CN102175869A - Tetragenous detection card of clenbuterol, ractopamine, salbutamol and tubertaline and preparation method thereof - Google Patents
Tetragenous detection card of clenbuterol, ractopamine, salbutamol and tubertaline and preparation method thereof Download PDFInfo
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- CN102175869A CN102175869A CN2011100399234A CN201110039923A CN102175869A CN 102175869 A CN102175869 A CN 102175869A CN 2011100399234 A CN2011100399234 A CN 2011100399234A CN 201110039923 A CN201110039923 A CN 201110039923A CN 102175869 A CN102175869 A CN 102175869A
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- Prior art keywords
- monoclonal antibody
- clenbuterol
- ractopamine
- salbutamol
- terbutaline
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- YJQZYXCXBBCEAQ-UHFFFAOYSA-N ractopamine Chemical compound C=1C=C(O)C=CC=1C(O)CNC(C)CCC1=CC=C(O)C=C1 YJQZYXCXBBCEAQ-UHFFFAOYSA-N 0.000 title claims abstract description 64
- 229960001117 clenbuterol Drugs 0.000 title claims abstract description 59
- STJMRWALKKWQGH-UHFFFAOYSA-N clenbuterol Chemical compound CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 STJMRWALKKWQGH-UHFFFAOYSA-N 0.000 title claims abstract description 58
- 229960002052 salbutamol Drugs 0.000 title claims abstract description 57
- NDAUXUAQIAJITI-UHFFFAOYSA-N albuterol Chemical compound CC(C)(C)NCC(O)C1=CC=C(O)C(CO)=C1 NDAUXUAQIAJITI-UHFFFAOYSA-N 0.000 title claims abstract description 56
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Abstract
The invention relates to a tetragenous detection card of clenbuterol, ractopamine, salbutamol and tubertaline and a preparation method thereof. In the invention, a test bar is arranged in a detection card housing and comprises a sample pad, a colloidal gold film, a coating film and an absorbent cotton which are bonded onto a PVC (Polyvinyl Chloride) supporting back plate in sequence; the colloidal gold film is a glass fiber film containing colloidal gold markers of an anti-clenbuterol monoclonal antibody, an anti-ractopamine monoclonal antibody, an anti-salbutamol monoclonal antibody and an anti-tubertaline monoclonal antibody; the coating film is an acetate fiber film on which four detection strips and a quality control strip are arranged; and the detection strips comprises a detection strip containing clenbuterol antigen, a detection strip containing ractopamine antigen, a detection strip containing salbutamol antigen and a detection strip containing tubertaline antigen in sequence, and the quality control strip contains a goat-anti-mouse antibody. By using the tetragenous detection card and the preparation method thereof, provided by the invention, the clenbuterol, the ractopamine, the salbutamol and the tubertaline can be simultaneously detected; and the tetragenous detection card has the advantages of simple, convenient and fast method and accurate result.
Description
Technical field
The present invention relates to a kind of utensil that detects the beta receptor activator, particularly relate to a kind of Clenbuterol, Ractopamine, salbutamol and Terbutaline tetrad test card and preparation method thereof.
Background technology
Current society, the importance of food security day by day comes into one's own.For example " clenbuterol hydrochloride " poisoning, China's " melamine " incident in 2008 or the like make food security become the focus that the whole world is paid close attention to.Live pig is as the animal-derived food of global maximum, and the quality problems of pork more cause the concern and the attention of a plurality of countries.
Pork quality is usually by the feeding quality decision, and in the past, Clenbuterol (clenbuterol hydrochloride) once was a kind of effective ways that are used for improving the live pig lean meat percentage and reduce fat deposition as feed addictive, has to promote quick growth of live pig and the effect that improves lean meat percentage.Last century the mid-80, American-European countries finds that there is serious toxic and side effect in " clenbuterol hydrochloride ".The poisoning that residual of kelengtelu causes occurs in Spain the earliest.In China, the poisoning of Pi Luing the earliest occurs in Hong Kong.Mainly show as after the people poisons: (1) myocardial contraction is strengthened, and heart rate is accelerated, and produces palpitaition, nervous; Can cause tachycardia to original cardiovascular and cerebrovascular disease, diabetes, coronary heart disease, hyperthyroidism, glaucoma, hypertrophy of the prostate person, premature ventricular beat, even toxic myocarditis, myocardial infarction, cardiogram can be the s-T section and press and be inverted with the T ripple or hang down the equality phenomenon.(2) face neck, limb muscle vibration, phenomenon such as nauseating, vomiting is arranged, dizziness is weak, hand is trembled even can not stand.(3) pregnant woman's poisoning can cause canceration, fetus teratogenesis.
In recent years, China departments at different levels have strengthened the control and monitoring to " clenbuterol hydrochloride " use, sternly hit the illegal behavior of using " clenbuterol hydrochloride " of pig-breeding industry.Recently, some illegal breed owners are in order to escape the monitoring of government, bring into use the Ractopamine that similar effect is arranged with " clenbuterol hydrochloride ", indivedual breed owners are in order to pursue bigger economic benefit, the amount of adding Ractopamine in feed is increasing, and Ractopamine more is far more than " clenbuterol hydrochloride " to the harm of human body.Ministries and commissions such as the Ministry of Agriculture of the Chinese government, the Ministry of Public Health expressly provided in 2002 and forbid to use Ractopamine.Ractopamine is residual in animal viscera, enters human body by food chain, and human body accumulative total is taken in the viscera tissue that surpasses certain value or eaten high residue, is prone to toxic and side effect.Symptoms such as showing as Skeletal Muscle Contraction increases, and destroys the fusion between quick muscle fiber and slow switch fibers, causes muscular tremor, and four limbs and facial muscles are particularly evident.Other toxicity symptoms comprise tachycardia, arrhythmia cordis, stomachache, myalgia epilepsy, feel sick and dizzy etc.The people toxicity symptom may occur after having eaten the residual tissue that Ractopamine arranged, be usually expressed as bad reactions such as flushed face, headache, dizziness, uncomfortable in chest, palpitaition, numb limb, to suffering from bigger, the serious possible threat to life of patient's harm of diseases such as hypertension, glaucoma, diabetes, hypertrophy of the prostate.
Along with country is increasing to Clenbuterol, Ractopamine supervision, some pig-breeding families are brought into use and violated similar salbutamol and the Terbutaline of thing effect of above-mentioned two classes, and this two classes material is not inferior to Clenbuterol and Ractopamine to the harm of human body at all.
At present, China's method that live pig is detected " clenbuterol hydrochloride " mainly contains three kinds of euzymelinked immunosorbent assay (ELISA) (ELISA), high performance liquid chromatography (HPLC) and gas chromatography-mass spectroscopies (GC-MS).In these three kinds of methods, chromatography (HPLC) and (GC-MS) be classical detection method, effective, be usually used in confirmatory analysis, but this method must be carried out pre-treatment to sample, more loaded down with trivial details.Enzyme-linked method (ELISA) is subjected to ectocine bigger though can simplify pre-treatment step, and its antibody all responds the right value that influence detects to " clenbuterol hydrochloride " and Ractopamine and metabolin thereof.More than three kinds of methods all must finish by the instrument and equipment of costliness, operating personnel must be the professionals, and testing process and time are all long.
Can, directly avoid the outflow of " clenbuterol hydrochloride " from the source? to this because the most layout in pig farm is at the small and medium-sized cities periphery, limited fund and professional are less after all, and conventional detection also is unfavorable for popularizing in small and medium-sized cities.Will thoroughly avoid " clenbuterol hydrochloride " to come into the market, the present invention wishes to develop a kind of fast and simple, with low cost and accurate test method as a result.
Summary of the invention
For overcoming the technological deficiency that now can not detect Clenbuterol, Ractopamine, salbutamol and four kinds of beta-agonists of Terbutaline simultaneously, the object of the present invention is to provide a kind of tetrad appliance that can detect Clenbuterol, Ractopamine, salbutamol and Terbutaline simultaneously.
Realize that technical scheme of the present invention is: a kind of Clenbuterol, Ractopamine, salbutamol and Terbutaline tetrad test card, be in the test card shell 4 of rectangular flat shelly, to place test-strips 3, the test card case surface has well 1 and detects hole 2, test-strips is by pasting sample pad 10 thereon successively by adhesive sticker on the PVC supporting backboard 13, collaurum film 11, coated film 12 and absorbent wool 14 are formed, the stacked stickup coated film 12 in supporting backboard 13 middle parts, one section stacked stickup absorbent wool 14 of supporting backboard, the stacked stickup sample pad 10 of the other end, absorbent wool 14 the inners overlap with coated film 12 respectively separately with sample pad 10 the inners, at the clinch of sample pad 10, insert and put between the two and paste several sections collaurum films 11 with coated film 12;
The collaurum film is the glass fibre membrane that contains Clenbuterol monoclonal antibody, Ractopamine monoclonal antibody, salbutamol monoclonal antibody, Terbutaline monoclonal antibody colloid gold label thing, there are four to detect band and a quality control band on the coated film, be to contain Clenbuterol Detection of antigen band 5 successively, contain Ractopamine Detection of antigen band 6, contain salbutamol Detection of antigen band 7, contain Terbutaline Detection of antigen band 8, quality control band 9, quality control band 9 is a sheep anti-mouse antibody, test-strips is inserted in the test card shell, sample pad 10 is over against well 1, and coated film 12 is over against detecting hole 2.
The preparation method of Clenbuterol of the present invention, Ractopamine, salbutamol and Terbutaline tetrad test card is to be realized by following steps:
(1) colloidal gold solution preparation: the conical flask of getting a cleaning, on balance, accurately take by weighing the 100g pure water, move to heating jacket and be heated with stirring to boiling fast, the boiling back adds 1% chlorauric acid solution 1ml, and the back that stirs adds 1% trisodium citrate 2ml fast, maintain the temperature at 90-100 ℃, continue to stir, after solution colour becomes aubergine, continue heating 10min, the cooling of taking-up room temperature, 4 ℃ of preservations;
(2) pre-service of antibody: the antibody of high concentration (〉=2mg/ml) gathering in various degree can take place in long-time freezing preservation, and these aggregations can influence the stability of mark, so need process before the mark.Clenbuterol that will mark or Ractopamine or salbutamol or Terbutaline monoclonal anti body and function 0.01mol/LPBS are diluted to 1mg/ml, cross the 0.22um filter membrane; Clenbuterol that perhaps will mark or Ractopamine or salbutamol or Terbutaline monoclonal antibody are at 10000r/min, and under 4 ℃ of conditions, centrifugal 60min gets supernatant, is diluted to 1mg/ml with 0.01mol/LPBS.
(3) preparation of colloid gold label thing: get the colloidal gold solution 40ml in the step (1), put, stir fast down, add 0.1mol/L K to room temperature
2CO
3Transfer to and be alkalescent, best pH value is 8.5, the concentration that adds step (2) then is that 1mg/ml Clenbuterol monoclonal antibody or Ractopamine monoclonal antibody or salbutamol monoclonal antibody or Terbutaline monoclonal antibody 400ul slowly are added drop-wise in the colloidal gold solution, control the speed that drips well, 1mg antibody adds in 5min, stirs 30min; Add 10%BSA 4.4ml, cessation reaction, the ultimate density that makes BSA is 1%, stirs 30min; Then at 10 ℃, under the 12000rpm/min, centrifugal 20min, collecting precipitation, to precipitate with golden labeling antibody dilution fixed moltenly, be prepared into Clenbuterol monoclonal antibody or Ractopamine monoclonal antibody or salbutamol monoclonal antibody or Terbutaline monoclonal antibody colloid gold label thing to 1ml.
(4) washing of colloid gold label thing and purifying: resulting Clenbuterol monoclonal antibody of step (3) or Ractopamine monoclonal antibody or salbutamol monoclonal antibody or Terbutaline monoclonal antibody colloid gold label thing are diluted to 40ml with golden labeling antibody lavation buffer solution, at 2000rpm, under 10 ℃ of conditions, centrifugal 20min discards precipitation, then with supernatant at 12000rpm, under 10 ℃ of conditions, centrifugal 25min abandons supernatant, collecting precipitation, fixed molten with golden labeling antibody dilution to 1ml;
(5) preparation of collaurum film: step (4) Clenbuterol monoclonal antibody or Ractopamine monoclonal antibody or salbutamol monoclonal antibody or Terbutaline monoclonal antibody colloid gold label thing evenly are sprayed on the carrier glass fibre membrane with drawing the concentration of film instrument with 5ul/cm, room temperature is dried or 37 ℃ of oven dry 2h naturally, makes the collaurum film that contains Clenbuterol monoclonal antibody, Ractopamine monoclonal antibody, salbutamol monoclonal antibody, Terbutaline monoclonal antibody colloid gold label thing;
(6) preparation of coated film: become 0.3mg/ml, Ractopamine antigen diluent to become 0.5mg/ml, salbutamol antigen diluent to become 1mg/ml to become 1.5mg/ml the Clenbuterol antigen diluent with the Terbutaline antigen diluent; After being attached to cellulose acetate film on the offset plate, the sheep anti-mouse igg that Clenbuterol, Ractopamine, salbutamol and Terbutaline antigen that dilution is good and concentration are 1mg/ml is sprayed on the cellulose acetate film with the concentration of 1.0ul/cm successively with drawing the film instrument, be to contain Clenbuterol Detection of antigen band 5 successively, contain Ractopamine Detection of antigen band 6, contain salbutamol Detection of antigen band 7, contain Terbutaline Detection of antigen band 8, quality control band 9,37 ℃ of bags are dried or 37 ℃ of oven dry naturally by after 2 hours;
(7) sample pad pre-treatment: the sample pad treating fluid is uniformly coated on the glass fibre membrane, is lower than in air humidity under 50% the condition, dry naturally;
(8) paste sample pad, collaurum film, coated film and absorbent wool thereon successively by adhesive sticker on the assembling of test card: the PVC supporting backboard, be assembled into test-strips, again test-strips is installed in the test card shell of rectangular flat shelly.
The described golden labeling antibody dilution of step (3) is by 5gBSA (bSA), and 10g sucrose adds 100ml 0.01mol/L PBS dissolving configuration and forms, and crosses the 0.22um filter membrane, and is now with the current;
The described golden labeling antibody lavation buffer solution of step (4) is by 10gBSA (bSA) and 1g PEG 20000, and is fixed molten to 100ml with 0.01mo/L PBS pH7.4;
The described sample pad treating fluid of step (7) is by 1gBSA (bSA) and 0.8g sodium chloride, and is fixed molten to 100ml with the 0.01mol/L PBS that contains 0.25%TRITON-100.
The present invention can detect Clenbuterol, Ractopamine, salbutamol and Terbutaline effectively simultaneously, and method is simple, convenient, fast, and the result is accurate.
Description of drawings
Fig. 1 is a test card structural drawing of the present invention;
Fig. 2 is the structural drawing of test card build-in test bar of the present invention.
Embodiment
Below in conjunction with accompanying drawing the specific embodiment of the invention is described
Embodiment 1
The preparation of tetrad test card:
(1) colloidal gold solution preparation: the conical flask of getting a cleaning, on balance, accurately take by weighing the 100g pure water, move to heating jacket and be heated with stirring to boiling fast, the boiling back adds 1% chlorauric acid solution 1ml, and the back that stirs adds 1% trisodium citrate 2ml fast, maintain the temperature at 90-100 ℃, continue to stir, after solution colour becomes aubergine, continue heating 10min, the cooling of taking-up room temperature, 4 ℃ of preservations;
(2) pre-service of antibody: the antibody of high concentration (〉=2mg/ml) gathering in various degree can take place in long-time freezing preservation, and these aggregations can influence the stability of mark, so need process before the mark.Clenbuterol that will mark or Ractopamine or salbutamol or Terbutaline monoclonal anti body and function 0.01mol/LPBS are diluted to 1mg/ml, cross the 0.22um filter membrane; Clenbuterol that perhaps will mark or Ractopamine or salbutamol or Terbutaline monoclonal antibody are at 10000r/min, and under 4 ℃ of conditions, centrifugal 60min gets supernatant, is diluted to 1mg/ml with 0.01mol/LPBS.
(3) preparation of colloid gold label thing: get the colloidal gold solution 40ml in the step (1), put, stir fast down, add 0.1mol/L K to room temperature
2CO
3Transfer to and be alkalescent, best pH value is 8.5, the concentration that adds step (2) then is that 1mg/ml Clenbuterol monoclonal antibody or Ractopamine monoclonal antibody or salbutamol monoclonal antibody or Terbutaline monoclonal antibody 400ul slowly are added drop-wise in the colloidal gold solution, control the speed that drips well, 1mg antibody adds in 5min, stirs 30min; Add 10%BSA 4.4ml, cessation reaction, the ultimate density that makes BSA is 1%, stirs 30min; Then at 10 ℃, under the 12000rpm/min, centrifugal 20min, collecting precipitation, to precipitate with golden labeling antibody dilution fixed moltenly, be prepared into Clenbuterol monoclonal antibody or Ractopamine monoclonal antibody or salbutamol monoclonal antibody or Terbutaline monoclonal antibody colloid gold label thing to 1ml.
(4) washing of colloid gold label thing and purifying: resulting Clenbuterol monoclonal antibody of step (3) or Ractopamine monoclonal antibody or salbutamol monoclonal antibody or Terbutaline monoclonal antibody colloid gold label thing are diluted to 40ml with golden labeling antibody lavation buffer solution, at 2000rpm, under 10 ℃ of conditions, centrifugal 20min discards precipitation, then with supernatant at 12000rpm, under 10 ℃ of conditions, centrifugal 25min abandons supernatant, collecting precipitation, fixed molten with golden labeling antibody dilution to 1ml;
(5) preparation of collaurum film: step (4) Clenbuterol monoclonal antibody or Ractopamine monoclonal antibody or salbutamol monoclonal antibody or Terbutaline monoclonal antibody colloid gold label thing evenly are sprayed on the carrier glass fibre membrane with drawing the concentration of film instrument with 5ul/cm, room temperature is dried or 37 ℃ of oven dry 2h naturally, makes the collaurum film that contains Clenbuterol monoclonal antibody, Ractopamine monoclonal antibody, salbutamol monoclonal antibody, Terbutaline monoclonal antibody colloid gold label thing;
(6) preparation of coated film: become 0.3mg/ml, Ractopamine antigen diluent to become 0.5mg/ml, salbutamol antigen diluent to become 1mg/ml to become 1.5mg/ml the Clenbuterol antigen diluent with the Terbutaline antigen diluent; After being attached to cellulose acetate film on the offset plate, the sheep anti-mouse igg that Clenbuterol, Ractopamine, salbutamol and Terbutaline antigen that dilution is good and concentration are 1mg/ml is sprayed on the cellulose acetate film with the concentration of 1.0ul/cm successively with drawing the film instrument, be to contain Clenbuterol Detection of antigen band 5 successively, contain Ractopamine Detection of antigen band 6, contain salbutamol Detection of antigen band 7, contain Terbutaline Detection of antigen band 8, quality control band 9,37 ℃ of bags are dried or 37 ℃ of oven dry naturally by after 2 hours;
(7) sample pad pre-treatment: the sample pad treating fluid is uniformly coated on the glass fibre membrane, is lower than in air humidity under 50% the condition, dry naturally;
(8) paste sample pad, collaurum film, coated film and absorbent wool thereon successively by adhesive sticker on the assembling of test card: the PVC supporting backboard, be assembled into test-strips, again test-strips is installed in the test card shell of rectangular flat shelly.
The test of minimum detectability amount
(1) Clenbuterol minimum detectability amount test
Accurately weighing 0.500g Clenbuterol antigen is dissolved in distilled water and is settled to 100mL, is designated as A
1Liquid is got A
1Liquid 1mL is in the volumetric flask adding distil water and be settled to 1000mL, forms A
2Liquid is got A
2Liquid 1mL is settled to 1000mL to the volumetric flask adding distil water, formation A
3Liquid, then A
3The concentration of liquid be 5/1000000000g/mL (be 5ng/mL (5 nanograms/mL, 5ppb).Get A
3Liquid 1mL is in 4 test tubes, and the difference label.Inject 9mL distilled water toward No. 1 test tube, inject 4mL distilled water toward No. 2 test tubes, inject 1mL distilled water in No. 3 test tubes, No. 4 test tube does not add any medicine.Other gets an empty test tube, and toward interior injection 5mL distilled water, is designated as test tube No. 5.No. 1 to No. 5 test tube Clenbuterol antigenic solution concentration is respectively: 0.5ppb, 1ppb, 2.5ppb, 5ppb, 0ppb.Getting above five kinds of liquid is added on the tetrad test card as sample drop, observations after 5 minutes, contain Ractopamine Detection of antigen band 6, contain salbutamol Detection of antigen band 7 and contain the color and the quality control band of Terbutaline Detection of antigen band 8 homochromy, the change color that contains Clenbuterol Detection of antigen band 5 sees Table 1.
The test of table 1 Clenbuterol minimum detectability amount
(2) Ractopamine minimum detectability amount test:
Accurately weighing 0.500g Ractopamine antigen is dissolved in distilled water and is settled to 100mL, is designated as B
1Liquid is got B
1Liquid 1mL is in the volumetric flask adding distil water and be settled to 1000mL, forms B
2Liquid is got B
2Liquid 1mL is settled to 1000mL to the volumetric flask adding distil water, formation B
3Liquid, then B
3The concentration of liquid is 5/1000000000g/mL, promptly 5ng/mL (5 nanograms/mL, 5ppb).Get B
3Liquid 1mL is in 4 test tubes, and the difference label.Inject 9mL distilled water toward No. 1 test tube, inject 4mL distilled water toward No. 2 test tubes, inject 1mL distilled water in No. 3 test tubes, No. 4 test tube does not add any medicine.Other gets an empty test tube, and toward interior injection 5mL distilled water, is designated as test tube No. 5.No. 1 to No. 5 test tube Ractopamine antigenic solution concentration is respectively: 0.5ppb, 1ppb, 2.5ppb, 5ppb, 0ppb gets above five kinds of solution and is added on the tetrad test card observations after 5 minutes as sample drop, contain Clenbuterol Detection of antigen band 5, contain salbutamol Detection of antigen band 7 and contain the color and the quality control band of Terbutaline Detection of antigen band 8 homochromy, the change color of Ractopamine Detection of antigen band 6 sees Table 2.
The test of table 2 Ractopamine minimum detectability amount
(3) salbutamol minimum detectability amount test:
Accurately weighing 0.500g salbutamol antigen is dissolved in distilled water and is settled to 100mL, is designated as C
1Liquid is got C
1Liquid 1mL is in the volumetric flask adding distil water and be settled to 1000mL, forms C
2Liquid is got C
2Liquid 1mL is settled to 1000mL to the volumetric flask adding distil water, formation C
3Liquid, then C
3The concentration of liquid is 5/1000000000g/mL, promptly 5ng/mL (5 nanograms/mL, 5ppb).Get C
3Liquid 1mL is in 4 test tubes, and the difference label.Inject 9mL distilled water toward No. 1 test tube, inject 4mL distilled water toward No. 2 test tubes, inject 1mL distilled water in No. 3 test tubes, No. 4 test tube does not add any medicine.Other gets an empty test tube, and toward interior injection 5mL distilled water, is designated as test tube No. 5.No. 1 to No. 5 test tube salbutamol antigenic solution concentration is respectively: 0.5ppb, 1ppb, 2.5ppb, 5ppb, 0ppb, getting above five kinds of solution is added on the tetrad test card as sample drop, observations after 5 minutes contains Clenbuterol Detection of antigen band 5, containing Ractopamine Detection of antigen band 6, to contain and contain the color and the quality control band of Terbutaline Detection of antigen band 8 homochromy, and the change color that contains salbutamol Detection of antigen band 7 sees Table 3.
The test of table 3 salbutamol antigen minimum detectability amount
(4) Terbutaline minimum detectability amount test:
Accurately weighing 0.500g Terbutaline antigen is dissolved in distilled water and is settled to 100mL, is designated as D
1Liquid is got D
1Liquid 1mL is in the volumetric flask adding distil water and be settled to 1000mL, forms D
2Liquid is got D
2Liquid 1mL is settled to 1000mL to the volumetric flask adding distil water, formation D
3Liquid, then D
3The concentration of liquid is 5/1000000000g/mL, promptly 5ng/mL (5 nanograms/mL, 5ppb).Get D
3Liquid 1mL is in 4 test tubes, and the difference label.Inject 9mL distilled water toward No. 1 test tube, inject 4mL distilled water toward No. 2 test tubes, inject 1mL distilled water in No. 3 test tubes, No. 4 test tube does not add any medicine.Other gets an empty test tube, and toward interior injection 5mL distilled water, is designated as test tube No. 5.No. 1 to No. 5 test tube Terbutaline antigenic solution concentration is respectively: 0.5ppb, 1ppb, 2.5ppb, 5ppb, 0ppb.Get above five kinds of solution and be added on " four inspections " test card as sample drop, observations after 5 minutes sees Table 4.
The test of table 4 Terbutaline minimum detectability amount
Through repeatedly check, the recall rate of Clenbuterol, Ractopamine, salbutamol and Terbutaline reaches 99.9%, and the minimum detectability amount is less than 5ppb.
Claims (2)
1. Clenbuterol, Ractopamine, salbutamol and Terbutaline tetrad test card, be in the test card shell (4) of rectangular flat shelly, to place test-strips (3), there is well (1) on test card shell (4) surface and detects hole (2), test-strips (3) is to be gone up by PVC supporting backboard (13) to paste sample pad (10) thereon successively by adhesive sticker, collaurum film (11), coated film (12) and absorbent wool (14) are formed, supporting backboard (13) middle part stacked stickup coated film (12), supporting one section stacked stickup absorbent wool of backboard (14), the stacked stickup sample pad of the other end (10), absorbent wool (14) is inner to overlap with coated film (12) respectively separately with sample pad (10) the inner, at the clinch of sample pad (10), insert and put between the two and paste several sections collaurum films (11) with coated film (12); It is characterized in that the collaurum film is for containing the Clenbuterol monoclonal antibody, the Ractopamine monoclonal antibody, the salbutamol monoclonal antibody, the glass fibre membrane of Terbutaline monoclonal antibody colloid gold label thing, there are four to detect band and a charge band on the coated film, be to contain Clenbuterol Detection of antigen band (5) successively, contain Ractopamine Detection of antigen band (6), contain salbutamol Detection of antigen band (7), contain Terbutaline Detection of antigen band (8), quality control band (9), quality control band (9) is a sheep anti-mouse antibody, test-strips is inserted in the test card shell, sample pad (10) is over against well (1), and coated film (12) is over against detecting hole (2).
2. the preparation method of Clenbuterol according to claim 1, Ractopamine, salbutamol and Terbutaline tetrad test card is characterized in that being realized by following steps:
(1) colloidal gold solution preparation: the conical flask of getting a cleaning, on balance, accurately take by weighing the 100g pure water, move to heating jacket and be heated with stirring to boiling fast, the boiling back adds 1% chlorauric acid solution 1ml, and the back that stirs adds 1% trisodium citrate 2ml fast, maintain the temperature at 90-100 ℃, continue to stir, after solution colour becomes aubergine, continue heating 10min, the cooling of taking-up room temperature, 4 ℃ of preservations;
(2) pre-service of antibody: Clenbuterol that will mark or Ractopamine or salbutamol or Terbutaline monoclonal anti body and function 0.01mol/LPBS are diluted to 1mg/ml, cross the 0.22um filter membrane; Clenbuterol that perhaps will mark or Ractopamine or salbutamol or Terbutaline monoclonal antibody are at 10000r/min, and under 4 ℃ of conditions, centrifugal 60min gets supernatant, is diluted to 1mg/ml with 0.01mol/LPBS;
(3) preparation of colloid gold label thing: get the colloidal gold solution 40ml in the step (1), put, stir fast down, add 0.1mol/L K to room temperature
2CO
3Transfer to and be alkalescent, best pH value is 8.5, the concentration that adds step (2) then is that 1mg/ml Clenbuterol monoclonal antibody or Ractopamine monoclonal antibody or salbutamol monoclonal antibody or Terbutaline monoclonal antibody 400ul slowly are added drop-wise in the colloidal gold solution, control the speed that drips well, 1mg antibody adds in 5min, stirs 30min; Add 10%BSA 4.4ml, cessation reaction, the ultimate density that makes BSA is 1%, stirs 30min; Then at 10 ℃, under the 12000rpm/min, centrifugal 20min, collecting precipitation, to precipitate with golden labeling antibody dilution fixed moltenly, be prepared into Clenbuterol monoclonal antibody or Ractopamine monoclonal antibody or salbutamol monoclonal antibody or Terbutaline monoclonal antibody colloid gold label thing to 1ml;
(4) washing of colloid gold label thing and purifying: resulting Clenbuterol monoclonal antibody of step (3) or Ractopamine monoclonal antibody or salbutamol monoclonal antibody or Terbutaline monoclonal antibody colloid gold label thing are diluted to 40ml with golden labeling antibody lavation buffer solution, at 2000rpm, under 10 ℃ of conditions, centrifugal 20min discards precipitation, then with supernatant at 12000rpm, under 10 ℃ of conditions, centrifugal 25min abandons supernatant, collecting precipitation, fixed molten with golden labeling antibody dilution to 1ml;
(5) preparation of collaurum film: step (3) Clenbuterol monoclonal antibody or Ractopamine monoclonal antibody or salbutamol monoclonal antibody or Terbutaline monoclonal antibody colloid gold label thing evenly are sprayed on the carrier glass fibre membrane with drawing the concentration of film instrument with 5ul/cm, room temperature is dried or 37 ℃ of oven dry 2h naturally, makes the collaurum film that contains Clenbuterol monoclonal antibody, Ractopamine monoclonal antibody, salbutamol monoclonal antibody, Terbutaline monoclonal antibody colloid gold label thing;
(6) preparation of coated film: become 0.3mg/ml, Ractopamine antigen diluent to become 0.5mg/ml, salbutamol antigen diluent to become 1mg/ml to become 1.5mg/ml the Clenbuterol antigen diluent with the Terbutaline antigen diluent; After being attached to cellulose acetate film on the offset plate, the sheep anti-mouse igg that Clenbuterol, Ractopamine, salbutamol and Terbutaline antigen that dilution is good and concentration are 1mg/ml is sprayed on the cellulose acetate film with the concentration of 1.0ul/cm successively with drawing the film instrument, be to contain Clenbuterol Detection of antigen band 5 successively, contain Ractopamine Detection of antigen band 6, contain salbutamol Detection of antigen band 7, contain Terbutaline Detection of antigen band 8, quality control band 9,37 ℃ of bags are dried or 37 ℃ of oven dry naturally by after 2 hours;
(7) sample pad pre-treatment: the sample pad treating fluid is uniformly coated on the glass fibre membrane, is lower than in air humidity under 50% the condition, dry naturally;
(8) paste sample pad, collaurum film, coated film and absorbent wool thereon successively by adhesive sticker on the assembling of test card: the PVC supporting backboard, be assembled into test-strips, again test-strips is installed in the test card shell of rectangular flat shelly.
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