CN2515679Y - Clenbuterol hydrochloride fast detecting test paper strip (II) - Google Patents

Abstract

The utility model relates to an apparatus for detecting receptor agonist, in particular to a hydrochloride clenbuterol quickly detecting test strip (II), which comprises a supporting layer and a reaction reagent carrier adsorption layer. The supporting layer is a no absorbing water thin film phonograph strip; the adsorption layer is fixed on the supporting layer. The sample terminal of the adsorption layer is sequentially provided with a fiber layer, and a colloidal-gold carrier protein Xi fiber layer adsorbed and coupled with a CL. A cellulose film and the end of a handle are water absorption material layers. The cellulose film is provided with a detection blot <I> that contains a CL monoclonal antibody Wi or a polyclonal antibody, a control blot <I> and a combined blot <II> that contain an antibody of anti-carrier protein Xi. The quickly detecting test strip (II) has advantages of strong specificity, high sensitivity, simple and quick operation, imaging, visual, and correct detected result, low cost and less investment without special instruments or professional staffs. Thus, the utility model is easy to be popularized and applied.

Description

Fast clenbuterol hydrochloride detecting test paper strip (II)

(1) technical field: the utility model relates to a kind of utensil that detects receptor agonist, particularly relates to a kind of clenobuterol hydrochloride (clenbuterol CL) quick detection test paper bar (II).

(2) background technology: the meat that extremely enriches, fowl, milk, egg etc. are of high nutritive value, colory various food for the mankind provide in modern animal husbandry, are the mankind's material life, improve the health of people level, prolong human longevity and have made very big contribution.Meanwhile, modern scientific research proves, because the animal husbandry modernization, uses feed addictive and diseases prevention in a large number, the needs of curing the disease, and uses microbiotic, hormone and other synthetic drug in a large number, causes the animal foods Chinese traditional medicine residual, hormone residues etc.Transmission by food chain, medicament residue and hormone residues material constitute a threat to health and life and endanger, increase as the cancer people incidence of disease, the lopsided ratio of human fertility increases, the human body resistance to the action of a drug strengthens, teenager's sex premature, person in middle and old age's angiocardiopathy spread and problem such as some food poisoning.Often closely related with medicament residue, the hormone residues of animal foods kind.Example hydrochloric acid Clenbuterol (Clenbuterol) is abbreviated as CL, is commonly called as clenbuterol hydrochloride, is a kind of adrenaline CNS stimulant of synthetic, belongs to beta-stimulants parahormone medicine.Be mainly used in the bronchial astehma and the bronchial spasm of control people, animal during low dosage, diseases such as pulmonary emphysema, but strict dose limitation should be arranged.The consumption people that exceeds standard knows from experience and produces palpitaition, vomiting, muscle symptom such as independently do not tremble, even is in peril of one's life, and long-term excessive absorption can be put aside poisoning, causes the possibility of chromosome aberration in addition.Because of it has the effect of the muscle strength of raising, there are some sportsmen illegally to use this medicine very early, forbid for a long time in the world in feed and herding production, using.If dosage is increased to 5～10 times of therapeutic dose, the fat redistribution that makes trunk is then arranged, promote protein synthesis and promote the growth effect.Be used as additive for farm animal feed so increase, add in the animal and fowl fodders such as pig, ox, sheep, rabbit, duck, goose, to reduce the trunk fat content, improve lean meat percentage, promote growth, food poisoning when containing the residual livestock and poultry meat of CL, egg, milk deli, takes place edible in people easily.Therefore, European and American countries forbids that all CL uses as growth promoter and feed addictive.China Ministry of Agriculture also formally hereinafter forbids CL to use as feed addictive.But some its feeding family and plant illegally use CL as feed addictive under the driving of economic interests secretly, contain the pig that the CL feed is raised as using, because lean meat percentage height, client is had powerful attractive force, make its duplicity and harmfulness bigger, the serious threat human health.Beta-stimulants such as while CL also are published as forbidden drug by the International Olympic Committee.

The current detection method that CL is used always mainly contains chromatographic technique and enzyme linked immunosorbent assay etc.Chromatographic technique is to be used for the commonly used classical technology that CL detects in the world, comprises high performance liquid chromatography (HPLC), liquid chromatography/mass spectrometry logotype (LC/MS), gas chromatography/mass spectrometry logotype (GC/MS), gas chromatography/fourier infrared coupling (GC/FTIR) etc.At present Hong Kong and in ground research unit adopt GC/MS and HPLC technology to carry out the residue detection of CL in animal tissue and the hair more, it is very effective, accurate, responsive should chromatographic technique CL being detected.But sample to be checked needs through a series of pre-service, and is loaded down with trivial details time-consuming, needs 2 day time from sample pretreatment at least to drawing testing result; This in addition detection method also needs expensive instrument and equipment, and needs professional's operation, can only detect a sample at every turn, has seriously hindered the penetration and promotion of this detection method, more can't realize on-the-spot the detection.ELISA also is a kind of detection technique commonly used, and existing commercial kit is sold.Be to serve as to detect principle with competitiveness enzyme-linked reaction, with beta-stimulants antibody sandwich ELISA Plate, during detection test sample and enzyme mark bond added ELISA Plate simultaneously, OD value is surveyed in the colour developing of reaction back, contains the CL content height of healing in the lower expression test sample of OD value.Detect overall process and need 2-4 hour approximately.ELISA once can detect a plurality of samples, gets final product qualitative detection, but also detection by quantitative.But exist false positive or false negative problem.Because ELISA is the very strong technology of a kind of susceptibility, the different people operation tends to occur Different Results, result's judgement is needed the professional of certain operating experience, so this detection method also usually causes artificial flase drop and omission.Simultaneously, the kind of beta-stimulants is more, also has certain antigenic specificity between each medicine, and this also is to cause one of omission reason, in addition detection time longer, be difficult to implement on-the-spot the detection.

Eaten the CL animal tissue of residual higher concentration as people after, symptoms such as tachycardia, muscular tremor, palpitaition and neuroticism can appear.For example the first half of the year in 1997,, symptoms such as finger trembles, dizziness, palpitaition, dry, insomnia, paralysis occur, cause that the Hong Kong Special Administrative Region and the Chinese government pay much attention at the edible pig lung decoction that contains CL of China Hong Kong 17 people.Ground such as Zhejiang, Beijing, Guangdong, Henan are also found many people and are caused food poisoning owing to eat the pork of residual Clenbuterol (CL clenbuterol hydrochloride), cause the great attention of the Ministry of Agriculture and local government.Food Inspection departments at different levels and foreign export unit all strengthen the inspecting force to GL, are able to eat quality-assured meat to guarantee the people.Therefore scientific and technical personnel develop sensitivity, fast, CL detection method accurately, and detecting whether contain CL in domestic animal tissue and the feed residual has crucial meaning.

(3) utility model content: the utility model purpose is: develop special, responsive, quick, easy fast clenbuterol hydrochloride detecting test paper strip (II).

The technical solution of the utility model is: a kind of hydrochloric acid is at Lun Teluo quick detection test paper bar (II), contain supporting layer, the reaction reagent adsorbed layer, supporting layer is the lamella that do not absorb water, adsorbed layer is fixed on the supporting layer, adsorbed layer is followed successively by fibrage from the sample end, the gold mark carrier protein Xi fibrage of absorption lotus root connection CL, the cellulose rete, handle end is an absorbent material layer, the detection trace " | " that contains CL monoclonal or polyclonal antibody is arranged on the cellulose rete and the contrast trace " | " that contains anti-carrier protein Xi antibody is arranged.

The supporting layer that does not absorb water can be used the hard plastic slip; or the cardboard bar that do not absorb water; fibrage useable glass cotton; absorbent material layer can be used thieving paper; the cellulose rete can be used nitrocellulose filter; the gold mark carrier protein Xi of lotus root connection CL is the carrier protein Xi of the lotus root connection CL of glue gold mark; at the gold mark carrier protein Xi of glass wool and lotus root connection CL glass wool; and carry out on the water accepting layer and be stamped diaphragm, print the sample mark line at the about 0.5cm of the gold mark carrier protein Xi glass wool deflection glass wool one side place of glass wool and lotus root connection CL.

The carrier protein Xi of lotus root connection CL can be bovine serum albumin(BSA) (BSA) X ₁, or be chicken egg white (OVA) X ₂, or be ferritin X ₃, or be hemocyanin X ₄Deng, detection trace on the cellulose rete and contrast trace assembled arrangement can be " ‖ ".Useful good effect of the present utility model, CL quick detection test paper bar (II) has following advantage:

(1) high specificity, the susceptibility height.CL quick detection test paper bar (II) is that the basis is prepared from the carrier protein Xi of colloid gold label lotus root connection CL, no covalent bond formation between gold grain and the carrier protein Xi molecule among the gold mark carrier protein Xi, the two combines by the Van der Waals force between the charges of different polarity, the specificity of collaurum antagonist and CL and affinity influence are very little, and have higher mark rate.Therefore, quick detection test paper bar (II) has higher specificity and susceptibility, can detect 0.1 nanogram.

(2) easy and simple to handle quick.Need not any other reagent when using quick detection test paper bar (II), as long as be inserted into sample to be checked about 10 seconds, was the decidable testing result in 2 minutes.

(3) show testing result image, directly perceived, accurate.Quick detection test paper bar (II) is to show that rufous " | " and " ‖ " trace are as the positive and the negative marker that detect, showing on cellulose membrane promptly that a brownish red " | " trace is illustrated in the detected sample liquid contains CL, article two, brownish red " ‖ " trace is illustrated in and does not contain CL in the test sample, the result judges image, directly perceived, accurate, simple and clear, be not prone to the erroneous judgement of false negative and false positive.

(4) cost is low, small investment.Use quick detection test paper bar (II), do not need to join in addition instrument and equipment and other reagent, the on-the-spot detection settled at one go, with low cost, small investment, instant effect.

(5) be easy to apply on a large scale.(II) is simple to operate for the quick detection test paper bar, everybody can operate, can better satisfy different levels personnel's needs, arrive individual breed etc. as specialty chemical examination, customs quarantine control, health and epidemic prevention, quality monitoring, livestock products processing, intensive culture, have vast market prospect and bigger economical, societal benefits.

(4) description of drawings:

Fig. 1. be fast clenbuterol hydrochloride detecting test paper strip (II) structural representation.

Fig. 2. be fast clenbuterol hydrochloride detecting test paper strip (II) plan structure synoptic diagram.

(5) specific embodiment:

Make fast clenbuterol hydrochloride detecting test paper strip (II), the carrier protein Xi that at first need prepare lotus root connection CL, be used to prepare the gold mark albumin X i glass wool of lotus root connection CL, and then monoclonal antibody or the polyclonal antibody of preparation CL, be used for printing detection trace " | ", secondly need preparation sheep or the anti-carrier protein Xi of rabbit antibody, be used for printing the contrast trace.

(1) lotus root of CL and carrier protein (Xi) connection

Adopt carbodiimide method, diazotising method or glutaraldehyde method, CL and carrier protein Xi are carried out lotus root connection preparation immunizing antigen.The 5mg clenobuterol hydrochloride is dissolved in the watery hydrochloric acid (pH3.0) of precooling (2-8 ℃), adds 10mg sodium nitrite (weight ratio is 2: 1), the room temperature lucifuge stirred 6 hours, and solution colour becomes yellow.With the carrier bovine serum albumin(BSA), or chicken egg white or be ferritin, or hemocyanin is dissolved in the PBS damping fluid, adds in 25: 1 in molar ratio to go up in the reaction, and the greenhouse stirs and spends the night, and reactant liquor is got final product with the dialysis of PBS damping fluid.

(2) anti-CL monoclonal antibody (W1) and Polyclonal Antibody Preparation

Carrier protein immunity BALB/c with 50 μ g-100 μ g/ lotus root connection CL only is mouse three times, each 15-30 days at interval; Behind the booster immunization 3-4 days for the third time, with the bloodletting of immune mouse eyeball, draw neck to cause death, in 75% alcohol-pickled 5-10min, aseptic its splenocyte of getting; Shred and through 100 order nylon net filters, the centrifugal 10min of 1000r/min collects splenocyte; With 1 * 10 ⁸Splenocyte and 2-5 * 10 ⁷NSO plasmacytoma mixing with cells, 1000r/min is centrifugal, and 10min abandons supernatant, cell precipitation slowly adds 40%-50%PEG4000 (PH8.5-9.0) the effect 1min of 0.7-1ml in 37 ℃ water-soluble, slowly add serum-free 1640 nutrient culture media 15ml then, to stop the effect of PEG, 37 ℃ of water-bath 5-10min, 1000r/min is centrifugal, and 10min abandons supernatant, cell precipitation is resuspended in HAT selects in the nutrient culture media, and add 96 well culture plates (100 μ l-200 μ l) hole, put 37 ℃ of 5%CO ₂Cultivate in the incubator.Cultivate after 7-10 days,, detect the culture supernatant of hybridoma, picking strong positive cell clone (OD with enzyme linked immunosorbent assay (ELISA) with the carrier protein coated elisa plate (40 holes/piece) of the lotus root of 5 μ g-10 μ g/ml connection CL ₄₉₂More than=0.8), carry out continuous three times limiting dilution assay cloning, the hybridoma chromosome number of being produced is 92-98, the monoclonal antibody (W of its secretion ₁) specifically with CL reaction, and not with other albumen generation cross reaction, affinity constant reaches 10 ^9-10, light chain subtype is κ or λ, the heavy chain hypotype is IgG ₁IgG _2aIgG _2bIgG ₃, at the monoclonal antibody (W1) of CL specific antigen determinant, the CL Polyclonal Antibody Preparation is carried out according to a conventional method, no longer repeats herein.

(3) preparation of the gold mark carrier protein Xi glass wool of lotus root connection CL

Prepare aurosol with the sodium citrate reducing process, promptly in the 50-100ml 0.01-0.05% aqueous solution of chloraurate of boiling, add the 0.5-2% citric acid three sodium solution of 2-4ml, obtain the collaurum about diameter 15nm.With 0.1mol/L K ₂CO ₃Transfer collaurum PH to 8.5-9.5, add in the pH8.5-9.5 aurosol than carrier protein Xi with 1: 1000～1: 1300 mark lotus root connection CL to be marked, behind the mark 10min, add 20%PEG10000 to final concentration 0.05%, 4 ℃ of centrifugal 20min of 1500-3000g, remove unconjugated gold grain, 4 ℃ of centrifugal 1h of 15000g, abandon supernatant, after obtaining preliminary purification gold protein mixture, with propylene glucosan S-400 column chromatography, separation and purification gold mark albumin X i, the lotus root that obtains colloid gold label joins the carrier protein Xi of CL.The carrier protein Xi of the golden mark lotus root connection of the glue of dilution in 1: 100～1: 1500 CL is adsorbed in the processed glass cotton 4 ℃ of low-temperature vacuum dryings, the gold mark carrier protein Xi glass wool of preparation CL.

(4) preparation of anti-carrier protein Xi antibody

With the pure carrier protein Xi of 50 μ g～100 μ g/kg body weight with immunologic adjuvant through subcutaneous and intramuscular injection immune health sheep or rabbit 3～4 times, the last immunity is after 10 days, venous blood collection, measure its serum antibody titer at 1: 2000 when above with ELISA, heart blood sampling or arteria carotis bloodletting, collect hyper-immune serum, extract preparation sheep or rabbit anteserum IgG with saturated ammonium sulfate, get 1 portion of sheep or rabbit anteserum and add 2 parts of PBS (7.2) mixing, add equal-volume saturated ammonium sulfate mixing, put 4 ℃ of refrigerator 2h, 4 ℃ of centrifugal 15min of 1200r/min abandon supernatant; With an amount of PBS (7.2) dissolution precipitation, add saturated ammonium sulfate to final concentration 33%, put 4 ℃ of refrigerator 2h, 4 ℃ of centrifugal 15min of 1200r/min abandon supernatant; With a small amount of PBS (7.2) dissolution precipitation, put in 4 ℃ of refrigerators and cross the liquid dialysis with PBS (7.2), change liquid 2～3 times, 4 ℃ of centrifugal 15min of 1200r/min collect supernatant, measure its protein concentration with ultraviolet spectrophotometer, and refrigerator is preserved standby.

(5) CL quick detection test paper bar (II) is implemented the detection reaction principle

After CL quick detection test paper bar (II) sample end inserts detected sample solution, solution to be checked spreads to nitrocellulose filter together by the gold mark carrier protein Xi that siphon drives CL to be checked and lotus root connection CL, CL then to be checked has suppressed lotus root connection CL by the competition combination principle and has combined with the CL antibody test marking on the cellulose membrane, can not show the detection trace, and anti-carrier protein Xi antibody capable combines with the gold mark carrier protein Xi of lotus root connection CL, form rufous contrast trace " | ", the i.e. positive mark of a rufous " | " trace, otherwise no CL then can not stop lotus root connection CL to combine with the CL antibody test marking on the cellulose membrane in the sample solution, show that rufous detects trace " | ", same anti-carrier protein Xi antibody capable combines with the gold mark carrier protein Xi of lotus root connection CL, show rufous contrast trace " | ", form two rufous " ‖ " negative marker.If do not have the rufous mark to show on the cellulose membrane, show that then test strips lost efficacy.

(6) clenobuterol hydrochloride (CL) quick detection test paper bar (II) detects the example operation method:

The preparation of test sample liquid, if detecting meat or meat products can shred sample, grind, make 1: 2～1: 10 detected sample suspension with physiological saline, if detect to as if egg or milk, can directly egg or milk be made 1: 2～1: 10 detected sample suspension with physiological saline.

If as testing sample, then extract serum with the blood of animal, as testing sample, or the urine of directly getting animal is as test sample with serum.

CL quick detection test paper bar (II) sample end is inserted in the detected sample liquid, and insertion depth is no more than mark line 9, takes out test strip (II) after about 10 seconds, about 2 minutes of horizontal positioned, observations.

Testing result is judged: if there is a rufous mark " | " to show on cellulose membrane, the expression testing result is positive, explanation contains clenobuterol hydrochloride in detected sample liquid, promptly this animal was fed and contained violated clenobuterol hydrochloride (CL) adjuvant feed, such animal flesh and meat products can not circulate on market, the people has eaten above-mentioned meat, and sitotoxismus will take place, and will be unhealthful.

If the cellulose membrane on the CL quick detection test paper bar has two rufous marks " ‖ ", the expression testing result is negative, promptly not hydrochloric Clenbuterol composition in sample to be checked does not contain the CL composition in the result's that then is negative animal flesh and the meat products, and it is edible to be safe.

Embodiment one, referring to Fig. 1, Fig. 2 is among the figure, 1 is supporting layer, make with the plastic slice bar, 2 is the fibrage of sample end, makes with glass wool, 3 gold mark carrier protein Xi fibrages for absorption lotus root connection CL, according to the foregoing description (3) preparation method, the gold mark carrier protein Xi glass wool of preparation absorption lotus root connection CL is called for short CL gold mark glass wool, 4 is the cellulose rete, present embodiment adopts nitrocellulose filter, and 5 for absorbent material layer is that handle end is made with filter paper, will number 2,3,4,5 each layers stick on the plastic slice bar 1 from left to right, on cellulose nitrate rete 4,6 for containing CL antibody test trace " | ", in addition, 7 for containing useful sheep or the contrast trace " | " of the anti-carrier protein Xi of rabbit antibody on nitrocellulose filter 4, and two traces form combination trace " ‖ " side by side.8-1 is white diaphragm; cover glass wool 2 and be adsorbed with lotus root and join on the gold mark carrier protein Xi cotton of CL; on the corresponding diaphragm 8-1 of 2 and 3 intersections position, be partial to glass wool 2 one side 0.5cm places and be printed on mark line 9; 9 right-hand member is printed on arrow and max printed words, and filter paper layer 5 is to be coated with yellow diaphragm 8-2 on the water accepting layer (handle end).The preparation of testing sample solution and detecting operation step see (6) in the specific embodiment for details, have not just repeated here.

Claims (3)

1. a fast clenbuterol hydrochloride detecting test paper strip (II), contain supporting layer, the reaction reagent adsorbed layer, supporting layer is the lamella that do not absorb water, adsorbed layer is fixed on the supporting layer, it is characterized in that: adsorbed layer is followed successively by fibrage from the sample end, the gold mark carrier protein Xi fibrage of absorption lotus root connection CL, the cellulose rete, handle end is an absorbent material layer, the detection trace " | " that contains CL monoclonal antibody W1 or polyclonal antibody is arranged on the cellulose rete and the contrast trace " | " that contains anti-carrier protein Xi antibody is arranged.

2. test strips according to claim 1 (II); it is characterized in that: the supporting layer that does not absorb water can be used the hard plastic slip; or the cardboard bar that do not absorb water; fibrage useable glass cotton; absorbent material layer can be used thieving paper; the cellulose rete can be used nitrocellulose filter; the gold mark carrier protein Xi of lotus root connection CL is the carrier protein Xi of the lotus root connection CL of glue gold mark; on the gold mark carrier protein Xi glass wool of glass wool and lotus root connection CL and water accepting layer, carry out and be stamped diaphragm, at glass wool and the about 0.5cm of lotus root connection CL gold mark carrier protein Xi glass wool deflection glass wool one side place's printing sample mark line.

3. test strips according to claim 1 and 2 (II) is characterized in that: the carrier protein Xi of lotus root connection CL can be bovine serum albumin(BSA) (BSA) X ₁Or be the pure albumen of ovum gallinaceum (OVA) X ₂, or be ferritin X ₃, or be hemocyanin X ₄Deng, detection trace on the cellulose rete and contrast trace assembled arrangement can be " ‖ ".

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