CN1264017C - Fast and semiquantitative detection method for Clenbuterol - Google Patents
Fast and semiquantitative detection method for Clenbuterol Download PDFInfo
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- CN1264017C CN1264017C CN 200310112423 CN200310112423A CN1264017C CN 1264017 C CN1264017 C CN 1264017C CN 200310112423 CN200310112423 CN 200310112423 CN 200310112423 A CN200310112423 A CN 200310112423A CN 1264017 C CN1264017 C CN 1264017C
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Abstract
The present invention relates to a semi-quantitative rapid detection method for detecting clenbuterol, which has the technical scheme that a sample liquid absorption part, a colloidal gold marking part, a detection reacting part and a water absorption part are sequentially pasted on the back lining of test paper; the detection reacting part is coated with 1 to 3 clenbuterol antibodies or 1 to 3 antigens used as detection lines for detection and is simultaneously coated with 1 to 3 IgG used as reference lines for resisting second type animal protein; the detection lines and the reference lines can not simultaneously more than one at the time of combination, and the number of combination lines is from 2 to 4. the rapid detection test paper has the advantage of strong specificity and can be used when the sensitivity reaches 0.09 ng/mL at the temperature of 4 to 40 DEG C; results can be observed after 2 min; the present invention is suitable for sanitation departments, quality monitoring departments, customs departments, meat processing factories, livestock cultivation farms, individuals, etc., to fast detect clenbuterol in samples of feed, meat, urine, dairy products, etc.
Description
Technical field
The present invention relates to a kind of detection Clenbuterol (clenbuterol, CLB) method.
Background technology
Clenbuterol claims clenobuterol hydrochloride again, or clenbuterol, is commonly called as " clenbuterol hydrochloride ", originally is used for the treatment of diseases such as asthma, after it can significantly improve the output and lean meat percentage of animal in discovery, once once is widely used in animal husbandry as feed addictive.But its toxicity is big, and easily residual in animal tissue, people Chang Yin eats its animal foodstuff and causes poisoning.Though China after the America and Europe, forbade that it uses in 1997 in feed, owing to ordered about by economic interests, illegal abuse phenomenon still exists.According to the data that the Ministry of Agriculture announces on the net, some illegal retailers make the inner wrapping retail to Clenbuterol or sell with feed collocation, and illegal sale goes underground, and more becomes hidden.Therefore, when promptly the source is controlled, must strengthen its safety detection.
To main at present high performance liquid chromatography (HPLC), gas chromatography mass spectrometry method (GC-MS) and the enzyme-linked immunosorbent assay (ELISA) of adopting of the detection of residual of kelengtelu.GC-MS method and HPLC method sensitivity, accurate, detect lower limit and can reach 0.25ng/mL, but because these two kinds of methods need expensive instrument, sample pre-treatments is loaded down with trivial details, analysis speed is slow, need several hours to tens hours, test sample amount few, be difficult to the batch sample is detected, could operate by the personnel of specialized training, the testing cost height, thousands of unit/the samples of hundreds of, so application surface is narrow, only be adopted as the master, adopt when usually the positive sample of Preliminary detection being confirmed with some important health and epidemic preventions and quality inspection superintendent office.The ELISA method is the present more Clenbuterol detection method of research, has highly sensitively, and detection limit is big, and sample pre-treatments is simple, and is shorter than the instrumental analysis time, cost is low, and detecting lower limit can reach below the 0.09ng/mL.The existing ELISA kit of American-European many companies is put on market, sells to the whole world as the kit of Britain Randox company.But this method still needs equipment components such as microplate reader, 1-3 hour analysis time, is difficult to adopt at the scene.
For overcoming its shortcoming, make more convenient to the detection of Clenbuterol, the time is shorter, cost is lower, some units have carried out colloidal gold test research, and the report of " fast clenbuterol hydrochloride detecting test paper strip ", " a kind of immunochromatography single stage method detect the method for adrenin agonist, medicine and the preparation of test paper " arranged, these the two described technology of report can only qualitative detection, can not half-quantitative detection, and combination line has only 2, and the described test paper of the application, be semiquantitative test paper, be added with prolongation or thickening, combination line is counted the 2-4 bar.The report of " colloidal gold strip of sxemiquantitative fast detecting clenobuterol hydrochloride and production and using method " is also arranged in addition, and itself and the application's difference are: the first, its no reference line; The second, on using method, clenobuterol hydrochloride content is high more in its sample, and detection line colour developing bar number is many, and the application's described test paper, Clenbuterol content is high more in the sample, and the detection line color is shallow more, and detection line colour developing bar number is few more.More than several then other researchers' report, between colloid gold label part and detection reaction part, all there is not any other material, and the described test paper of the application is added with prolongation between colloid gold label part and detection reaction part, or under the colloid gold label part or on be added with thickening.Therefore, the application has overcome above-mentioned several then shortcoming of report, has developed Clenbuterol half-quantitative detection test paper.
Summary of the invention
The design's purpose: develop easy, sensitive, cheap Clenbuterol rapid semi-quantitative detection method.
Now in conjunction with the accompanying drawings invention is described:
Accompanying drawing 1 is a structural representation of the present invention.1 is that sample liquid absorption portion, 2 is that colloid gold label part, 3 is that detection reaction part, 4 is that detection line, 5 is that reference line, 6 is the part that absorbs water among the figure.They stick on the backing according to following order: sample liquid absorption portion, colloid gold label part, detection reaction part, suction part.Colloid gold label is glass fibre or acetate fiber or nylon membrane partly, and material of mark is the potpourri of the second kind animal protein and antibody of clenbuteral on it, or the potpourri of the second kind animal protein and Clenbuterol detection usefulness antigen.The mixed method of potpourri: the blending ratio of the second kind animal protein and antibody of clenbuteral or antigen: the Clenbuterol sample with standard is a standard, and when negative sample detected, reference line and detection line macroscopic redness just occurred and be as the criterion.Be coated with Clenbuterol above the detection reaction part and detect with antigen 1～3 as detection line, or be coated with 1～3 of antibody of clenbuteral as detection line, 1～3 of the IgG that also is coated with the anti-second kind animal protein simultaneously is as reference line.Can not be when detection line and reference line combination simultaneously more than 1, the combination number of lines is 2～4, and array mode is 1 detection line and 1 reference line, 1 detection line and 2 reference lines, 1 detection line and 3 reference lines, 2 detection lines and 1 reference line, 3 detection lines and five kinds of forms of 1 reference line.According to the accuracy requirement of half-quantitative detection, the combination line number is many more, and sxemiquantitative is accurate more.
When colloid gold label part tagged object is the second kind animal protein and antibody of clenbuteral potpourri, in order to improve the sensitivity that detects test paper, be added with all-glass paper or filter paper or thieving paper between colloid gold label part and the detection reaction part prolongation or on the colloid gold label part or under be added with thickening.
Wherein detect with antigen and be meant the conjugates that Clenbuterol and protein substance or polysaccharose substance form, said protein is bovine serum albumin(BSA), keyhole limpet hemocyanin, oralbumin.Polysaccharide is glucosan, shitosan, cellulose.The object that this potpourri serves as a mark with colloid gold label antigen the time, or with colloid gold label antibody the time this potpourri as the encrusting substance matter on the detection line; The second kind animal protein refers to that the non-antibody source belongs to the albumen of animal.
The each several part of test paper described in the invention is handled with function as follows:
Backing:, play fixing other ingredients of test paper of supporting for simultaneously scribbling the toughness material that does not absorb water of adhesive sticker.
The preparation of sample liquid absorption portion: with 2min among the PBS of filter paper or all-glass paper immersion pH7.0～8.4, take out, 80 ℃ of oven dry or other mode dryings promptly as sample liquid absorption portion, play a part during detection to absorb sample solution, are convenient to sample solution and move up.
The preparation of colloid gold label part: this part plays the fixedly antigen or the antibody of colloid gold label.Preparation process comprises preparation, colloid gold label antibody of clenbuteral or Clenbuterol antigen, the colloid gold label section processes of collaurum colloidal sol.
(1) preparation of collaurum colloidal sol: with gold chloride (HAuCL
4) be configured to 1% mother liquor with ultrapure water, get the mother liquor of 1mL, use the ultrapure water constant volume to 100mL, be made into 0.01% solution, be heated to boiling, add 1% trisodium citrate aqueous solution of 1～5mL, continue to be heated to occur transparent orange red till, be collaurum colloidal sol.
(2) colloid gold label antibody of clenbuteral: Clenbuterol monoclonal antibody or polyclonal antibody and the second kind animal protein are used PBS (0.01mol/L respectively, pH7.0～7.5) dissolved dilution to 2～4mg/mL, every 100mL collaurum colloidal sol adds antibody of clenbuteral and 1～1.5mL, 2～4mg/mL, the second kind animal protein of 1～3mL, 2～4mg/mL, concussion 2min is with the K of 0.2mol/L
2CO
3Regulate pH to 8.4, vibration 5min adds 11%PEG-10000 2mL, vibration 5min, the centrifugal 15min of 8000～15000r/min removes supernatant, with PBS (0.01mol/L, pH7.0～7.5) redissolve, with the centrifugal 15min of 6000～13000r/min, remove supernatant, will precipitate (0.01mol/L with PBS, pH7.0～7.5) dilution is 500～2000 times, and product is marked antibody of clenbuteral (GAb) as gold.
(3) colloid gold label Clenbuterol antigen: the Clenbuterol detection is used PBS (0.01mol/L respectively with the antigen and the second kind animal protein, pH7.0～8.4) dissolved dilution to 2～4mg/mL, every 100mL collaurum colloidal sol adds detection antigen and 1～1.5mL, 2～4mg/mL, the second kind animal protein of 1～3mL, 2～4mg/mL, concussion 2min is with the K of 0.2mol/L
2CO
3Regulate pH to 8.4, vibration 5min adds 11%PEG-100002mL, vibration 5min, the centrifugal 15min of 8000 ~ 15000r/min removes supernatant, with PBS (0.01mol/L, pH7.0～7.5) redissolve, with the centrifugal 15min of 6000～13000r/min, remove supernatant, will precipitate (0.01mol/L with PBS, pH7.0～7.5) dilution is 500～2000 times, and product is as the Clenbuterol antigen (GAg) of golden mark.
(4) colloid gold label section processes: GAb or GAg are poured in the groove, all-glass paper or filter paper are immersed 1min, take out, after the drying at room temperature, promptly as the colloid gold label part.
Detection reaction partly prepares: glutaraldehyde solution with 0.8% or 0.2% carbodiimide solution soak nitrocellulose membrane or cellulose acetate film or nylon membrane 30min, take out, 37 ℃ of oven dry, the top bag is detected by the Clenbuterol of 1～3 variable concentrations uses antigen line or antibody of clenbuteral line as detection line, wraps simultaneously by the IgG line of the anti-second kind animal protein of 1～3 variable concentrations as the reference line.When colloid gold label part tagged object was the second kind animal protein and antibody of clenbuteral potpourri, detection line then wrapped by Clenbuterol detection antigen; When colloid gold label part tagged object is that the second kind animal protein and Clenbuterol detect when using antigen mixture, detection line then wraps by antibody of clenbuteral; Detection line and reference line can not be simultaneously more than 1, several 2～4 of combination line.This is the detection reaction part, and it is that reaction result is come out with macroscopic characterization that this part mainly acts on.
Suction part preparation: after all-glass paper or filter paper or thieving paper drying at room temperature, promptly as the suction part.This part mainly acts on the unnecessary sample solution that is moving up and absorbs.
The preparation of prolongation or thickening: after all-glass paper or filter paper or thieving paper drying at room temperature, be added between colloid gold label part and the detection reaction part, be prolongation; Cover on the collaurum part or stick under the collaurum part on the backing, be thickening.
Test paper assembling: on backing, paste sample liquid absorption portion, colloid gold label part, prolongation, detection reaction part and the part that absorbs water successively, Clenbuterol half-quantitative detection test paper.When colloid gold label part tagged object is the antibody of clenbuteral and the second kind animal protein, increases on the backing and paste prolongation or thickening.
Detect principle: detect the object difference of principle because of colloid gold label, and variant slightly.
When the label of colloid gold label part is the second kind animal protein and antibody of clenbuteral potpourri, if contain Clenbuterol in the sample, sample solution is absorbed by the sample liquid absorption portion of test paper and reaches the colloid gold label part by moving on on the capillary action, Clenbuterol in the sample solution and the reaction of the antibody of clenbuteral of colloid gold label form bond, bond moves on to detection line on continuing, because of the antibody of clenbuteral of colloid gold label has only a binding site, Clenbuterol in the sample solution with it in conjunction with after, detection on the detection line just can not combine with the antibody of clenbuteral of colloid gold label with antigen again, so detection line is colourless; When not having Clenbuterol in the sample, the detected antigen capture of the using when antibody of clenbuteral of colloid gold label arrives detection line then forms the naked eyes red color visible, and this is promptly negative.No matter whether contain Clenbuterol in the sample, move on on the second kind animal protein of colloid gold label all can be caught by the IgG of the anti-second kind animal protein of bag quilt on the reference line when reaching reference line and form macroscopic redness, this is reference line.
When the label of colloid gold label part is that the second kind animal protein and Clenbuterol detect when using antigen mixture, if contain Clenbuterol in the sample, sample solution is by the absorption of the sample liquid absorption portion of test paper and by moving on the capillary action, the little translational speed of free Clenbuterol molecular weight in the sample is fast, arrive detection line earlier, earlier with detection line on wrap quilt antibody of clenbuteral combine, because of the antibody of clenbuteral that wraps quilt on the detection line has only a binding site, and the Clenbuterol binding ability of dissociating in the sample is stronger with antigen than the Clenbuterol detection of parataxic, so the detection of colloid gold label is caught with the antibody of clenbuteral of antigen on can not tested again survey line, so detection line is colourless, this is promptly positive; If there is not Clenbuterol in the sample, then the Clenbuterol of colloid gold label detects with catching with regard to the antibody of clenbuteral of bag quilt on the tested survey line behind the antigen arrival detection line, forms macroscopic redness, and this is promptly negative.No matter whether contain Clenbuterol in the sample, move on on the second kind animal protein of colloid gold label all can be caught by the IgG of the anti-second kind animal protein of bag quilt on the reference line when reaching reference line and form macroscopic redness, this is reference line.
The test paper of above dual mode can be regulated the colour developing depth by regulating detection line and reference line encrusting substance concentration, by many combination lines are set, and combination line color or colour developing bar number and standard substance concentration is mapped, and can reach the half-quantitative detection purpose.
Using method:
Article (1) 1, the test paper of detection line+1 a reference line form
Detection line is coated with Clenbuterol monoclonal antibody or Clenbuterol detection the antigen 1 bar, wide 1mm that concentration is 0.1-30 μ g/mL; Reference line is coated with 1 of the IgG that concentration is the anti-second kind animal protein of 0.01-30 μ g/mL, wide 1mm; Article two, 2-6mm at interval between the line.When the detection line color was identical with the reference line color, then sample was negative; When detection line was identical with the reference line color, then sample was positive, and concentration is greater than Ang/mL, and the detectability of A for setting can be a certain concentration more than or equal to 0.1ng/mL.Reference line develops the color all the time, if reference line does not develop the color, shows that test paper lost efficacy.
Article (2) 2, the test paper of detection line+1 a reference line form
Article 2, detection line is coated with 0.01-30 μ g/mL Clenbuterol monoclonal antibody or Clenbuterol and detects and use antigen, and the concentration of encrusting substance is less than the 1st detection line reference line encrusting substance concentration on the 2nd detection line; Reference line is coated with the IgG that concentration is the anti-second kind animal protein of 0.01-30 μ g/mL, wide 1mm; Interval 2-6mm between each bar line.When two detection lines all presented color, then sample was negative; When the 1st detection line colour generation of detection line, the 2nd detection line be during colour generation, then in the sample Clenbuterol concentration greater than Ang/mL, less than Bng/mL; When two detection lines not during colour generation, Clenbuterol concentration is greater than Bng/mL in the sample.A, the B detectability for setting can be a certain concentration more than or equal to 0.1ng/mL.Reference line develops the color all the time, if reference line does not develop the color, shows that test paper lost efficacy.
Article (3) 3, the test paper of detection line+1 a reference line form
Article 3, detection line is coated with 0.01-30 μ g/mL Clenbuterol monoclonal antibody or Clenbuterol and detects and use antigen, and the concentration of encrusting substance is successively decreased successively on three detection lines; Reference line is coated with the IgG that concentration is the anti-second kind animal protein of 0.01-30 μ g/mL; Interval 2-6mm between each bar line.When 3 detection lines all presented color, then sample was negative; When the 1st, 2 detection line colour generation of detection line, the 3rd detection line be during colour generation, then in the sample Clenbuterol concentration greater than Ang/mL, less than Bng/mL, when the 1st detection line colour generation, the 2nd, article 3, not during colour generation, Clenbuterol concentration is greater than Bng/mL, less than Cng/mL in the sample for detection line; When 3 detection lines not during colour generation, Clenbuterol content is greater than Cng/mL in the sample bottle; A, B, the detectability of C for setting can be a certain concentration more than or equal to 0.1ng/mL, and B is greater than A, and C is greater than B.Reference line develops the color all the time, if reference line does not develop the color, shows that test paper lost efficacy.
Article (4) 1, detection line+3 reference line:
Be coated with 0.01-30 μ g/mL Clenbuterol monoclonal antibody or Clenbuterol detection antigen on the detection line, be coated with 3 reference lines after the detection line, reference line is coated with the IgG that concentration is the anti-second kind animal protein of 0.01-30 μ g/mL, regulate the label concentration of the 2nd kind animal protein of reference line encrusting substance concentration and colloid gold label, the color that makes 3 reference lines respectively with standard model in Clenbuterol content when being C, B, Ang/mL the color of detection line identical, determine reference line encrusting substance concentration according to this; Interval 2-6mm between each bar line; When the detection line color is deeper than the 3rd reference line color, show that sample is negative; Be shallower than the 2nd when detecting when the detection line color is deeper than the 3rd reference line color, show that Clenbuterol concentration is greater than Ang/mL, less than Bng/mL in the sample; When the detection line color is deeper than the 2nd reference line color and is shallower than the 1st reference line, show that Clenbuterol concentration is greater than Bng/mL, less than Cng/mL in the sample; When detection line not during colour generation, show that Clenbuterol concentration is greater than Cng/mL in the sample.
Article (5) 1, detection line+2 reference line:
Be coated with 0.01-30 μ g/mL Clenbuterol monoclonal antibody or Clenbuterol detection antigen on the detection line, be coated with 2 reference lines after the detection line, reference line is coated with the IgG that concentration is the anti-second kind animal protein of 0.01-30 μ g/mL, regulate the label concentration of the 2nd kind animal protein of reference line encrusting substance concentration and colloid mark, the color that makes 2 reference lines respectively with standard model in Clenbuterol content when being B, Ang/mL the color of detection line identical, determine reference line encrusting substance concentration according to this; Interval 2-6mm between each bar line; When the detection line color is deeper than the 2nd reference line color, show that sample is negative; Be shallower than the 1st when detecting when the detection line color is deeper than the 2nd reference line color, show that Clenbuterol concentration is greater than Ang/mL, less than Bng/mL in the sample; When the detection line color is deeper than the 1st reference line color and is shallower than the 1st reference line, show that Clenbuterol concentration is greater than Bng/mL in the sample.
Advantage of the present invention:
1. high specificity.Owing to adopted high-quality Clenbuterol monoclonal antibody, this detection paper specificity is extremely strong, with structural similarity material and veterinary drug commonly used and feed addictive no cross reactions such as terramycin, aureomycin, tylosin, Organic Chromium, sulfamethazine, bromhexine hydrochloride, chloromycetin, ethopabate, Ractopamine, Kitasamycin, fluorobenzeneization are examined, Thiamphenicol such as adrenaline, norepinephrines, can avoid false positive.
2. highly sensitive.This detection paper lower limit can reach 0.09ng/mL, also can be limited to certain concentration more than the 0.09ng/mL according to detecting to require to improve under the detection when preparing test paper, has avoided false positive and false negative, and the result is guaranteed reliably.
3. accuracy is good, can sxemiquantitative.Testing result correlativity with this detection paper result and the German r-Biopharm ELISA of company kit and instrumental analysis meets the requirements fully.According to detection line color and reference line color contrast or be the vitta number according to detection line and reach the half-quantitative detection purpose.
4. operation is convenient especially, need not any instrument and equipment, be suitable for face width.This test paper does not need any professional training, does not need any instrument and equipment, and the ordinary person can operate, as long as test paper is inserted in the detection liquid interpretation with the naked eye.Various samples all can be measured easily.Therefore, this test paper is suitable for face width, and sanitary inspection superintendent office, animal-breeding unit, slaughterhouse and consumer are individual can be used per capita.
5. detection speed is fast, and the colour stable time is long.Test paper inserts in the sample liquid, if negative, 30s begins colour developing, but 2min just observations later on is faster more than 40～160 times than the Clenbuterol ELISA kit detection speed that generally uses at present, and its color can forever be preserved.
6. the detected temperatures optimum range is wide.All can use at 4～40 ℃, the result is normal, does not need to carry out at low temperatures, need not take the insulation measure, and indoor field all can be used.
7. test paper long shelf-life.According to the accelerated aging test result, the test paper shelf-life can reach 2 years.
8. with low cost: this test paper method detects cost and is significantly less than Clenbuterol ELISA kit detection method, and after large-scale production, its cost also can reduce significantly.
Specific embodiment
Clenbuterol during embodiment 1:1 bar detection line+2 reference line detection paper pigs urinate
1, Clenbuterol coupling antigen is synthetic
Clenbuterol coupling antigen is divided into immunity and uses antigen with antigen and detection, and the former is used for immune animal, and the latter is used for colloid gold label or the detection line bag quilt in screening of Antibody Preparation detection of antibodies and the test paper preparation.
Antigen preparation is used in immunity: accurately take by weighing Clenbuterol 22mg, be dissolved in 2mL, in the hydrochloric acid solution of pH1.5～2.0.The sodium nitrite solution 2mL that adds pH2.0, reaction 20min.Take by weighing the 100mg bovine serum albumin simultaneously and be dissolved in 5mL pH7.4, in the phosphate buffer solution of 0.1mol/L, add wherein reacting good Clenbuterol diazo salt, with the NaOH adjust pH to 7.5 of 1mol/L, 4 ℃ are reacted after 12 hours down.With this reactant liquor pH7.4, the PBS solution of 0.01mol/L dialysis is packed as to exempt to establish and uses antigen.
Detect and use antigen preparation: adopt oralbumin to replace bovine serum albumin(BSA), all the other steps are used antigen preparation with immunity, and products therefrom is promptly as detection antigen.
2, Clenbuterol Monoclonal Antibody
Get 6 of health female secondary Balb/c mouse in 5 age in week and carry out immunity.Fundamental immunity uses antigen and equivalent complete Freund's adjuvant with the abundant mixing emulsification of stirrer the immunity of 0.1mL Clenbuterol for the first time, carries out lumbar injection, and injection volume 0.2mL/ only.Begin all around to carry out booster immunization, adjuvant is changed to incomplete Freund's adjuvant, every two all booster immunizations once, gets spleen behind the last immunity 3d and merges.
With HAT nutrient solution, the incomplete nutrient solution of IMDM, IMDM complete culture solution and 50%PEG-6000 solution pre-temperature in 37 ℃ of water-baths, the beaker that another is filled with water is put into the pre-temperature of 37 ℃ of water-baths simultaneously earlier.The above-mentioned splenocyte for preparing and myeloma cell and splenocyte are mixed in 1: 5 ratio, and the full nutrient solution that toos many or too much for use in the 50mL plastic centrifuge tube is washed 1 time, and the centrifugal 8min of 1200r/min abandons supernatant, with the dropper residual liquid that exhausts.Put in 37 ℃ of water-baths, at the bottom of the suction pipe tubular stinger, in 90s, add 1mL, the 50%PEG-6000 of preheating while stirring.After leaving standstill 90s, in 37 ℃ of water-baths, add the complete culture solution of 10mL preheating in the 60s.Suspend once gently, with the centrifugal 6min of 1000r/min, supernatant discarded suspends gently with 6mL left and right sides complete culture solution earlier.According to the quantity of used 96 well culture plates, add the HAT nutrient solution to 76.8mL, mixing gently, in 96 orifice plates that feeder cells are arranged, every hole 0.1mL once inoculates 8 blocks of plates with the mixing suspension inoculation.Culture plate is placed 37 ℃, 5%CO
2Cultivate in the incubator.Merge back 7d, change liquid 1 time, behind 13d, use the complete culture solution of 20%NBS instead according to the propagation situation with HT nutrient solution half amount.Occur the hybridoma colony behind about 7d, cell is big, round and bright.Treat that colony grows at the bottom of the hole at 1/3 o'clock, draw the mark of clonal growth on the cover plate of culture plate, be that desirable supernatant detects corresponding specific antibody this moment.After the complete cloning of cell, cell is injected mouse peritoneal, when ascites such as certain interval of time are abundant, get ascites, behind ascites usefulness albumin A immunoaffinity chromatography purifying, promptly obtain the Clenbuterol monoclonal antibody.
3, sample liquid absorption portion is handled:
Filter paper or all-glass paper etc. are immersed 2min among the PBS of 0.1mol/L pH7.4, take out, 80 ℃ of oven dry or other mode dryings, sample liquid absorption portion.
4, the preparation and the processing of colloid gold label part:
The preparation of colloid gold label part and processing comprise the preparation of collaurum colloidal sol, collaurum mark antibody of clenbuteral, colloid gold label section processes.
Collaurum colloidal sol preparation: with gold chloride (HAuCL
4) be configured to 1% mother liquor with ultrapure water, get the mother liquor of 1mL, use the ultrapure water constant volume to 100mL, be made into 0.01% solution, be heated to boiling, add a certain amount of 1% trisodium citrate aqueous solution, continue to be heated to occur transparent orange red till, be collaurum colloidal sol.
Colloid gold label Clenbuterol Monoclonal Antibody: Clenbuterol monoclonal antibody and porcine hemoglobin are used PBS (0.01mol/L respectively, pH7.0～7.5) dissolved dilution is to 3mg/mL, every 100mL collaurum colloidal sol adds Clenbuterol monoclonal antibody and the 1mL 2mg/mL porcine hemoglobin of 1mL 4mg/mL, concussion 2min is with the K of 0.2mol/L
2CO
3Regulate pH to 8.4, vibration 5min adds 11%PEG-10000 2mL, vibration 5min, the centrifugal 15min of 15000r/min removes supernatant, with PBS (0.01mol/L, pH7.4) redissolve, with the centrifugal 15min of 6000～13000r/min, remove supernatant, will precipitate (0.01mol/L with PBS, pH7.5) dilution is 1000 times, and product is as gold mark Clenbuterol monoclonal antibody.
The colloid gold label section processes: gold is marked the Clenbuterol monoclonal antibody pour in the groove, all-glass paper or filter paper are immersed 1min, take out, drying at room temperature or vacuum freeze drying promptly become the colloid gold label part.
5, detection reaction section processes:
Glutaraldehyde solution with 0.8% or 0.2% carbodiimide solution soak nitrocellulose membrane 30min, takes out, and 37 ℃ of oven dry, the top is detected with the antigen line as detection line by 1 Clenbuterol with flush coater spraying bag, and concentration is 3 μ g/mL; As the reference line, the concentration of close lower end is 2 μ g/mL to bag by the IgG line of 2 goat-anti porcine hemoglobin, and another is 1 μ g/mL.This is the detection reaction part.
6, the processing of prolongation: after the all-glass paper drying at room temperature, promptly as prolongation.
7, suction section processes: after the thieving paper drying at room temperature, promptly as the suction part.
8, test paper assembling: on backing, paste sample liquid absorption portion, colloid gold label part, prolongation, detection reaction part and the part that absorbs water successively, Clenbuterol half-quantitative detection test paper.
9, detect
Urine sample is directly used as detecting liquid.Test strips is directly inserted detection liquid, behind the 2min, observe color.If 2 detection lines all present color, show that sample is negative; If the 1st detection line colour generation, the 2nd colour generation not shows that then Clenbuterol content is 1ng/mL in the sample; If the 1st detection line and the 2nd detection line be colour generation not, show that then Clenbuterol content is 5ng/mL in the sample.Regardless of Clenbuterol content in the sample, reference line should develop the color, if reference line does not develop the color, shows that test paper lost efficacy.
The sample detection of other forms of test paper preparation and other kinds is similar to Example 1.
Claims (7)
1, the Clenbuterol rapid semi-quantitative detection method, on the backing of test paper, be stained with sample liquid absorption portion, the colloid gold label part, detection reaction part and suction part, it is characterized in that: the material of colloid gold label part mark is the potpourri of the second kind animal protein and antibody of clenbuteral, or second kind animal protein and Clenbuterol detect potpourri with antigen, being coated with Clenbuterol above the detection reaction part detects with antigen 1-3 as detection line, or be coated with antibody of clenbuteral 1-3 bar as detection line, the IgG1-3 bar that is coated with the anti-second kind animal protein simultaneously is as reference line, detection line and reference line can not be simultaneously more than 1, and the combination line number is the 2-4 bar.
2, according to the said detection method of claim 1, it is characterized in that: it is the conjugates of Clenbuterol and protein substance or polysaccharose substance formation that Clenbuterol detects with antigen.
3, detection method according to claim 1 is characterized in that: the second kind animal protein belongs to the albumen of animal for referring to the non-antibody source.
4,, it is characterized in that said protein substance is bovine serum albumin(BSA), keyhole limpet hemocyanin, oralbumin according to the said detection method of claim 2.
5,, it is characterized in that said polysaccharose substance is glucosan, shitosan, cellulose according to the said detection method of claim 2.
6, according to the said detection method of claim 1, it is characterized by: the array mode of detection line and reference line is 1 detection line and 1 reference line, 1 detection line and 2 reference lines, 1 detection line and 3 reference lines, 2 detection lines and 1 reference line, 3 detection lines and 5 kinds of forms of 1 reference line.
7, detection method according to claim 1, it is characterized in that: when colloid gold label part tagged object is the antibody of clenbuteral and the second kind animal protein, be added with prolongation between colloid gold label part and the detection reaction part, or under the colloid gold label part or on be added with thickening.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200310112423 CN1264017C (en) | 2003-12-03 | 2003-12-03 | Fast and semiquantitative detection method for Clenbuterol |
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| CN 200310112423 CN1264017C (en) | 2003-12-03 | 2003-12-03 | Fast and semiquantitative detection method for Clenbuterol |
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| CN1547024A CN1547024A (en) | 2004-11-17 |
| CN1264017C true CN1264017C (en) | 2006-07-12 |
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Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100449315C (en) * | 2005-06-20 | 2009-01-07 | 华南农业大学 | Streptomycin immune chromatographic semiquantitative test paper detecting method |
| CN101290315B (en) * | 2008-06-06 | 2013-08-07 | 华南农业大学 | Clenbuterol ELISA method and reagent kit and method for making same |
| CN102109524A (en) * | 2009-12-24 | 2011-06-29 | 上海市农业科学院 | Preparation method of test paper strip for Clenbuterol hydrochloride with high sensitivity |
| CN102520180B (en) * | 2011-12-13 | 2014-06-18 | 青岛汉唐生物科技有限公司 | Detection reagent, reagent strip and kit for semiquantitative detection of clenobuterol hydrochloride through colloidal gold fusion latex method, and preparation methods thereof |
| CN102565407A (en) * | 2011-12-22 | 2012-07-11 | 正元盛邦(天津)生物科技有限公司 | Method for semi-quantitative diagnosis of clenbuterol hydrochloride through double-indicating line immunochromatography |
| CN102879584A (en) * | 2012-09-26 | 2013-01-16 | 中国人民解放军军事医学科学院基础医学研究所 | Human blood HSP70 antibody colloidal gold-labeled detection test strip and preparation method thereof |
| CN105277665A (en) * | 2014-07-24 | 2016-01-27 | 江苏维赛科技生物发展有限公司 | Test paper strip for rapidly detecting overstandard preservative benzoic acid |
| CN105527438A (en) * | 2015-12-25 | 2016-04-27 | 广州甘蔗糖业研究所 | Colloidal gold test strip for semi-quantitative detection of alpha-glucan, and detection method thereof |
| CN107918015A (en) * | 2017-07-14 | 2018-04-17 | 王镕 | Immune chromatographic semiquantitative test paper bar, kit and detection method |
| CN111458525A (en) * | 2020-01-16 | 2020-07-28 | 卢氏实验室公司 | Test strip and kit for detecting human body physiological substance concentration and preparation method |
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