liquid in the hole behind the 15s with 10 times of distilled water dilutings, pat dry with thieving paper, so repetitive operation is washed plate 3 times altogether;
5) colour developing: every hole adds developer 100 μ l, the mixing that vibrates gently, room temperature lucifuge colour developing 20min;
6) measure: every hole adds stop buffer 100 μ l, and the mixing that vibrates is gently set microplate reader in the 450nm place, measures every hole absorbance.
10. the detection method of the enzyme-linked immunologic detecting kit that Clenbuterol according to claim 9, Ractopamine, salbutamol are three-in-one, testing result is judged:
1) qualitatively judge: mean light absorbency value and standard value with detected sample relatively can draw the contained Clenbuterol of detected sample, Ractopamine, salbutamol concentration range, and concentration is the ng/ml of unit;
2, quantitatively judge: with standard items percentage absorptance is ordinate, the logarithm value of standard items concentration is a horizontal ordinate, the drawing standard curve, in the percentage absorptance substitution typical curve with detected sample, read the pairing concentration of detected sample from typical curve, multiply by the actual concentrations that its corresponding extension rate is Clenbuterol in the detected sample, Ractopamine, salbutamol.
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