CN1232825C - Reference immuno chromatographic test paper for detecting clenbuterol hydrochloride and detection method thereof - Google Patents
Reference immuno chromatographic test paper for detecting clenbuterol hydrochloride and detection method thereof Download PDFInfo
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- CN1232825C CN1232825C CN 03153346 CN03153346A CN1232825C CN 1232825 C CN1232825 C CN 1232825C CN 03153346 CN03153346 CN 03153346 CN 03153346 A CN03153346 A CN 03153346A CN 1232825 C CN1232825 C CN 1232825C
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Abstract
The present invention relates to reference immune chromatographic test paper for detecting clenbuterol (Cl) and a detecting method thereof. The test paper comprises two forms: A. the test paper is composed of a bottom card, a surface card and reference immune chromatographic test paper, wherein a sample adding hole and an observation window are arranged on the surface card in advance; B. the test paper is composed of a bottom card, a surface card, reference immune chromatographic test paper and a sample cup, wherein an observation window is arranged on the surface card in advance. One to four clenbuterol antibodies or detection lines of linear point samples of clenbuterol carrier protein conjugates are positioned on a chromatographic membrane of a unit test paper strip. Conjugates of trace particle labeled clenbuterol-carrier protein or conjugates of trace particle labeled clenbuterol antibodies are positioned on a trace particle combining pad. The test paper of the present invention has the advantages of simple structure, convenient use, long storage time, few detecting steps, rapidness and accuracy is used for the rapid field screening and monitoring of a small number of samples and multiple samples of suspected objects in the fields of commodity inspection, epidemic prevention and animal husbandry.
Description
Technical field
The present invention relates to biomedicine field, specifically relate to detect the reference immune chromatography test paper and the detection method thereof of clenobuterol hydrochloride.
Background technology
Clenobuterol hydrochloride (clenbuterol, Cl) (be commonly called as " clenbuterol hydrochloride "), chemical name is α-[(uncle's fourth amino) methyl]-4-amino-3,5-dichlorbenzyl alcohol hydrochloride, have another name called two chloramines, chlorine roar plain, gram breathe heavily peaceful, a kind of for beta-adrenaline excitant.Feed live pig with Cl, can improve lean meat percentage, contain the excessive meat of Cl, the neural bad reaction of unifying of cardiovascular systems such as tachycardias, palpitaition, muscular tremor can occur, the serious harm health but the people is edible.Departments such as the Ministry of Agriculture, the Ministry of Foreign Trade and Economic Cooperation and The State Administration for Entry-Exit Inspection and Quarantine repeatedly send the documents and forbid strictly to use beta-adrenaline excitant in animal produces.But still there is the phenomenon of illegal production, use beta-2-agonists series products such as " clenbuterol hydrochlorides " in domestic some areas, and a lot of poisonings take place.Many countries have implemented strict monitoring to the Cl in feed and the food.In plant, the Cl monitoring live body sample of frequent collection is a urine, effective sample such as blood plasma that other is easy to gather, and milk, ight soil and feed are also often used.
At present, the method for mensuration Cl has high performance liquid chromatography-mass spectrometry method (HPLC-MS), GC-MS(gas chromatography-mass spectrography) (GC-MS), high performance liquid chromatography (HPLC), enzyme linked immunosorbent assay method (ELISA) and radioimmunoassay (RIA) etc.The sensitivity of immunoassay, ELISA and GC-MS at present can be satisfied Cl retention analysis requirement preferably, uses morely, and detectability is usually less than 0.5 μ g/kg, and the former is main screening technique, and the latter then is the conclusive evidence method of maturation.Because above method all needs the instrument and equipment by top grade, the cost height needs Special Training to operating personnel simultaneously, and experimental result can't show immediately.Therefore be not suitable for commodity inspection, epidemic prevention, the herding producer rapid screening and monitoring to object of suspicion.
ELISA detects the existing many pieces of bibliographical informations of Cl, and present disclosed patent is " a kind of clenobuterol hydrochloride enzyme immunoassay kit and detection method thereof " (application number, 02137941.6).
Immunochromatography is the immunoassay mode that comes across a kind of uniqueness at the initial stage eighties, it is a solid phase with the fibre strip chromatographic material usually, make sample solution swimming on chromatography strip by capillary action, and make on determinand in the sample and the chromatographic material acceptor (as antibody or antigen) at determinand that the immune response of high special, high-affinity takes place simultaneously.Immune complex is by enrichment or be trapped in certain zone (detection line) of chromatographic material in the chromatography process, by enzyme reaction or directly the experimental phenomena intuitively (as colour developing) seen of the label (as collaurum) that can estimate of utilization obtain measurement result.Free label (i.e. the label that does not combine with determinand) is then crossed and is detected band, reaches the purpose of separating automatically with binding label.The common trace particle of immunochromatography technique has collaurum, latex, electroselenium, gelatin etc., and wherein the most successful label of utilization is a collaurum.
Immunochromatography is used to measure Cl, and disclosed relevant patent is as follows at present:
1. the Cl on the Cl/ chromatographic film in the sample competes the antibody of limiting the quantity of of trace particle mark jointly, judged result whether occurs by test strip.As the previous invention disclosed patent of the inventor " a kind of method of immunochromatography single stage method detection beta-adrenin agonist, medicine and the preparation of test paper " (application number, 02131321.0).In addition, patent of invention " fast clenbuterol hydrochloride detecting test paper strip " (application number, 02101928.2) and utility model patent " fast clenbuterol hydrochloride detecting test paper strip " (application number, 02202033.0) have also been used similar method.
2. the Cl of the Cl/ trace particle mark in the sample competes the antibody of limiting the quantity of on the chromatographic film jointly, judged result whether occurs by test strip.For example, disclosed utility model patent " fast clenbuterol hydrochloride detecting test paper strip (II) " (application number, 02228104.5).
3. the Cl of the Cl/ trace particle mark in the sample competes the antibody of limiting the quantity of on the chromatographic film jointly, counts judged result by the bar that test strip occurs.For example, and disclosed patent " colloidal gold strip of sxemiquantitative fast detecting clenobuterol hydrochloride and production and using method " (application number, 02139704.X).
Above-mentioned 3 kinds of immunochromatography detecting patterns have a common characteristic, be used for not having standard items to do reference when quantitative exactly, essential by amount that adds the anti-Cl antibody of Cl/ in the detection architecture or the test material of selecting strictness, come quantitatively by the control detection sensitivity of method.Consequent shortcoming is: complex manufacturingization, or quantitative accuracy changes along with the prolongation of holding time.In addition, above-mentioned 3 kinds of immunochromatography detecting patterns can not detect a plurality of samples simultaneously, and detecting pattern 1 and 2 can not be done a preliminary judgement to the concrete content of clenobuterol hydrochloride.And ELISA detects the Cl complicated operation, and it is many to detect step, and the reagent that needs is many, can not preserve by normal temperature, can only batch detection, be not suitable for commodity inspection, epidemic prevention, the herding producer rapid screening and monitoring to object of suspicion.Therefore, press for a kind of simple, fast, be convenient to preserve, be suitable for the detection test paper of non-batch, a plurality of sample field screening Cl, and set up the method for its detection.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art part, and provide a kind of simple in structure, easy to use, holding time long, can open a kind of detection clenobuterol hydrochloride (clenbuterol, Cl) the reference immune chromatography test paper of concentration that add sample and standard items on the unit test paper bar respectively and carry out non-batch, a plurality of sample detection simultaneously at 2-10 side by side.
Another object of the present invention provide a kind of step simple, fast, sensitive, utilize reference immune test paper of the present invention to detect the method for clenobuterol hydrochloride accurately.
Purpose of the present invention realizes by following scheme:
Preparation detects the reference immune chromatography test paper of clenobuterol hydrochloride, and it is one of following A, two kinds of forms of B:
A. test paper is made up of end card, face card and reference immunity-chromatography test refill, wherein the reference immunity-chromatography test refill of being made up of side by side 2-10 bar unit test paper bar is fixed between end card and the face card, reserve well and view window on the face card, the position of well is corresponding with the sample pad of unit test paper bar, and the position of view window is corresponding with the chromatographic film of unit test paper bar;
B. test paper is formed the reference immunity-chromatography test refill of wherein being made up of side by side 2-10 bar unit test paper bar by end card, face card, reference immunity-chromatography test refill and sample cup and is fixed between end card and the face card, the sample pad of unit test paper bar is stretched out outside end card and the face card, reserve view window on the face card, the position of view window is corresponding with the chromatographic film of unit test paper bar, sample cup is positioned under the reference immunity-chromatography test refill, and the position of each sample cup is corresponding with every unit test paper bar.
The detection line that 1-4 bar antibody of clenbuteral hydrochloride linear spotting is arranged on the chromatographic film of unit test paper bar has the bond of the clenobuterol hydrochloride-carrier protein couplet thing of trace particle mark on the trace particle pad of unit test paper bar; Perhaps, the detection line of 1-4 bar clenobuterol hydrochloride-carrier protein couplet thing linear spotting is arranged on the chromatographic film of unit test paper bar, the bond of the antibody of clenbuteral hydrochloride of trace particle mark is arranged on the trace particle pad.The detection line of the detection line of the antibody of clenbuteral hydrochloride linear spotting on the unit test paper bar chromatographic film or clenobuterol hydrochloride-carrier protein couplet thing linear spotting is preferably the 1-2 bar.
Detect the method for clenobuterol hydrochloride with the immune chromatography test paper of detection clenobuterol hydrochloride of the present invention, comprise the step of carrying out in the following order:
[1] sample preparation;
[2] add clenobuterol hydrochloride standard and sample respectively on the well of the reference immunity-chromatography test refill of form A, adding the Cl standard is arbitrary proportion with the unit test paper bar number that adds sample; Perhaps, the sample pad of the unit test paper bar of the reference immunity-chromatography test refill of form B is inserted in the corresponding sample cup, sample cup Central Plains is added with clenobuterol hydrochloride standard and sample, and to add the clenobuterol hydrochloride standard be arbitrary proportion with the sample cup number that adds sample;
[3] with the concentration of clenobuterol hydrochloride in any detection sample in following two kinds of methods:
1. relatively in identical position, the time of band appears in sample unit test strips and standard block test strips, the sample unit test strips evening time that the time ratio standard block test strips of band occurs occurs in same position, then in the sample clenobuterol hydrochloride concentration greater than certain normal concentration;
2. in the identical time, the depth of the band color that comparative sample unit test paper bar and standard block test strips occur, in the identical time, band appears in the sample unit test strips in same position color than certain standard block test strips occur of light color, then in the sample clenobuterol hydrochloride concentration greater than certain normal concentration.
When detecting clenobuterol hydrochloride with method of the present invention, it is limited to 1ng/ml under detecting.
Detection clenobuterol hydrochloride reference immune chromatography test paper method for making of the present invention is as follows:
[1] making of unit test paper
Detection line on the chromatographic film of unit test paper bar prepares with following pattern 1 or pattern 2:
Pattern 1: with anti-antibody of clenbuteral hydrochloride (ClenbuteroAntibody, Cl-Ab) on chromatographic film linear spotting as detection line.Nitrocellulose filter (NC film) is cut out by the wide size of 10-35mm.Cl-Ab is adjusted into 0.01-5mg/ml with concentration after fully dialysing with damping fluid, and respectively linear spotting is as detection line on film, and detection line point sample position is from film base 4-16mm, and the distance between two detection lines is 2-12mm.
Pattern 2: with clenobuterol hydrochloride-carrier protein couplet thing (Clenbuterol-carrier protein, Cl-Cp) on chromatographic film linear spotting as detection line.The NC film is cut out by the wide size of 10-35mm.Cl-Cp solution is adjusted into 0.01-5mg/ml with concentration after fully dialysing with damping fluid, and respectively linear spotting is as detection line on film, and detection line point sample position is from film base 4-16mm, and the distance between two detection lines is 2-12mm.
According to the principle of chromatography, the distance of chromatography is far away more from well, and the speed of chromatography is slow more, and resolution is high more.The present invention has utilized the principle of chromatography, adopts many chromatographic film detection lines, and to improve resolution, the present invention is according to the scope of Cl content in the sample, and the chromatographic film detection line of employing is the 1-4 bar, is preferably the 1-2 bar.
The trace particle bond pad of unit test paper bar prepares with following mode 3 or pattern 4:
Mode 3: the Cl-Cp bond with the trace particle mark is dispersed on the all-glass paper, as trace particle bond pad;
Pattern 4: the Cl-Ab bond with the trace particle mark is dispersed on the all-glass paper, as trace particle bond pad.
The trace particle of preparation trace particle bond pad is selected from: colloid gold particle, electroselenium particle, latex particle, and its preparation and labeling method are as follows:
(a) colloid gold particle:
Collaurum colloidal sol preparation: get 0.01% tetrachloro Jinsui River solution 200ml, be heated to boiling, add 1-20ml 1% sodium citrate aqueous solution, boil 5min, occur orange redly, the colloid gold particle diameter is determined as 10-80nm by Electronic Speculum;
Colloid gold label Cl-Cp/Cl-Ab: get the 50ml collaurum, (mark Cl-Cp selects pH6.0-7.5 for use to transfer pH with 0.1mol/L sal tartari, mark Cl-Ab selects pH7.5-9.0 for use), stir down collaurum colloidal sol and Cl-Cp/Cl-Ab are mixed, add the polyglycol aqueous solution again, making its final concentration is 0.05%.With semifinished product 4,000-20, centrifugal 45min under 000 * g, the precipitation be suspended to 1.5ml with physiological saline, 4 ℃ of preservations.
(b) electroselenium particle:
The preparation of electroselenium colloidal sol: add deionized water 550ml and polyacrylic acid 5-20g in four neck round-bottomed flasks of 1 liter of capacity, logical nitrogen stirring at room also adds hydrazine hydrate 40-100ml, continues to stir 10-30 minute.Selenous acid 5-15g is dissolved in the 280ml water, splashes into reaction mixture under the stirring at room and obtain the red selenium sol of 50-250nm.
(mark Cl-Cp selects pH6.0-7.5 for use to electroselenium mark Cl-Cp/Cl-Ab:Cl-Cp/Cl-Ab with the 20mmol/L phosphate buffer, mark Cl-Ab selects pH7.5-9.0 for use) be made into 1-8mg/ml concentration solution, getting 25 μ l adds in the 25ml selenium sol, stirring at room adds the PEG 8000 of 1ml1% after 10 minutes, mixing, in 4 ℃ with 3,000-10, centrifugal 5 minutes of 000rpm obtains soft red precipitate, with containing 0.05%NaN
3Phosphate buffer be made into the 1ml suspension.
(e) latex particle:
The color latex particle is available from Bangs Laboratories company.
Latex particle mark Cl-Cp/Cl-Ab: with phosphate buffer latex is diluted to 1% concentration, stirs and to add a certain amount of Cl-Cp/Cl-Ab solution down, stirring at room in 37 ℃ of water bath heat preservations 60 minutes, was placed 4-5 hour for 4 ℃ after 30 minutes.15, centrifugal 30 minutes of 000rpm, resuspended with an amount of phosphate buffer.
Unit test paper is assembled with following pattern 5 or pattern 6, and liner wherein is the plastic plate or the cardboard of coating viscose glue:
Pattern 5:, adding with the chromatographic film of pattern 1 preparation detection line on the liner earlier, add trace particle bond pad with the mode 3 preparation, sample pad then downwards successively, on chromatographic film, adding adsorptive pads at last with reference to Fig. 3;
Pattern 6:, adding with the chromatographic film of pattern 2 preparation detection lines on the liner earlier, add the trace particle bond pad for preparing with pattern 4, sample pad then downwards successively, on chromatographic film, adding adsorptive pads at last with reference to Fig. 4.
Cut into proper width after the test paper assembling is finished, be the unit test paper bar.
[2] making of reference immune chromatography test paper
2-10 is opened the unit test paper bar to be fixed on the end card side by side; form reference immunity-chromatography test refill; end card is generally plastic clip; it can make chromatographic film, trace particle bond pad, sample pad and adsorptive pads on the liner combine closely; the surface of unit test paper bar side by side compresses with the face card; the face card also is a plastic clip generally, and it can protect test paper, and it is not damaged.Reference immune chromatography test paper according to the present invention is applicable to the feature of non-batch, a plurality of sample determinations, and it is suitable selecting for use 2-10 to open the unit test paper bar.
Make the reference immune chromatography test paper that detects the special human relations in hydrochloric acid Crow with following mode 7 or pattern 8:
Mode 7: with reference to Fig. 1, the unit test paper bar is fixed on the end card, forms reference immunity-chromatography test refill, the test paper surface compresses with the face card, reserves well and view window on the face card; The position of well is corresponding with the sample pad of unit test paper bar, and the position of view window is corresponding with the chromatographic film of unit test paper bar.From the well application of sample, the result reads from view window during detection.
Pattern 8: with reference to Fig. 2, the reference immune chromatography test paper is divided into upper and lower two parts, top is unit test paper bar side by side, the bottom is a sample cup, partial fixing more than the unit test paper bar sample pad is on end card, the test paper surface compresses with the face card, and sample pad is stretched out outside end card and the face card, and view window is reserved in the position corresponding to chromatographic film on the face card.Be respectively charged into Cl standard items and sample during detection in sample cup, stretch out the sample pad of end card and face card and insert simultaneously in the sample cup of bottom, the result reads from view window.
The method that detection clenobuterol hydrochloride reference immune chromatography test paper of the present invention detects clenobuterol hydrochloride is as follows:
[1] processing of sample to be checked:
1) urine sample need not to handle:
Get an amount of urine sample and directly test (the centrifugal or filtration of suggestion when urine sample is muddy).
2) liver of low-fat content, meat, tissue:
Get the sample 5g of rubbing, adding 5mM Hcl 25ml shakes effect 1.5h makes its abundant homogenate; Get 6g homogenate (being equivalent to 1.0 livers) and place centrifuge tube centrifugal, get supernatant and put another centrifuge tube, added 1M NaOH 300 μ l immixtures 15 minutes, add 0.5M KH
2PO
4(pH3.0) 45ml, rapid mixing was placed 1.5 hours or the processing of spending the night under 4 ℃ at least.Centrifugal 1 minute of 4000 * g or 10-15 ℃ of high speed centrifugation are got supernatant (almost limpid), and balance is to room temperature.
3) higher fatty acid meat tissue:
Get and rub sample 5 gram adding PH8.5,50mM Tris damping fluid 25ml, oscillation action made its abundant homogenate in 0.5 hour; Add the 15ml normal heptane, vibrate 5 minutes, 4000 * g or 10-15 ℃ high speed centrifugation 5 minutes is removed upper strata heptane liquid and middle level fat liquor with the sterilization suction pipe; Repeat to extract once with the 15ml normal heptane, in water-soluble meat homogenate, add 0.5ml HCl, vibrated 1 hour; It is centrifugal to get 6 gram meat homogenate (being equivalent to 1.0g meat or tissue), and supernatant is put another centrifuge tube, adds 1M NaOH 300 μ l immixtures 15 minutes; Add 0.5M KH
2PO
4(pH3.0) 45ml, rapid mixing was placed 1.5 hours or the processing of spending the night under 4 ℃ at least.Centrifugal 1 minute of 4000 * g or 10-15 ℃ of high speed centrifugation are got supernatant (almost limpid), and balance is to room temperature.
5) feed sample treatment
Grind the feed sample with mortar, claim the feed sample that 2.0g grinds, add 2ml 1M HCl, add the 16ml distilled water.Eddy flow 3 minutes is put on the oscillator and was vibrated 15 minutes.Centrifugal 20 minutes of 2000rpm migrates out supernatant and adds 2ml 1M NaOH and mixes to supernatant, checks that the pH value is whether between 6.5-7.5.Centrifugal 20 minutes of 2000rpm migrates out supernatant.(if supernatant still muddiness can improve rotating speed or use filter paper filtering).With 10 times of supernatant dilutions (add 100 μ l supernatants in 900 μ l distilled waters and mix), the total extension rate of sample this moment is 100 with distilled water.Getting an amount of dilution supernatant detects.
[2] application of sample:
Structure at the reference immune chromatography test paper is the occasion of mode 7, opens at 2-10 on the well of cell stripes test strips to add Cl standard and sample respectively, and adding the Cl standard is arbitrary proportion with the unit test paper bar number that adds sample; Structure at the reference immune chromatography test paper is the occasion of pattern 8, in 2-10 sample cup, add Cl standard and sample respectively, adding the Cl standard is arbitrary proportion with the sample cup number that adds sample, and the sample pad that will stretch out the outer unit test paper bar of end card and face card is then inserted in the sample cup and tested.
[3] detect:
Behind the application of sample, according to following two kinds of detection modes, according to the detection line time of occurrence, or the color distortion of detection line comes judged result in the certain hour.
1. the sample unit test strips evening time that the time ratio standard block test strips of band occurs occurs in same position, then in the sample Cl concentration greater than certain normal concentration.
2. in the identical time, band appears in the sample unit test strips in same position color than certain standard block test strips occur of light color, then in the sample Cl concentration greater than certain normal concentration.
The present invention has compared following advantage with the method, particularly ELISA that detected Cl in the past:
1. easy and simple to handle, only need 1-2 operation steps;
2. detection speed is fast, can obtain the result in 5-15 minute;
3. can single part the also denumerable part of mensuration mensuration, room temperature preservation, holding time are long simultaneously, and can carry.
The present invention has compared following advantage with the method, particularly immunochromatographic method that detected Cl in the past:
1. use the reference of standard block test paper row culture, do not need extra nature controlling line antibody (anti-trace particle marker antibody);
2. quantitative accuracy is along with the growth of holding time can not change;
3. detection mode is flexible, can come judged result according to the color distortion of detection line time of occurrence or certain hour detection line;
4. a Cl standard can be added, also several Cl standards can be made simultaneously;
5. a sample can be detected, also several samples can be detected simultaneously.
The present invention can be applicable to detect the Cl concentration of pig urine, people's urine, dairy produce, ight soil, feed, meat product, pluck etc.The applying unit of test paper of the present invention is mainly national monitoring such as hospital, department of public security organs, sportsman's drug-testing department, epidemic prevention station department etc., and the individual also can use.
Description of drawings
Fig. 1. be the structural representation of reference immune chromatography test paper (mode 7).
Among the figure: 1. end card; 2. face card; 3. reference immunity-chromatography test refill; 4. well; 5. view window; 7. chromatographic film; 10. detection line; 1-1,1-2,1-3,1-4,1-5 are the unit test paper bar.
Fig. 2. be the structural representation of reference immune chromatography test paper (pattern 8).
Among the figure: 1. end card; 2. face card; 3. reference immunity-chromatography test refill; 5. view window; 6. sample pad; 7. chromatographic film; 10. detection line; 2-1,2-2,2-3,2-4,2-5 are the unit test paper bar; 8-1,8-2,8-3,8-4,8-5 are sample cup.
Fig. 3. be the structural representation of reference immunochromatography (pattern 5) test paper core.
Among the figure: 6. sample pad; 7. chromatographic film; 9.Cl-Ab; 10. detection line; 11. trace particle bond pad; 12.Cl-Cp conjugate; 13 trace particle mark Cl-Cp conjugate bonds; 15. adsorptive pads.
Fig. 4. be the structural representation of reference immunochromatography (pattern 6) test paper core.
Among the figure: 6. sample pad; 7. chromatographic film; 9.Cl-Ab; 10. detection line; 11. trace particle bond pad; 12.Cl-Cp conjugate; 14. trace particle mark Cl-Ab bond; 15. adsorptive pads.
Reference immune chromatography test paper of the present invention utilizes the Cl of the Cl/ trace particle mark in the sample to compete the antibody of limiting the quantity of on the chromatographic film jointly, or the Cl on the Cl/ chromatographic film in the sample competes the antibody of limiting the quantity of of trace particle mark jointly, according to the detection line time of occurrence, or the color distortion of the interior detection line of certain hour comes, and the principle of Cl is described as follows in the test sample:
With reference to Fig. 3, after dripping sample or Cl standard on the sample pad, sample or Cl standard spread to the adsorptive pads end, make trace particle mark Cl-Cp conjugate bond on the trace particle bond pad enter the liquid phase of sample or Cl standard, and therewith spread to the adsorptive pads end, on chromatographic film, Cl in sample or the Cl standard and the competition of the Cl-Cp in the trace particle bond stop the reaction of trace particle bond and Cl-Ab.Because rate of propagation is very fast, reaction is dynamic and incomplete.After adding the Cl standard on the sample pad of reference cell test strips, mark Cl ratio on Cl in the standard and the trace particle compound is certain, therefore, the speed of the reaction of standard items prevention trace particle bond and Cl-Ab is certain, and it is also just certain that the required time of visible color appears in detection line.On the sample pad of detecting unit test strips, add sample simultaneously, if the Cl concentration overgauge in the sample, Cl stops the Cl-Cp of trace particle bond and the speed of Cl-Ab9 reaction to increase in the sample, and Cl-Ab enrichment trace particle bond to the required time of visible color on the detection line also increases; Perhaps, in the regular hour, the detection line band color of detecting unit test strips is more shallow than reference cell test strips.Otherwise, if the Cl concentration in the sample less than standard, the Cl-Ab enrichment trace particle bond on the detection line is to the required time decreased of visible color; Perhaps, in the regular hour, the detection line band color of detecting unit test strips is darker than reference cell test strips.The priority that occurs according to visible color, but or the shade of the band that occurs in the same time just the Cl concentration in the judgement sample be greater than or less than standard.
With reference to Fig. 4, on sample pad, drip sample or Cl standard, sample or Cl standard spread to the adsorptive pads end, make trace particle bond on the trace particle bond pad enter the liquid phase of sample or Cl standard, and therewith spread to the adsorptive pads end, on chromatographic film, Cl in sample or the Cl standard and the competition of the Cl-Cp on the detection line, the Cl-Cp on the prevention detection line and the reaction of trace particle bond.Because rate of propagation is very fast, reaction is dynamic and incomplete.After adding the Cl standard on the sample pad of reference cell test strips, the Cl in the Cl prevention detection line in the standard and the speed of trace particle bond reaction are certain, and it is also just certain that the required time of visible color appears in detection line.On the sample pad of detecting unit test strips, add sample simultaneously, if the Cl concentration overgauge in the sample, the Cl-Cp in the sample in the Cl prevention detection line and the speed of the reaction of the Cl-Ab in the trace particle bond increase, and Cl-Cp enrichment trace particle bond to the required time of visible color increases on the detection line.Perhaps, in the regular hour, the detection line band color of detecting unit test strips is more shallow than reference cell test strips.Otherwise, if the Cl concentration in the sample less than standard, Cl-Cp enrichment trace particle bond is to the required time decreased of visible color on the detection line.Perhaps, in the regular hour, the detection line band color of detecting unit test strips is darker than reference cell test strips.The priority that occurs according to visible color, but or the shade of the band that occurs in the same time just the Cl concentration in the judgement sample be greater than or less than standard.
Embodiment
Embodiment one: electroselenium reference immune chromatography test paper detects the clenobuterol hydrochloride concentration of urine
1. the making of unit test paper
[1] preparation of fine little plain chromatographic film (NC film) detection line 10 of nitric acid:
The NC film is cut out by the wide size of 25mm.Clenobuterol hydrochloride-carrier protein (Clenbuterol-carrier protein, Cl-Cp) after 12 solution is fully dialysed with phosphate buffer, concentration is adjusted into 1mg/ml, linear spotting is as detection line on film, detection line point sample position is from film base 8mm, and detection line is 1.The at room temperature dry 30min of NC film behind the point sample places the physiological saline of 1% bovine serum albumin(BSA) to soak 30min again, takes out suck dry moisture, in 37 ℃ of insulation 30min, puts drying at room temperature place hermetically storing.
[2] preparation of electroselenium bond pad 11:
1. electroselenium colloidal sol preparation: add deionized water 550ml and polyacrylic acid 10g in four neck round-bottomed flasks of 1 liter of capacity, logical nitrogen stirring at room also adds hydrazine hydrate 69.5ml, continues to stir 20 minutes.The 9.63g selenous acid is dissolved in the 280ml water, splashes in the reaction mixture under the stirring at room, obtain particle and directly be the red selenium sol of 120nm.
2. the anti-antibody of clenbuteral hydrochloride (clenbuterolantibody of electroselenium mark, Cl-Ab) 9:IgG type Cl-Ab is made into the solution that concentration is 4.6mg/ml with the 20mmol/LpH7.3 phosphate buffer, get in the 25ml selenium sol of 25 μ l adding pH7.3, stirring at room adds 1% PEG80001ml after 10 minutes, mixing is under 4 ℃, with 5, centrifugal 5 minutes of 000rpm obtains soft red precipitate, with containing 0.05% NaN
3Phosphate buffer be made into the 1ml suspension.
3. the Cl-Ab bond 13 with the electroselenium mark is dispersed on the all-glass paper that thickness is 1mm, and the freeze-drying hermetically drying is preserved.
[3] assembling of unit test paper:
At the NC film 7 that adds the Cl-Cp detection line on the liner earlier, then add successively downwards electroselenium bond pad 11, sample pad 6, on the NC film, add adsorptive pads 15 at last.
[4] shearing of unit test paper bar:
Cut into the width of 4mm after the unit test paper assembling is finished, be the unit test paper bar.
2. the reference immune chromatography test paper is made
10 unit test paper bars are installed on the plastic bottom card 1 side by side, promptly become reference immunity-chromatography test refill 3, the test paper surface compresses with face card 2, and the sample pad 6 of unit test paper bar is stretched out outside end card and the face card, and view window 5 is reserved at the position of corresponding unit test strips chromatographic film 7 on the face card.The test paper bottom is the sample cup 8 with upper unit test strips position correspondence.
3. detection method
Add concentration respectively and be 0 and the Cl standard items of 10ng/ml in 2 sample cups, all the other 8 add urine specimens, in the sample cup of the sample pad 6 parallel insertion bottoms of upper unit test strips, and judged result with the following methods.
1. react 15min, observation band shade.Red stripes appears in standard 0ng/ml unit test paper, and more weak light red band appears in standard 10ng/ml unit test paper.Cl concentration is not greater than 10ng/ml if band does not appear in the urine sample unit test paper; If the urine sample unit test paper occur light red band color 0 and 10ng/ml standard block test paper between, then Cl concentration 0 and 10ng/ml between; If urine sample unit test paper band color and 0ng/ml standard block test paper band solid colour, then the result is negative.
2. react about 5min, more weak red stripes has just appearred in standard 0ng/ml unit test paper, and band does not appear in the 10ng/ml unit test paper, observes the urine sample unit test paper simultaneously, if band is negative, it is positive band not occur.
Embodiment two: collaurum reference immune chromatography test paper detects clenobuterol hydrochloride concentration in the pig liver sample
1. the making of unit test paper
[1] preparation of NC film detection line 10:
The NC film is cut out by the wide size of 25mm.After Cl-Ab 9 usefulness phosphate buffers are fully dialysed, concentration is adjusted into 0.1mg/ml, linear spotting is as detection line on film, and detection line point sample position is from film base 8mm, and detection line is 1, and the distance between two detection lines is 6mm.The at room temperature dry 30min of NC film behind the point sample places the physiological saline of 1% bovine serum albumin(BSA) to soak 30min again, takes out suck dry moisture, in 37 ℃ of guarantor 30min, puts drying at room temperature place hermetically storing.
[2] preparation of collaurum bond pad:
1. collaurum colloidal sol preparation: get 0.01% tetrachloro Jinsui River solution 200ml, be heated to boiling, add 2ml 1% sodium citrate aqueous solution, boil 5min, occur orange redly, the colloid gold particle diameter is 35-45nm;
2. the carrier protein of colloid gold label Cl-Cp 12:Cl-Cp is selected bovine serum albumin(BSA) for use, get the 50ml collaurum, transfer pH to 6.5, stir the Cl-Cp 5ml that adds affinity purification down with 0.1mol/L sal tartari, add the polyglycol aqueous solution again, making its final concentration is 0.05%.With semifinished product centrifugal 45min under 12000 * g, precipitation is suspended to 1.5ml and is diluted to OD520 with physiological saline is 3,4 ℃ of preservations.
3. the Cl-Cp bond 14 with colloid gold label is dispersed on the all-glass paper that thickness is 1mm, and the freeze-drying hermetically drying is preserved.
[3] assembling of unit test paper:
At the NC film 7 that adds the Cl-Ab detection line on the liner earlier, then add successively downwards collaurum bond pad 11, sample pad 6, on the NC film, add adsorptive pads 15 at last.
[4] shearing of unit test paper bar:
Cut into the width of 4mm after the unit test paper assembling is finished, be the unit test paper bar.
2. the reference immune chromatography test paper is made
3 unit test paper bars are installed on the plastic bottom card 1 side by side, promptly become reference immunity-chromatography test refill 3, the test paper surface compresses with face card 2, and face is stuck in the sample pad 6 of corresponding unit test paper and the position of chromatographic film 7 is reserved well 4 and view window 5 respectively.
3. detection method
The pig liver sample is handled by the described method of this instructions, and the supernatant that obtains adds 0ng/ml, 5ng/mlCl standard items respectively and detects sample as detecting sample in well 4 during detection, and the result reads from view window, with the following methods judged result.
1. react 15min, the depth of observation band color.This moment, band all appearred in 0ng/ml, 5ng/ml Cl standard block test paper, band do not occur as the detecting unit test paper, or band color shallow than 5ng/ml Cl standard block test paper, then detected the Cl＞5ng/ml of sample; The band color that occurs as the detecting unit test paper is darker than 5ng/ml Cl standard block test paper, but more shallow than 0ng/ml, the Cl that then detects sample is between 0ng/ml and 5ng/ml.
2. react 5-10min, the observation band occurs successively.0ng/ml, do not see the detecting unit band when detection line appears in 5ng/ml standard block test paper, then detect the Cl＞5ng/ml of sample; Do not see when detection line appears in the 0ng/ml standard block that band appears in 5ng/ml standard block and detecting unit, the Cl that then detects sample is between 0ng/ml and 5ng/ml.
Embodiment three: collaurum reference immune chromatography test paper detects clenobuterol hydrochloride concentration in the pork sample
Removing detection line is Cl-Cp, and detection line is beyond 2, and the 1. making of unit test paper, 2. the reference immune chromatography test paper is made with embodiment two.
3. detection method:
The pork sample is handled by the described method of this instructions, and the supernatant that obtains adds 0ng/ml, 1ng/mlCl standard items respectively and detects sample as detecting sample in well 4 during detection, and the result reads from view window, with the following methods judged result.
1. react 15min, the depth of observation band color.This moment, band all appearred in 0ng/ml, 1ng/ml Cl standard block test paper, band do not occur as the detecting unit test paper, or band color shallow than 1ng/ml Cl standard block test paper, then detected the Cl＞1ng/ml of sample; The band color that occurs as the detecting unit test paper is darker than 1ng/ml Cl standard block test paper, but more shallow than 0ng/ml, the Cl that then detects sample is between 0ng/ml and 1ng/ml; The band color that the detecting unit test paper occurs and the color no significant difference of 0ng/ml standard block test paper, the Cl that then detects sample is negative.
2. react 5-10min, when the detection line that standard block test paper and detecting unit test paper occur was following situation, article one line appearred in the Cl＞1ng/ml:0ng/ml standard block that detects sample, and band does not appear in 1ng/m standard block and detecting unit; First, second line appears in the 0ng/ml standard block, and article one line appears in the 1ng/ml standard block, and band does not appear in detecting unit; First, second line all appears in 0ng/ml standard block, 1ng/ml standard block, and article one line appears in detecting unit.When the detection line that standard block test paper and detecting unit test paper occur was following situation, the Cl that detects sample was between 0ng/ml and 1ng/ml: the 0ng/ml standard block, and detecting unit article one line all appears, but band does not appear in the 1ng/ml standard block; First, second line appears in the 0ng/ml standard block, and detecting unit article one line occurs but band does not appear in the 1ng/ml standard block; First, second line all appears in 0ng/ml standard block detecting unit, and article one line all appears in the 1ng/ml standard block.
The band number that the detecting unit test paper occurs and the color no significant difference of color and 0ng/ml standard block test paper, the Cl that then detects sample is negative.
Embodiment four: latex reference immune chromatography test paper detects clenobuterol hydrochloride concentration in the feed sample
1. the making of unit test paper
[1] preparation of detection line 10 on the NC film 7:
The NC film is cut out by the wide size of 25mm.After Cl-Ab 9 usefulness phosphate buffers are fully dialysed, concentration is adjusted into 0.05mg/ml, linear spotting is as detection line on film, and detection line point sample position is from film base 6mm, and detection line is 4, and the distance between two detection lines is 2.5mm.The at room temperature dry 30min of NC film behind the point sample places the physiological saline of 1% bovine serum albumin(BSA) to soak 30min again, takes out suck dry moisture, in 37 ℃ of insulation 30min, puts drying at room temperature place hermetically storing.
[2] preparation of latex bond pad:
1. latex mark Cl-Cp: the color latex particle is available from BangsLaboratories company, and the carrier protein of Cl-Cp is selected bovine serum albumin(BSA) for use.Getting 100ml, to be diluted to concentration with phosphate buffer be 1% latex, stirs the Cl-Cp solution that adds 500 μ g down, and stirring at room in 37 ℃ of water bath heat preservations 60 minutes, was placed 4-5 hour for 4 ℃ after 30 minutes.15, centrifugal 30 minutes of 000rpm, resuspended with an amount of phosphate buffer.
2. the Cl-Cp bond 14 with the latex mark is dispersed on the all-glass paper that thickness is 1mm, and the freeze-drying hermetically drying is preserved.
[3] assembling of unit test paper:
In the chromatographic film 7 that adds Cl-Ab detection line 10 on the liner earlier, then add successively downwards collaurum bond pad 11, sample pad 6, on chromatographic film, add adsorptive pads 15 at last.
[4] shearing of unit test paper bar:
Cut into the width of 4mm after the unit test paper assembling is finished, be the unit test paper bar.
2. the reference immune chromatography test paper is made
4 unit test paper bars are installed on the plastic bottom card 1 side by side, promptly become reference immunity-chromatography test refill 3, the test paper surface compresses with face card 2, and face is stuck in the sample pad 6 of corresponding unit test paper and the position of chromatographic film 7 is reserved well 4 and view window 5 respectively.
3. testing process.
The feed sample process is handled by the described method of this instructions, and the supernatant of gained is as detecting sample.
Add 0ng/ml Cl standard items and sample during detection in well 4 respectively, the result reads from view window, with the following methods judged result.
1. react 15min, observation band shade.Strong positive (＞10ng/ml): band appears in the standard block test paper, and band does not appear in the detecting unit test paper.Positive (＞5ng/ml): article one detection line all appears in standard block test paper and detecting unit test paper, and the second detection line appears in the standard block test paper and the detecting unit test paper does not occur.Weak positive (＞1ng/ml): first, second detection line all appears in standard block test paper and detecting unit test paper, and the 3rd detection line appears in the standard block test paper and the detecting unit test paper does not occur.Weak positive (＞0.5ng/ml): first, second, third detection line all appears in standard block test paper and detecting unit test paper, and the 4th detection line appears in the standard block test paper and the detecting unit test paper does not occur.Negative (0ng/ml): 2 detection line color no significant differences appear in standard block test paper and detecting unit test paper.
2. react 5-10min, the observation band occurs successively.Positive (＞5ng/ml): when appearring in standard block, do not see article one detection line the detecting unit band; Positive (＞2ng/ml): when appearring in standard block, do not see the second detection line detecting unit second band.The weak positive (＞1ng/ml): when appearring in standard block, do not see the 3rd detection line detecting unit the 3rd band.The weak positive (＞0.5ng/ml): when appearring in standard block, do not see the 4th detection line detecting unit the 4th band.Negative (0ng/ml): indifference appears in standard block and detecting unit band.
The inventive method is used for the detection by quantitative of biological sample clenobuterol hydrochloride.
Claims (4)
1. detect the reference immune chromatography test paper of clenobuterol hydrochloride, it is characterized in that it is one of following A, two kinds of forms of B:
A. test paper is made up of end card (1), face card (2) and reference immunity-chromatography test refill (3), wherein the reference immunity-chromatography test refill of being made up of side by side 2-10 bar unit test paper bar (3) is fixed between end card and the face card, reserve well (4) and view window (5) on the face card, the position of well is corresponding with the sample pad (6) of unit test paper bar, and the position of view window is corresponding with the chromatographic film (7) of unit test paper bar;
B. test paper is made up of end card (1), face card (2), reference immunity-chromatography test refill (3) and sample cup (8), wherein the reference immunity-chromatography test refill of being made up of side by side 2-10 bar unit test paper bar (3) is fixed between end card and the face card, the sample pad of unit test paper bar (6) is stretched out outside end card and the face card, reserve view window (5) on the face card, the position of view window is corresponding with the chromatographic film (7) of unit test paper bar, sample cup is positioned under the reference immunity-chromatography test refill, and the position of each sample cup is corresponding with every unit test paper bar;
The detection line (10) of 1-4 bar with antibody of clenbuteral hydrochloride (9) linear spotting arranged on the chromatographic film of unit test paper bar (7), the bond (13) of the clenobuterol hydrochloride-carrier protein couplet thing (12) of trace particle mark is arranged on the trace particle pad (11) of unit test paper bar; Perhaps, the detection line (10) that 1-4 bar clenobuterol hydrochloride-carrier protein couplet thing (12) linear spotting is arranged on the chromatographic film of unit test paper bar has the bond (14) of the antibody of clenbuteral hydrochloride (9) of trace particle mark on the trace particle pad (11) of unit test paper bar.
2. the reference immune chromatography test paper of detection clenobuterol hydrochloride according to claim 1, the detection line that it is characterized in that the antibody of clenbuteral hydrochloride linear spotting on the unit test paper bar, or the detection line of clenobuterol hydrochloride-carrier protein couplet thing linear spotting is the 1-2 bar.
3. one kind is detected the method for clenobuterol hydrochloride with the immune chromatography test paper of the described detection clenobuterol hydrochloride of claim 1, it is characterized in that it comprises the step of carrying out in the following order:
[1] sample preparation;
[2] add clenobuterol hydrochloride standard and sample respectively on the well (4) of the reference immunity-chromatography test refill of form A, adding the clenobuterol hydrochloride standard is arbitrary proportion with the unit test paper bar number that adds sample; Perhaps, the sample pad (6) of the unit test paper bar of the reference immunity-chromatography test refill of form B is inserted in the corresponding sample cup (8), be added with clenobuterol hydrochloride standard and sample in sample cup (8) Central Plains, adding the clenobuterol hydrochloride standard is arbitrary proportion with the sample cup number that adds sample;
[3] with the concentration of clenobuterol hydrochloride in any detection sample in following two kinds of methods:
1. relatively in identical position, the time of band appears in sample unit test strips and standard block test strips, the sample unit test strips evening time that the time ratio standard block test strips of band occurs occurs in same position, then in the sample clenobuterol hydrochloride concentration greater than certain normal concentration;
2. in the identical time, the depth of the band color that comparative sample unit test paper bar and standard block test strips occur, in the identical time, band appears in the sample unit test strips in same position color than certain standard block test strips occur of light color, then in the sample clenobuterol hydrochloride concentration greater than certain normal concentration.
4. method according to claim 3 wherein is limited to 1ng/ml under the detection of clenobuterol hydrochloride.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 03153346 CN1232825C (en) | 2003-08-11 | 2003-08-11 | Reference immuno chromatographic test paper for detecting clenbuterol hydrochloride and detection method thereof |
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| CN 03153346 CN1232825C (en) | 2003-08-11 | 2003-08-11 | Reference immuno chromatographic test paper for detecting clenbuterol hydrochloride and detection method thereof |
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| CN1232825C true CN1232825C (en) | 2005-12-21 |
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101398424A (en) * | 2007-09-26 | 2009-04-01 | 上海乾隽生物技术有限公司 | Rapid detection kit for brown meat essence and method for manufacturing same |
| CN101470115A (en) * | 2007-12-28 | 2009-07-01 | 万积成 | Apparatus for simultaneously detecting 10 kinds of food microorganism and screening diagnosis method thereof |
| CN101993486B (en) * | 2009-08-27 | 2014-10-08 | 深圳市三方圆生物科技有限公司 | Clenbuterol immunogen, coatingen and application thereof in colloidal gold test paper |
| 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 |
| CN104569408B (en) * | 2014-12-30 | 2016-06-22 | 无锡中德伯尔生物技术有限公司 | A kind of colloidal-gold detecting-card of fenoterol and preparation method thereof |
| CN107167615B (en) * | 2017-05-16 | 2019-01-15 | 河南大学 | It is a kind of for clenobuterol hydrochloride detection electroselenium, colloidal selenium marked object, detection card and preparation method thereof, application |
| CN109100303A (en) * | 2017-06-21 | 2018-12-28 | 上海溯源生物技术有限公司 | Test strips |
| CN109100346A (en) * | 2017-06-21 | 2018-12-28 | 上海溯源生物技术有限公司 | Test paper bar assembly |
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