CN105334323A - Method and test strip for detecting zilpaterol, and application of test strip - Google Patents
Method and test strip for detecting zilpaterol, and application of test strip Download PDFInfo
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- CN105334323A CN105334323A CN201410387216.8A CN201410387216A CN105334323A CN 105334323 A CN105334323 A CN 105334323A CN 201410387216 A CN201410387216 A CN 201410387216A CN 105334323 A CN105334323 A CN 105334323A
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Abstract
The invention discloses a method and test strip for detecting zilpaterol, and application of the test strip. The method is a combination of colloidal-gold immunochromatography assay and competitive immunochromatography assay; an antibody used in the method is a colloidal-gold-labeled monoclonal antibody against zilpaterol, and competitive antigen zilpaterol is immobilized on a detection line of the test strip; a to-be-tested sample reacts with the colloidal-gold-labeled monoclonal antibody against zilpaterol at first and then with the competitive antigen; when a red strip does not appear on the detection line, it is determined that the to-be-tested sample contains zilpaterol; and when a red strip appears on the detection line, it is determined that the to-be-tested sample does not contain zilpaterol or has low zilpaterol content which does not exceed detection limit. The test strip comprises a base plate, and a sample absorption pad, a bonding pad, a chromatographic membrane and a water absorbing pad which are all adhered on the base plate and closely connected in sequence, wherein the bonding pad is provided with the colloidal-gold antibody, and the chromatographic membrane is provided with a detection line, a quality control line and the detection line. When the test strip is used to detect a positive sample, no color is displayed on the detection line.
Description
Technical field
The invention belongs to field of detection of food safety and technical field of immunoassay, particularly a kind of detect Zilpaterol method and test strips and application thereof.
Background technology
Food security is great livelihood issues, is related to healthy, the life security of broad masses of the people, is also the important foundation of national economic development, social stability.In recent years the problem that the various foodsafety of China is low causes bad impact to the international fame of the common people, society, economy, local government's image, China, and the monitoring of food security and the exploitation of detection technique especially need to pay attention to.
Zilpaterol (Zipaterol) is a kind of beta 2 adrenoreceptor agonists, the beta 2 receptor of the exciting smooth muscle of energy selectivity, nutritional labeling in the many animals bodies such as ox, pig, sheep and poultry is shifted to muscle by fat, remarkable increase ketoboidies lean meat element, has " clenbuterol hydrochloride " effect.More than 20 the state approval adrenoceptor agonists such as the U.S., Canada comprises Zilpaterol and is used as novel hormonal class additive for farm animal feed, (Ministry of Agriculture announces No. 176 for European Union (96/22/EC) and China, 2002) prohibit the use, reality is the serious existence of illegal abuse both at home and abroad.
Zilpaterol is that livestock and poultry muscle and internal organ remain.Because Zilpaterol has the increase lean meat percentage effect similar to Ractopamine (" clenbuterol hydrochloride "), but toxic and side effect is relatively lower, disguise as feed addictive is strong, in addition the domestic reagent not detecting Zilpaterol, the residual detection of Zilpaterol is not carried out to livestock and poultry meat etc., Zilpaterol is come with spreading by as " clenbuterol hydrochloride " substitute illegal use, works the mischief to the healthy of the people and life security.
Remain for Zilpaterol, main detection method comprises instrumental method and immunoassay two kinds.
Instrumental method: instrumental method mainly uses chromatographic technique to carry out separation and purification to sample, then adopt various detecting device to detect the single component separated.Isolation technics mainly contains thin-layer chromatography, liquid chromatography, gas chromatography, Capillary Electrophoresis etc., and detecting device then adopts UV-detector, infrared detector, fluorescence detector, mass spectrum etc.Adopt instrumental method to have the features such as accurate, sensitive, false positive rate is low, but sample pre-treatments is loaded down with trivial details time-consuming, need expensive instrument, the professional needed through specialized training operates in special laboratory.Once can only analyze a sample, analysis time is long, and analysis cost is high, is difficult to the analysis of applicable grass-roots unit for batch samples, limits it and apply.
High performance liquid chromatography: Zilpaterol molecule can be combined with C18 or C8 reversed-phase column, therefore this pillar can be utilized to be separated sample, mobile phase adopts the potpourri of phosphoric acid and acetonitrile, and isolated component mainly adopts UV-detector, diode array detector or mass spectrum to detect.
Combined gas chromatography mass spectrometry (GC-MS): gas chromatography mass spectrometry method be generally acknowledge now to the sensitiveest detection method the most accurately of Zilpaterol.Its Stationary liquid adopts nonpolar capillary column to be separated, and with two trimethyl silane trifluoroacetamide for derivatization reagent, detecting device then adopts mass spectrum to detect.
Immunoassay: immunoassay is one of " medical test technology of 21 st Century ", be widely used in endocrinology, clinical medicine, microbiology, botany and other field now, be listed in the analytical technology of the priority research nineties in 20th century, development and utilization.It is simple that immunoassay has sample pre-treatments, high specificity, and highly sensitive, analysis time is short, and analysis throughput is large, the features such as cost is low, is a kind of cheap and analysis and detection technology fast.But, the antibody used in immunoassay has higher cross reaction to a series of analogue usually, causes higher false positive rate, is generally suitable for multi-residue analysis, and as initial screening means, the further confirmation of single component also must use instrumental method.And some material immunogenicity difference (as haptens material), antibody preparation is more difficult, and manufacturing cycle is long, is very restricted.The monoclonal antibody technique grown up in the last few years and genetic engineering antibody technology are then continuous to be improved these problems, immuno analytical method is had new progress.According to the difference of antibody labeling thing, immunoassay mainly comprises radio immunoassay (RIA), enzyme immunoassay (EIA) (EIA), fluoroimmunoassay (FIA) and gold-marking immunity analytic approach (GIA) etc.
Because Zilpaterol is still very serious in the phenomenon of the illegal abuse of China.Although government prohibites the use of Clenbuterol, poisoning does not still happen occasionally, and one of them major reason is exactly that detection means is delayed, and the quick detection means being applicable to basic unit and consumer's use is delayed especially.Therefore, develop sensitive, accurate, with low cost, quick and be suitable for the Clenbuterol analyzing detecting method of onsite application, the residual monitoring tool strengthening Zilpaterol is of great significance.
Summary of the invention
Primary and foremost purpose of the present invention is that the shortcoming overcoming prior art is with not enough, provides a kind of method detecting Zilpaterol.
Another object of the present invention is to provide the test strips of the method realizing above-mentioned detection Zilpaterol.
Another object of the present invention is the application providing described test strips.
Object of the present invention is achieved through the following technical solutions: a kind of method detecting Zilpaterol, that colloidal gold immunochromatographimethod technology and competitive immunization chromatographic technique combine the method obtained, antibody is the Zilpaterol monoclonal antibody of colloid gold label, and competitive antigen Zilpaterol is fixed on the detection line of test strip; By the Zilpaterol monoclonal antibody reactive of measuring samples and colloid gold label, then with competitive antigen-reactive: when detection line does not occur red stripes, illustrate that measuring samples contains Zilpaterol; When red stripes appears in detection line, illustrate that measuring samples does not contain Zilpaterol or Zilpaterol content is very low, do not exceed detectability;
The method of described detection Zilpaterol, specifically comprises following steps:
(1) Zilpaterol monoclonal antibody is prepared: adopt ascites revulsion in body to prepare Zilpaterol monoclonal antibody;
(2) golden labeling antibody is prepared: the Zilpaterol monoclonal antibody obtain step (1) and collaurum combine, and obtain golden labeling antibody;
(3) fixing competitive antigen: Zilpaterol is fixed on the detection line of test strip;
(4) react: measuring samples and golden labeling antibody are reacted, then react with the Zilpaterol be fixed on detection line;
(5) judge: when detection line does not occur red stripes, illustrate that measuring samples contains Zilpaterol; When red stripes appears in detection line, illustrate that measuring samples does not contain Zilpaterol or Zilpaterol content is very low, do not exceed detectability;
Zilpaterol monoclonal antibody described in step (1) prepares preferably by following method: by the splenocyte of the Balb/c mouse through Zilpaterol immunity and SP2/0 myeloma cell fusion, HAT selective medium is adopted to cultivate screening hybridoma, obtain the monoclonal cell strain of the hybridoma of energy stably excreting Zilpaterol antibody, purifying, obtains Zilpaterol monoclonal antibody;
The grain size of the collaurum described in step (2) is preferably 20 ~ 30mm, is more preferably 29mm;
Golden labeling antibody described in step (2) prepares preferably by following steps:
1. with ultrapure water, chlorauride is mixed with the solution of mass percent 0.01%, is heated to boiling; Then add the trisodium citrate aqueous solution that concentration is mass volume ratio 1%, continue to be heated to occur transparent orange red till, obtain colloidal gold solution;
2. the pH value of colloidal gold solution is adjusted to 8 ~ 9, under stirring, adds Zilpaterol monoclonal antibody, reaction;
3. under stirring, add bovine serum albumin(BSA), obtain reaction solution, the final concentration of bovine serum albumin(BSA) in reaction solution is mass volume ratio 1 ~ 5%; Leave standstill, centrifugal, abandon supernatant, get precipitation, be precipitated as golden labeling antibody;
Step 1. described in chlorauride and described trisodium citrate 1:1 ~ 2.4 proportioning in mass ratio;
Step 2. described in collaurum and described Zilpaterol monoclonal antibody by 3.5 × 10
-3mol:40g proportioning;
Step 2. described in pH value regulate preferably by BAS;
The concentration of described BAS is preferably 0.2 ~ 0.5mol/L;
Step 2. described in pH value be preferably 8.5;
Step 2. described in time of reaction be preferably 30min ~ 1h; Be more preferably 1h;
Step 3. described in the final concentration of bovine serum albumin(BSA) in reaction solution be preferably mass volume ratio 1%;
Step 3. described in leave standstill time be preferably 30 ~ 60min; Be more preferably 60min;
Step 3. described in centrifugal condition be preferably 8000 ~ 15000rpm centrifugal 5 ~ 15 minutes; Be more preferably the centrifugal 15min of 15000rpm;
Realize the test strips of the method for above-mentioned detection Zilpaterol, comprise base plate, be attached to absorption of sample pad, pad, chromatographic film and adsorptive pads that base plate is closely connected successively; Wherein: pad is provided with golden labeling antibody, chromatographic film is provided with detection line and nature controlling line, detection line, nature controlling line, adsorptive pads are arranged successively, and detection line is fixed with Zilpaterol, and nature controlling line is fixed with against murine two resist;
The material of described base plate is preferably PVC board;
The material of described pad is preferably glass fibre membrane or polyester film;
The material of described chromatographic film is preferably nitrocellulose filter;
The material of described adsorptive pads is preferably thieving paper;
Described golden labeling antibody prepares preferably by following steps:
1. with ultrapure water, chlorauride is mixed with the solution of mass percent 0.01%, is heated to boiling; Then add the trisodium citrate aqueous solution that concentration is mass volume ratio 1%, continue to be heated to occur transparent orange red till, obtain colloidal gold solution;
2. the pH value of colloidal gold solution is adjusted to 8 ~ 9, under stirring, adds Zilpaterol monoclonal antibody, reaction;
3. under stirring, add bovine serum albumin(BSA), obtain reaction solution, the final concentration of bovine serum albumin(BSA) in reaction solution is mass volume ratio 1 ~ 5%; Leave standstill, centrifugal, abandon supernatant, get precipitation, be precipitated as golden labeling antibody;
Two anti-preferably sheep anti-mouse igg or rabbit against murine two anti-igg of described against murine;
The preparation method of described test strips, comprises the following steps:
(1) golden labeling antibody dissolution homogeneity is sprayed on pad;
(2) on chromatography pad, arrange detection line and nature controlling line, detection line fixes Zilpaterol, nature controlling line is fixed against murine two resist;
(3) adsorptive pads, chromatography pad, pad, absorption of sample pad are pasted toward bottom successively from the top of base plate, wherein the nature controlling line of chromatography pad is near adsorptive pads, and detection line is then near pad; Slitting, must detect the test strips of Zilpaterol;
Described golden labeling antibody solution prepares as follows:
1. with ultrapure water, chlorauride is mixed with the solution of mass percent 0.01%, is heated to boiling; Then add the trisodium citrate aqueous solution that concentration is mass volume ratio 1%, continue to be heated to occur transparent orange red till, obtain colloidal gold solution;
2. the pH value of colloidal gold solution is adjusted to 8 ~ 9, under stirring, adds Zilpaterol monoclonal antibody, reaction;
3. under stirring, add bovine serum albumin(BSA), obtain reaction solution, the final concentration of bovine serum albumin(BSA) in reaction solution is mass volume ratio 1 ~ 5%; Leave standstill, centrifugal, abandon supernatant, get precipitation, be precipitated as golden labeling antibody;
4. by TBS solubilize precipitation, golden labeling antibody solution is obtained;
The concentration of described TBS solution is preferably 0.02 ~ 0.05mol/L, is more preferably 0.02mol/L;
The pH value of described TBS solution is preferably 8.2;
The application of described test strips, comprises the following steps:
(1) on absorption of sample pad, add testing sample, under capillary action, sample liquids is to the swimming of adsorptive pads one end;
(2) judgement of result: negative: red stripes appears in nature controlling line (C line), and detection line (T line) also occurs red stripes, to show in sample containing Zilpaterol item or Zilpaterol content lower than detectability; Positive: red stripes appears in nature controlling line (C line), and detection line (T line) redfree band occurs, shows that detection content corresponding in sample is higher than detectability; Invalid: nature controlling line (C line) and detection line (T line) all redfree band occur or nature controlling line (C line) redfree band occurs but detection line (T line) has red stripes to occur, illustrate that this reagent card lost efficacy.
Principle of the present invention:
Colloidal gold immunochromatographimethod technology is a kind of solid phase labelling immunoassay technology multiple methods such as colloidal gold-labeled method, immunoassay technology and Chromatographic techniques organically combined.Its principle is using collaurum as trace labelling thing, take miillpore filter as solid phase carrier, bag is known antigen or antibody by oneself, after adding sample to be checked, through filter membrane capillarity, the antibody in sample or antigen envelope antigen or antibody on film are combined, when colloidal gold conjugate is assembled in a large number, naked eyes red color visible or pink spot, thus in qualitative or semiquantitative tachysynthesis detection method.
Paper slip provided by the present invention, adopt the principle of Competitive assays immunochromatography, when after sample solution instillation sample well, Zilpaterol antigen in sample solution combines with golden labeling antibody, and then close the antigen binding site of thing to be checked on golden labeling antibody, stop golden labeling antibody thing protein conjugate to be checked on cellulose membrane to be combined.When content of material to be checked in sample is higher than detectability, detection line (T line) redfree band occurs, result is positive; Otherwise, when in sample containing thing to be checked or content lower than detectability time, detection line (T line) has red stripes to occur, and result is feminine gender.
The present invention has following advantage and effect relative to prior art:
(1) first, the present invention adopts the principle of Competitive assays immunochromatography, high specificity;
(2) immunologic detection method provided by the invention, specifically provide the test strips realizing the method, simple to operate, detection time is short (within 15 minutes), highly sensitive feature, can make up the shortcomings such as running time length, expensive equipment such as instrumental method, high performance liquid chromatography, combined gas chromatography mass spectrometry.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
The preparation of embodiment 1 Zilpaterol monoclonal antibody
(1) Zilpaterol antigen synthesis fundamental test step
1. accurately take Zilpaterol 22mg, be dissolved in the ice-cold hydrochloric acid solution of 2mlpH3.0,0.1mol/L.Slowly add the ice-cold sodium nitrite solution 2mL of pH2.0,30mg/mL, diazo-reaction 20min at 0 ~ 5 DEG C, obtains Zilpaterol diazo salt.Accurately take 100mg bovine serum albumin is dissolved in the phosphate buffer solution of 5mlpH8.6,0.1mol/L simultaneously, is dropwise added wherein by completely reacted Zilpaterol diazo salt, and with the sodium hydroxide solution adjust pH to 7.5 of 1mol/L, at 4 DEG C, reaction is spent the night.Dialyse the PBS solution (containing 0.15mol/LNaCl) of this reactant liquor pH7.4,0.01mol/L 3d, changes water every day 4 times, collects the reactant liquor after dialysis, obtain Zilpaterol antigen.Adopt visible spectrophotometry to measure it and combine ratio.
2. envelope antigen adopts ovalbumin (OVA) to be prepared for carrier, and method with step 1..
(2) Zilpaterol mice immunized with antigen is used
Get health (Guangdong Medical Lab Animal Center) 6 in female secondary Balb/c mouse in 5 week age and carry out immunity.The Zilpaterol antigen (concentration counts 1mg/mL with albumen) that 1. 0.1mL step is prepared by fundamental immunity for the first time fully mixes emulsification with equivalent complete Freund's adjuvant stirrer, carries out lumbar injection (injection volume 0.2mL/ only).Start after surrounding to carry out booster immunization, adjuvant is changed to incomplete Freund's adjuvant, once (abdominal cavity, foot pad, muscle is taked in conjunction with injection system) every two weeks booster immunizations, about 10d after each booster shots takes a blood sample from the afterbody of Balb/c mouse, the blood collected places 1h at 37 DEG C, centrifugal 4min under 10000rpm rotating speed, draws the serum separated out with suction pipe, with the centrifugal tubule packing of 0.5ml, frozen in order to detecting use in refrigerator.
(3) cell fusion and hybridization knurl technology screening Zilpaterol monoclonal antibody
1. by 1 × 10
8splenocyte (obtaining from the mouse spleen after step (2) immunity) and 2 × 10
7sP2/0 myeloma cell mixes in the ratio of 1:5, and the full nutrient solution that cannots be used up in 50mL plastic centrifuge tube washes 1 time, and the centrifugal 8min of 1200rpm, abandons supernatant, to exhaust residual liquid, in order to avoid affect the concentration of PEG with dropper.
2. use bottom finger tapping down centrifuge tube, make cell precipitation loosening slightly, become pasty state.Then put in 37 DEG C of water-baths, at the bottom of suction pipe Inserting Tube, in 60s, add the PEG-4000 (DMSO containing percent by volume 5%) of the 1mL of preheating, mass percent 50% while stirring.After leaving standstill 60s, in 37 DEG C of water-baths, in 90s, add the complete DMEM nutrient solution (i.e. DMEM+10%v/v calf serum) of 10mL preheating, to dilute PEG termination.Slowly add during beginning, after can be slightly fast.Suspend gently once, with the centrifugal 6min of 1000rpm, supernatant discarded,
3. suspend gently with about 6mL complete culture solution, make sure to keep in mind firmly to blow and beat in order to avoid make the cell merged scatter.According to the quantity of 96 well culture plates used, add HAT nutrient solution to 76.8mL, mix gently, by mixing suspension inoculation in 96 orifice plates having feeder cells, every hole 0.1ml, once inoculates 8 blocks of plates.Culture plate is placed in 37 DEG C, 5%CO
2cultivate in incubator.
4. 6 ~ 9d after merging, changes liquid 1 time by HT nutrient culture media half amount, uses the complete DMEM nutrient solution of 15 ~ 20% after 12 ~ 14d according to proliferative conditions instead.Occur hybridoma colonies after about 6 ~ 7d, cell is large, round and bright.When colony to grow at the bottom of hole 1/3, the cover plate of culture plate draws the mark of clonal growth, now namely desirable supernatant detects corresponding specific antibody.
5. use Competitive assays ELISA method, filter out the hybridoma cell strain of secretion Zilpaterol antibody.By hybridoma cell strain high for a strain affinity, called after hybridoma cell strain 1A5.
(4) ascites produce and Zilpaterol monoclonal antibody-purified
1. selection standard BALB/c mouse (Guangdong Medical Lab Animal Center), first carries out mouse peritoneal injection with norphytane, and after one week, every mouse is according to 5 × 10
6hybridoma 1A5 is inoculated in mouse peritoneal and goes.Gather ascites after 1 week, ascites is placed in 37 DEG C and leaves standstill after 2 hours, the centrifugal 10min of 13000rpm, removing cell component and other sediment, collect supernatant, after glass fiber pellets, collect the ascites of clarification.
2. Zilpaterol is monoclonal antibody-purified: adopt sad-ammonium sulfate precipitation method to carry out preliminary purification, get ascites and be about 3mL, add the sodium-acetate buffer of 2 times of volumes 0.06mol/L, pH4.5.Dropwise slowly added in sample by caprylic acid (33 μ g/mL ascites), limit edged stirs, and adds rear continuation and stirs 30min, the centrifugal 30min of 10000rpm at 4 DEG C, goes precipitation (albumin and other non-IgG albumen).Get supernatant through 0.45 μm of micro-pore-film filtration, mix (10 × PBS:80gNaCl, 2gKCl, 11.5gNa with 1/10 volume 10 × PBS
2hPO
4, 2gKH
2pO
4, 0.5845gEDTA, 1000ml distilled water, pH7.4), with 1mol/LNaOH solution adjust pH to 7.4.Supernatant is cooled to 4 DEG C, adds ammonium sulfate (0.277g/mL makes final saturation degree be 45%).Stir 30min, at 4 DEG C, the centrifugal 30min of 10000rpm, abandons supernatant.With a small amount of PBS solution dissolution precipitation, by the PBS dialysed overnight of 50 ~ 100 times of volumes, change liquid 3 times.Solution PEG-6000 after dialysis suitably concentrates, and preserves for subsequent use at 4 DEG C.Adopt SDS-PAGE its purity of electrophoresis detection and concentration, use ELISA to detect antibody purification simultaneously and tire.Zilpaterol antibody purity reaches more than 85%, and concentration is 10.25mg/mL, tires and reaches 1: 2.56 × 10
5.
The preparation of embodiment 2 pad
1. the preparation of collaurum
(1) adopt sodium citrate prepare respectively mean grain size be about 20,30, the collaurum of 40nm.The preparation method of 20nm collaurum is as follows: utilize condensation reflux unit by 50mL massfraction be 0.01% chlorauric acid solution be heated to boiling after add rapidly the citric acid three sodium solution 1.2ml that massfraction is 1%, continue to boil 10min after colour stable, cooling, 4 DEG C save backup.Prepare 30 in the same way, the collaurum of 40nm, difference be add 0.75 respectively, the massfraction of 0.5ml is the citric acid three sodium solution of 1%.By distribution and the shape characteristic of spectral scan, nanometer dynamics laser, transmission electron microscope observing 3 kinds of collaurums.After testing, obtain respectively mean grain size be 20,29, the collaurum of 40nm.
2. colloidal gold labeled monoclonal antibody albumen
(1) process of antibody protein: by phosphate buffer 4 DEG C of dialysed overnight of antibody protein solution pH7.2,0.1mol/L to be marked, remove wherein unnecessary salt ion, with 12000rpm in 4 DEG C of centrifugal 1h, removes polymkeric substance wherein.
(2) preparation of colloidal gold solution: the mean grain size of the collaurum of the present invention is 29nm, concentration is 0.35mol/L.Make it blocking because gold grain is easily adsorbed on electrode, therefore the pH value of gold solution can not be measured with pH meter, the general pH test paper using precision.Colloidal gold solution pH to 8.5 is regulated with 0.2 ~ 0.5mol/L boric acid.
(3) colloidal gold labeled monoclonal antibody albumen: 10mL colloidal gold solution is added in the poly-plug centrifuge tube of 50mL, shake up concussion in oscillator, return to room temperature; With diluting monoclonal antibody albumen in the phosphate buffer solution of pH8.6,0.1mol/L to suitable concn, the antibody diluent containing 40g antibody protein is added in colloidal gold solution, concussion mixing 30min under room temperature condition; Dropwise add 1mL mass percent 10%BSA as confining liquid to close unnecessary collaurum binding site, sustained response concussion 15min.
(4) purifying of golden labeling antibody: adopt low-temperature and high-speed centrifuge method purifying gold mark monoclonal antibody, to remove the polymkeric substance formed in wherein unlabelled antibody, unlabelled collaurum, unlabelled BSA and labeling process.Concrete operations are: by golden labeling antibody 2000rpm, and 4 DEG C of centrifugal 20min, discard precipitation; By supernatant with 10000rpm, 4 DEG C of centrifugal 30min, supernatant discarded; Precipitation dissolved with the gold mark dilution of original volume, repeated centrifugation 2 ~ 3 times, precipitation is dissolved in original volume 1/10 gold medal mark dilution, and 4 DEG C save backup.
3. pad preparation
Get the glass fibre element film of import, be cut into the gold pad that specification size is 0.7 × 6cm, then 50 μ L gold labeling antibody bond dissolution homogeneity are sprayed on glass fibre element film, for subsequent use after drying.
The preparation of embodiment 3 Test paper
1. production testing test strips material requested
1. nitrocellulose filter (NC film): by U.S. Millipore/NC import, the specification of nitrocellulose membrane is 30cm × 3m/HAHY00010:
Article one, needed for Immunofluorescence test paper strip, cellulose nitrate membrane area is: 2.5cm × 0.6cm.
Producing the nitrocellulose membrane total area required for 100,000 part test strips is:
2.5cm × 0.6cm × 100,000=1.56 × 10
5cm
2;
Produce the total Volumes of nitrocellulose membrane needed for 100,000 part test strips:
2.25 × 10
5cm
2/ 30 × 300cm
2=20 volumes.
2. the total amount of required antigen: 4mg/mL × 2.0 μ l/cm × 0.25cm × 100,000=20mg
3. the total amount of required goat anti-rabbit igg: 1mg/ml × 2.0 μ l/cm × 0.25cm × 100,000=50mg
2. the C (nature controlling line) on tunica fibrosa, the spray of T (detection line)
Selected two anti-concentration are 1mg/mL, and antigen concentration is 4mg/mL, and draw on different NC films with drawing film machine, two lines, at a distance of 4mm, are made into test strips.
3. the pad that embodiment 2 prepares is assembled into the position (now nitrocellulose membrane has sprayed C line, T line) of pad on above-mentioned nitrocellulose membrane.Finally, same principle, is assembled into the position of sample pad in test strips by sample pad.
4. cut and finished product assembling
Absorption of sample pad, pad, be coated with nitrocellulose membrane (, near pad, nature controlling line is near adsorptive pads for detection line), the adsorptive pads of detection line and nature controlling line, head and the tail are connected mutually, optimize convergence condition, are fixed on adhesive sticker base plate in order; Be cut into the wide test paper rule of 4mm with cutting machine, load the assembling namely completing test strips in test card.
The determination of embodiment 4 ELISA test strip limit
By Zilpaterol concentration be the standard items negative sample of 1mg/mL be mixed with negative blank, 1,2,3,4,5,8, the normal gradients serial solution such as 10 μ g/kg.Draw 80 μ L standard series sample liquid in the well of test strips with suction pipe, judged result after reaction 3 ~ 6min, only have nature controlling line to occur red for positive, detection line and nature controlling line all occur red for negative.Testing result is as shown in table 1: the 3rd minute that tests time, test strips starts to occur color, and obviously, after the 5th minute, colour developing reaches stable in colour developing in 4 minutes.Same batch and different batches revision test, sensitivity is 2 μ g/kg.
Observation test test strips sensitivity experiment result in table 1 different time
Note: "; " left and right expression C line T line colour developing degree respectively; + represent colored intensity;-represent and do not develop the color; +/-represents mays be seen indistinctly
Embodiment 5
1. Sample pretreatment
(1) urine sample pre-treatment
Directly get (the attention: urine sample does not detect immediately after refrigeration to be checked of the fresh pollution-free urine sample of clarification.Urine sample sample must be collected in cleaning, drying, the plastics urine cup not containing any antiseptic or glass container, as urine sample is muddy, and can be to be checked after centrifugal 5min process under room temperature 4000rpm condition.As urine sample sample can not detect in time, can in 2 ~ 8 DEG C of stored refrigerated 24 hours, long-term preservation needs freezing-20 DEG C, must guard against multigelation sample.)
(2) tissue pre-treatment
The musculature sample taking 5g homogeneous is put in clean bottle, is placed in more than 90 DEG C water-bath water-baths 10 minutes; After being cooled to normal temperature, getting 1 ~ 2 solution cooked and detect (drawing supernatant in gravy) as far as possible.
(3) feed pre-treatment
Take 5g feed, add 10ml deionized water, then add 5ml ethyl acetate, the centrifugal 10min of vibration 3 ~ 5min, 4000rpm.Get 2ml supernatant dry in 50 DEG C of water-baths in air/nitrogen, add 0.3ml deionized water and 2ml normal hexane, the centrifugal 5min of vibration 1min, 4000rpm, removing supernatant liquid, takes off layer liquid 2 detection.
2. Detection results
(1) urine sample beneficial effect inspection
According to urine sample pre-treating method process urine sample, get urine sample liquid 2, to be added drop-wise to below test card on aperture, after 5 minutes, if show 2 lines (T+C) be namely judged to be feminine gender, if show 1 line (C) be namely judged to be the positive, if occur, other results (occurring as there not being index line) machine is judged to be that test card lost efficacy.Show that the detection of Zilpaterol colloidal gold strip of the present invention to urine sample is limited to 2 μ g/kg, testing result is as shown in table 2:
Table 2
(2) tissue beneficial effect inspection
According to organizing pre-treating method process tissue, sampling liquid 2, to be added drop-wise to below test card on aperture, after 5 minutes, if show 2 lines (T+C) be namely judged to be feminine gender, if show 1 line (C) be namely judged to be the positive, if occur, other results (occurring as there not being index line) machine is judged to be that test card lost efficacy.Show that the detection of Zilpaterol colloidal gold strip of the present invention to tissue is limited to 2 μ g/kg, testing result is as shown in table 3:
Table 3
(3) feed beneficial effect inspection
According to embodiment 2 feed pre-treating method process feed, sampling liquid 2, to be added drop-wise to below test card on aperture, after 5 minutes, if show 2 lines (T+C) be namely judged to be feminine gender, if show 1 line (C) be namely judged to be the positive, if occur, other results (occurring as there not being index line) machine is judged to be that test card lost efficacy.Show that the detection of Zilpaterol colloidal gold strip of the present invention to feed is limited to 2 μ g/kg, testing result is as shown in table 4:
Table 4
3. stability test
Sealing is stored in normal temperature test paper to detect once every 7 days, carry out stability test, result shows still can detect the positive in 120 days.
Find out from above-mentioned experimental result, test strips provided by the present invention is simple to operate, highly sensitive, high specificity.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from Spirit Essence of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. one kind is detected the method for Zilpaterol, it is characterized in that: described method is that colloidal gold immunochromatographimethod technology and competitive immunization chromatographic technique combine the method obtained, antibody is the Zilpaterol monoclonal antibody of colloid gold label, and competitive antigen Zilpaterol is fixed on the detection line of test strip; By the Zilpaterol monoclonal antibody reactive of measuring samples and colloid gold label, then with competitive antigen-reactive: when detection line does not occur red stripes, illustrate that measuring samples contains Zilpaterol; When red stripes appears in detection line, illustrate that measuring samples does not contain Zilpaterol or Zilpaterol content is very low, do not exceed detectability.
2. detect the method for Zilpaterol according to claim 1, it is characterized in that comprising following steps:
(1) Zilpaterol monoclonal antibody is prepared: adopt ascites revulsion in body to prepare Zilpaterol monoclonal antibody;
(2) golden labeling antibody is prepared: the Zilpaterol monoclonal antibody obtain step (1) and collaurum combine, and obtain golden labeling antibody;
(3) fixing competitive antigen: Zilpaterol is fixed on the detection line of test strip;
(4) react: measuring samples and golden labeling antibody are reacted, then react with the Zilpaterol be fixed on detection line;
(5) judge: when detection line does not occur red stripes, illustrate that measuring samples contains Zilpaterol; When red stripes appears in detection line, illustrate that measuring samples does not contain Zilpaterol or Zilpaterol content is very low, do not exceed detectability.
3. detect the method for Zilpaterol according to claim 2, it is characterized in that comprising following steps:
Zilpaterol monoclonal antibody described in step (1) prepares by the following method: by the splenocyte of the Balb/c mouse through Zilpaterol immunity and SP2/0 myeloma cell fusion, HAT selective medium is adopted to cultivate screening hybridoma, obtain the monoclonal cell strain of the hybridoma of energy stably excreting Zilpaterol antibody, purifying, obtains Zilpaterol monoclonal antibody;
The grain size of the collaurum described in step (2) is 20 ~ 40mm.
4. detect the method for Zilpaterol according to claim 3, it is characterized in that comprising following steps:
Golden labeling antibody described in step (2) prepares as follows:
1. with ultrapure water, chlorauride is mixed with the solution of mass percent 0.01%, is heated to boiling; Then add the trisodium citrate aqueous solution that concentration is mass volume ratio 1%, continue to be heated to occur transparent orange red till, obtain colloidal gold solution;
2. the pH value of colloidal gold solution is adjusted to 8 ~ 9, under stirring, adds Zilpaterol monoclonal antibody, reaction;
3. under stirring, add bovine serum albumin(BSA), obtain reaction solution, the final concentration of bovine serum albumin(BSA) in reaction solution is mass volume ratio 1 ~ 5%; Leave standstill, centrifugal, abandon supernatant, get precipitation, be precipitated as golden labeling antibody.
5. detect the method for Zilpaterol according to claim 4, it is characterized in that:
Step 1. described in chlorauride and described trisodium citrate 1:1 ~ 2.4 proportioning in mass ratio;
Step 2. described in collaurum and described Zilpaterol monoclonal antibody by 3.5 × 10
-3mol:40g proportioning;
Step 2. described in pH value regulated by BAS;
Step 2. described in time of reaction be 30min ~ 1h;
Step 3. described in the final concentration of bovine serum albumin(BSA) in reaction solution be mass volume ratio 1%;
Step 3. described in leave standstill time be 30 ~ 60min;
Step 3. described in centrifugal condition be centrifugal 5 ~ 15 minutes of 8000 ~ 15000rpm.
6. realize the test strips of the method for the detection Zilpaterol described in any one of Claims 1 to 5, comprise base plate, be attached to absorption of sample pad, pad, chromatographic film and adsorptive pads that base plate is closely connected successively, it is characterized in that: pad is provided with golden labeling antibody, chromatographic film is provided with detection line and nature controlling line, detection line, nature controlling line, adsorptive pads are arranged successively, detection line is fixed with Zilpaterol, and nature controlling line is fixed with against murine two resist.
7. test strips according to claim 6, is characterized in that:
The material of described base plate is PVC board;
The material of described pad is glass fibre membrane or polyester film;
The material of described chromatographic film is nitrocellulose filter;
The material of described adsorptive pads is thieving paper;
Described golden labeling antibody prepares as follows:
1. with ultrapure water, chlorauride is mixed with the solution of mass percent 0.01%, is heated to boiling; Then add the trisodium citrate aqueous solution that concentration is mass volume ratio 1%, continue to be heated to occur transparent orange red till, obtain colloidal gold solution;
2. the pH value of colloidal gold solution is adjusted to 8 ~ 9, under stirring, adds Zilpaterol monoclonal antibody, reaction;
3. under stirring, add bovine serum albumin(BSA), obtain reaction solution, the final concentration of bovine serum albumin(BSA) in reaction solution is mass volume ratio 1 ~ 5%; Leave standstill, centrifugal, abandon supernatant, get precipitation, be precipitated as golden labeling antibody;
Two of described against murine resists for sheep anti-mouse igg or rabbit against murine two anti-igg.
8. the preparation method of the test strips described in claim 6 or 7, is characterized in that comprising the following steps:
(1) golden labeling antibody dissolution homogeneity is sprayed on pad;
(2) on chromatography pad, arrange detection line and nature controlling line, detection line fixes Zilpaterol, nature controlling line is fixed against murine two resist;
(3) adsorptive pads, chromatography pad, pad, absorption of sample pad are pasted toward bottom successively from the top of base plate, wherein the nature controlling line of chromatography pad is near adsorptive pads, and detection line is then near pad; Slitting, must detect the test strips of Zilpaterol;
Described golden labeling antibody solution prepares as follows:
1. with ultrapure water, chlorauride is mixed with the solution of mass percent 0.01%, is heated to boiling; Then add the trisodium citrate aqueous solution that concentration is mass volume ratio 1%, continue to be heated to occur transparent orange red till, obtain colloidal gold solution;
2. the pH value of colloidal gold solution is adjusted to 8 ~ 9, under stirring, adds Zilpaterol monoclonal antibody, reaction;
3. under stirring, add bovine serum albumin(BSA), obtain reaction solution, the final concentration of bovine serum albumin(BSA) in reaction solution is mass volume ratio 1 ~ 5%; Leave standstill, centrifugal, abandon supernatant, get precipitation, be precipitated as golden labeling antibody;
4. by TBS solubilize precipitation, golden labeling antibody solution is obtained.
9. preparation method according to claim 8, is characterized in that: described TBS solution is the TBS solution of 0.02 ~ 0.05mol/L, pH8.2.
10. the application of the test strips described in claim 8 or 9, is characterized in that comprising the following steps:
(1) on absorption of sample pad, add testing sample, under capillary action, sample liquids is to the swimming of adsorptive pads one end;
(2) judgement of result: negative: red stripes appears in nature controlling line, and detection line also occurs red stripes, to show in sample containing Zilpaterol item or Zilpaterol content lower than detectability; Positive: red stripes appears in nature controlling line, and detection line redfree band occurs, shows that detection content corresponding in sample is higher than detectability; Invalid: nature controlling line and the equal redfree band of detection line occur or nature controlling line redfree band occurs but detection line has red stripes to occur, illustrate that this reagent card lost efficacy.
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| CN201410387216.8A CN105334323A (en) | 2014-08-07 | 2014-08-07 | Method and test strip for detecting zilpaterol, and application of test strip |
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| CN111909903A (en) * | 2020-08-06 | 2020-11-10 | 江南大学 | Zilpaterol monoclonal antibody hybridoma cell strain and application thereof |
| CN112630207A (en) * | 2020-12-24 | 2021-04-09 | 江南大学 | Method for rapidly detecting zilpaterol residue in pork |
| CN112679509A (en) * | 2021-01-21 | 2021-04-20 | 新乡学院 | Preparation and application of zilpaterol hapten, complete antigen and monoclonal antibody |
| CN114184782A (en) * | 2021-12-13 | 2022-03-15 | 深圳容金科技有限公司 | Colloidal gold detection kit and detection method for zilpaterol |
| CN114264812A (en) * | 2021-12-13 | 2022-04-01 | 深圳容金科技有限公司 | Kit and method for rapidly detecting zilpaterol content of food |
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| CN112630207A (en) * | 2020-12-24 | 2021-04-09 | 江南大学 | Method for rapidly detecting zilpaterol residue in pork |
| CN112679509A (en) * | 2021-01-21 | 2021-04-20 | 新乡学院 | Preparation and application of zilpaterol hapten, complete antigen and monoclonal antibody |
| CN114184782A (en) * | 2021-12-13 | 2022-03-15 | 深圳容金科技有限公司 | Colloidal gold detection kit and detection method for zilpaterol |
| CN114264812A (en) * | 2021-12-13 | 2022-04-01 | 深圳容金科技有限公司 | Kit and method for rapidly detecting zilpaterol content of food |
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Application publication date: 20160217 |