CN1727899A - Method for detecting Enrofloxacin through semiquantitative test paper of side stream immunity chromatography - Google Patents
Method for detecting Enrofloxacin through semiquantitative test paper of side stream immunity chromatography Download PDFInfo
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Abstract
A method of using immune chromatography semi - quantitative test paper to detect ennorxacin sticks sample solution absorbing portion, colloidal gold labeled portion, detecting reaction portion and water absorbing portion in sequence on backing of test paper. The detecting reaction portion is prepared as coating 1 - 3 bar of ennorxacin antibody or detection antigen as detection line, coating 1 - 3 bar of IgG for anti - second generic animal protein as reference line and using 2 - 4 bar of combination line formed by said two lines being used no more than one bar at the same time in combination.
Description
Technical field
The present invention relates to a kind of method for antibiotic residue detection in the animal food, particularly a kind of method for detecting Enrofloxacin through semiquantitative test paper of side stream immunity chromatography.
Background technology
Antibiotic residue is a ubiquitous problem in the whole world livestock and poultry cultivation, and wherein Enrofloxacin is a kind of more common residual antibiotic.Enrofloxacin is a kind of fluoroquinolone antibiotics, it has brought into play vital role in prevention and treatment fowl bacterial disease and Mycoplasma disease, but its residue toxic and side effect is bigger, the human long-term because edible food that contains enrofloxacin residual and unnecessary this microbiotic of absorption, to damage nervous system, cause arthritis, disease such as phlebitis, and may cause the appearance of drug-fast bacteria.In order to strengthen the residue of veterinary drug management work, guarantee the livestock products quality safety, China Ministry of Agriculture has revised " animal food herbal medicine maximum residue limit(MRL) " in 2002, wherein the maximum residue limit(MRL) of Enrofloxacin is 100 μ g/kg in the regulation milk, and enrofloxacin residual in other animal muscles, fat, the kidneys such as sheep, pig, chicken is also had phase limes reacting dose regulation.
To the main at present high performance liquid chromatography (HPLC) that adopts of the detection of enrofloxacin residual.Yet sample pre-treatments is very loaded down with trivial details when using HPLC, and assay method is quite complicated, could operate by the personnel of specialized training, and it is very little to detect flux.Under the situation of the present animal-breeding decentralized management of China, be difficult to unified management, therefore cause the quality of animal derived food to be difficult to unanimity, adopt large-scale instrument to detect the monitoring product quality merely, very unrealistic, and instrument detecting expense height, be difficult to popularize and use.The external method that generally adopts the rapid screening detection to prove conclusively in conjunction with instrument to the detection of enrofloxacin residual, China is studied seldom on aspect a large amount of sample rapid screening detection techniques.Microorganism inhibition method is a kind of antibiotic-screening detection method comparatively commonly used, its cardinal principle is according to antimicrobial the inhibiting effect of special microorganism to be detected to be subjected to antimicrobial residual in the sample product, mainly comprise cylinder plate method, paper disk method, agar diffusion method and triphenyl tetrazolium chloride method, though still using at present, but these method poor specificity, time and effort consuming, the measurement result error is very big.The ELISA method is the more antibiotic-screening detection method of another kind of research at present, have highly sensitive, detection limit big, low cost and other advantages, but this method also needs part laboratory equipments such as microplate reader, general 1-4 hour analysis time, still has certain limitation.Therefore, the present corresponding Enrofloxacin detection technique of China and not really complete, necessary development is simple and fast testing product easily more.
Summary of the invention
Purpose of the present invention promptly is to develop easy, sensitive, cheap Enrofloxacin rapid semi-quantitative test paper detecting method.
The present invention posts sample liquid absorption portion, colloid gold label part, detection reaction part, suction part successively on the backing of test paper.Colloid gold label is glass fibre or acetate fiber or nylon membrane partly, and material of mark is the potpourri of the second kind animal protein and Enrofloxacin antibody on it, or the potpourri of the second kind animal protein and Enrofloxacin detection usefulness antigen.Be coated with Enrofloxacin above the detection reaction part and detect with antigen 1-3 as detection line, or be coated with Enrofloxacin antibody 1-3 bar as detection line, the IgG 1-3 bar that also is coated with the anti-second kind animal protein simultaneously is as reference line.Can not be when detection line and reference line combination simultaneously more than 1, the combination number of lines is the 2-4 bar, and array mode is 1 detection line+1 reference line, 1 detection line+2 reference line, 1 detection line+3 reference line, 2 detection line+1 reference lines, five kinds of forms of 3 detection line+1 reference lines.According to the accuracy requirement of half-quantitative detection, the combination line number is many more, and sxemiquantitative is accurate more.
Detect among the present invention with antigen and be meant the conjugates that Enrofloxacin and carrier mass form, wherein carrier mass comprises protein, protein fragments, synthetic polypeptide, semi-synthetic polypeptide, polysaccharide, as seralbumin, globulin, lipoprotein, polyamino acid, glucosan, exemplary carrier mass comprises bovine serum albumin(BSA), oralbumin, keyhole limpet hemocyanin, thyroglobulin, L-poly-D-lysine etc.In addition, carrier mass also can be other the synthetic or natural polymers with reactive group, as diphtheria toxin, tetanus toxin, yeast, nylon, dextrorotation glucosan, cellulose etc., equally also can be used for preparing detecting and use antigen.The second kind animal protein refers to that the non-antibody source belongs to the albumen of animal, and for example, antibody is mouse source property, and then the second kind animal protein can be chicken, rabbit or other non-mouse source property animal proteins such as oralbumin, albumin rabbit serum.
The each several part of test paper described in the invention is handled with function as follows:
Backing:,, play fixing other ingredients of test paper of supporting as the PVC plate for simultaneously scribbling the toughness material that does not absorb water of adhesive sticker.
The preparation of sample liquid absorption portion: with 2min among the PBS of filter paper or all-glass paper immersion pH7.0-8.4, take out, 80 ℃ of oven dry or other mode dryings promptly as sample liquid absorption portion, play a part during detection to absorb sample solution, are convenient to sample solution and move up.
The preparation of colloid gold label part: this part plays the fixedly antigen or the antibody of colloid gold label.Preparation process comprises preparation, colloid gold label Enrofloxacin antibody or Enrofloxacin antigen, the colloid gold label section processes of collaurum colloidal sol.
(1) preparation of collaurum colloidal sol: with gold chloride (HAuCL
4) be configured to 1% mother liquor with ultrapure water, get the mother liquor of 1mL, use the ultrapure water constant volume to 100mL, be made into 0.01% solution, be heated to boiling, add 1% trisodium citrate aqueous solution of 1-5mL, continue to be heated to occur transparent orange red till, be collaurum colloidal sol.
(2) colloid gold label Enrofloxacin antibody: enrofloxacin monoclonal antibody or polyclonal antibody and the second kind animal protein are used PBS (0.01mol/L respectively, pH7.0-7.5) dissolved dilution is to 2-4mg/mL, every 100mL collaurum colloidal sol adds Enrofloxacin antibody and the 1-1.5mL 2-4mg/mL second kind animal protein of 1-3mL 2-4mg/mL, concussion 2min is with the K of 0.2mol/L
2CO
3Regulate pH to 8.4, vibration 5min adds 11%PEG-100002mL, vibration 5min, the centrifugal 15min of 8000-15000r/min removes supernatant, with PBS (0.01mol/L, pH7.0-7.5) redissolve, with the centrifugal 15min of 6000-13000r/min, remove supernatant, will precipitate (0.01mol/L with PBS, pH7.0-7.5) dilution is 500-2000 times, and product is as gold mark Enrofloxacin antibody (GAb).
(3) colloid gold label Enrofloxacin antigen: the Enrofloxacin detection is used PBS (0.01mol/L respectively with the antigen and the second kind animal protein, pH7.0-8.4) dissolved dilution is to 2-4mg/mL, every 100mL collaurum colloidal sol adds detection antigen and the 1-1.5mL 2-4mg/mL second kind animal protein of 1-3mL 2-4mg/mL, concussion 2min is with the K of 0.2mol/L
2CO
3Regulate pH to 8.4, vibration 5min adds 11%PEG-100002mL, vibration 5min, the centrifugal 15min of 8000-15000r/min removes supernatant, with PBS (0.01mol/L, pH7.0-7.5) redissolve, with the centrifugal 15min of 6000-13000r/min, remove supernatant, will precipitate (0.01mol/L with PBS, pH7.0-7.5) dilution is 500-2000 times, and product is as the Enrofloxacin antigen (GAg) of golden mark.
(4) colloid gold label section processes: GAb or GAg are poured in the groove, all-glass paper or filter paper are immersed 1min, take out, after the drying at room temperature, promptly as the colloid gold label part.
Detection reaction partly prepares: glutaraldehyde solution with 0.8% or 0.2% carbodiimide solution soak nitrocellulose membrane or cellulose acetate film or nylon membrane 30min, take out, 37 ℃ of oven dry, the top bag is detected by the Enrofloxacin of 1-3 bar variable concentrations uses antigen line or Enrofloxacin antibody line as detection line, wraps simultaneously by the IgG line of the anti-second kind animal protein of 1-3 bar variable concentrations as the reference line.When colloid gold label part tagged object was the Enrofloxacin antibody and the second kind animal protein, detection line then wrapped by Enrofloxacin detection antigen; When colloid gold label part tagged object was the Enrofloxacin detection usefulness antigen and the second kind animal protein, detection line then wrapped by Enrofloxacin antibody; Detection line and reference line can not be simultaneously more than 1, and combination line is counted the 2-4 bar.This is the detection reaction part, and it is that reaction result is come out with macroscopic characterization that this part mainly acts on.
Suction part preparation: after all-glass paper or filter paper or thieving paper drying at room temperature, promptly as the suction part.This part mainly acts on the unnecessary sample solution that is moving up and absorbs.
Test paper assembling: on backing, paste sample liquid absorption portion, colloid gold label part, detection reaction part and suction part successively, Enrofloxacin half-quantitative detection test paper.
Detect principle: detect the object difference of principle because of colloid gold label, and variant slightly.
When the tagged object of colloid gold label part is the Enrofloxacin antibody and the second kind animal protein, if contain Enrofloxacin in the sample, sample solution is absorbed by the sample liquid absorption portion of test paper and reaches the colloid gold label part by moving on on the capillary action, the Enrofloxacin antibody response of Enrofloxacin in the sample solution and colloid gold label forms bond, bond moves on to detection line on continuing, because of the Enrofloxacin antibody of colloid gold label has only a binding site, Enrofloxacin in the sample solution with it in conjunction with after, detection on the detection line just can not be again and the Enrofloxacin antibodies of colloid gold label with antigen, so detection line is colourless; When not having Enrofloxacin in the sample, the detected antigen capture of using when the Enrofloxacin antibody of colloid gold label arrives detection line then forms the naked eyes red color visible, and this is promptly negative.No matter whether contain Enrofloxacin in the sample, move on on the second kind animal protein of colloid gold label all can be caught by the IgG of the anti-second kind animal protein of bag quilt on the reference line when reaching reference line and form macroscopic redness, this is reference line.
When the tagged object of colloid gold label part is the Enrofloxacin detection usefulness antigen and the second kind animal protein, if contain Enrofloxacin in the sample, sample solution is by the absorption of the sample liquid absorption portion of test paper and by moving on the capillary action, the little translational speed of free Enrofloxacin molecular weight in the sample is fast, arrive detection line earlier, the Enrofloxacin antibodies of bag quilt on elder generation and the detection line, because of the Enrofloxacin antibody that wraps quilt on the detection line has only a binding site, and the Enrofloxacin binding ability of dissociating in the sample is stronger with antigen than the Enrofloxacin detection of parataxic, so the detection of colloid gold label is with the Enrofloxacin antibody capture of antigen on can not tested again survey line, so detection line is colourless, this is promptly positive; If there is not Enrofloxacin in the sample, then the Enrofloxacin of colloid gold label detects with antigen and arrives behind the detection line with regard to the Enrofloxacin antibody capture of bag quilt on the tested survey line, forms macroscopic redness, and this is promptly negative.No matter whether contain Enrofloxacin in the sample, move on on the second kind animal protein of colloid gold label all can be caught by the IgG of the anti-second kind animal protein of bag quilt on the reference line when reaching reference line and form macroscopic redness, this is reference line.
The test paper of above dual mode, when preparing, passes through by test paper to regulate detection line and reference line encrusting substance concentration, the colour developing depth of detection line and reference line during control detection, and its colour developing depth or colour developing bar number and standard substance concentration be mapped, can reach the half-quantitative detection purpose.
Description of drawings:
Fig. 1 is a structural representation of the present invention.1 is that sample liquid absorption portion, 2 is that colloid gold label part, 3 is that detection reaction part, 4 is that detection line, 5 is that reference line, 6 is the part that absorbs water among the figure.
Embodiment:
Article (1) 1, the test paper of detection line+1 a reference line form
Detection line is coated with enrofloxacin monoclonal antibody or Enrofloxacin detection the antigen 1 bar, wide 1mm that concentration is 0.1-30 μ g/mL; Reference line is coated with 1 of the IgG that concentration is the anti-second kind animal protein of 0.01-30 μ g/mL, wide 1mm; Article two, 2-6mm at interval between the line.When the detection line color was identical with the reference line color, then sample was negative; When detection line was more of light color than reference line, then sample was positive, and concentration is greater than A μ g/kg, and the detectability of A for setting can be a certain concentration more than or equal to 10 μ g/kg.Reference line develops the color all the time, if reference line does not develop the color, shows that test paper lost efficacy.
Article (2) 2, the test paper of detection line+1 a reference line form
Article 2, detection line is coated with 0.01-30 μ g/mL enrofloxacin monoclonal antibody or Enrofloxacin and detects and use antigen, and the concentration of encrusting substance is less than the 1st detection line reference line encrusting substance concentration on the 2nd detection line; Reference line is coated with the IgG that concentration is the anti-second kind animal protein of 0.01-30 μ g/mL, wide 1mm; Interval 2-6mm between each bar line.When two detection lines all presented color, then sample was negative; When the 1st detection line colour generation of detection line, the 2nd detection line be during colour generation, then in the sample Enrofloxacin concentration greater than A μ g/kg, less than B μ g/kg; When two detection lines not during colour generation, Enrofloxacin concentration is greater than B μ g/kg in the sample.A, the B detectability for setting can be a certain concentration more than or equal to 10 μ g/kg.Reference line develops the color all the time, if reference line does not develop the color, shows that test paper lost efficacy.
Article (3) 3, the test paper of detection line+1 a reference line form
Article 3, detection line is coated with 0.01-30 μ g/mL enrofloxacin monoclonal antibody or Enrofloxacin and detects and use antigen, and the concentration of encrusting substance is successively decreased successively on three detection lines; Reference line is coated with the IgG that concentration is the anti-second kind animal protein of 0.01-30 μ g/mL; Interval 2-6mm between each bar line.When 3 detection lines all presented color, then sample was negative; When the 1st, 2 detection line colour generation of detection line, the 3rd detection line be during colour generation, then in the sample Enrofloxacin concentration greater than A μ g/kg, less than B μ g/kg; When the 1st detection line colour generation, not during colour generation, Enrofloxacin concentration is greater than B μ g/kg, less than C μ g/kg in the sample for the 2nd, 3 detection line; When 3 detection lines not during colour generation, determining enrofloxacin content is greater than C μ g/kg in the sample bottle; A, B, the detectability of C for setting can be a certain concentration more than or equal to 10 μ g/kg.Reference line develops the color all the time, if reference line does not develop the color, shows that test paper lost efficacy.
Article (4) 1, the test paper of detection line+3 a reference line form
Be coated with 0.01-30 μ g/mL enrofloxacin monoclonal antibody or Enrofloxacin detection antigen on the detection line, be coated with 3 reference lines after the detection line, reference line is coated with the IgG that concentration is the anti-second kind animal protein of 0.01-30 μ g/mL, regulate the label concentration of the 2nd kind animal protein of reference line encrusting substance concentration and colloid gold label, the color that makes 3 reference lines respectively with standard model in determining enrofloxacin content when being C, B, A μ g/kg the color of detection line identical, determine reference line encrusting substance concentration according to this; Interval 2-6mm between each bar line; When the detection line color is deeper than the 3rd reference line color, show that sample is negative; Be shallower than the 2nd when detecting when the detection line color is deeper than the 3rd reference line color, show that Enrofloxacin concentration is greater than A μ g/kg, less than B μ g/kg in the sample; When the detection line color is deeper than the 2nd reference line color and is shallower than the 1st reference line, show that Enrofloxacin concentration is greater than B μ g/kg, less than C μ g/kg in the sample; When detection line not during colour generation, show that Enrofloxacin concentration is greater than C μ g/kg in the sample.
Article (5) 1, the test paper of detection line+2 a reference line form
Be coated with 0.01-30 μ g/mL enrofloxacin monoclonal antibody or Enrofloxacin detection antigen on the detection line, be coated with 2 reference lines after the detection line, reference line is coated with the IgG that concentration is the anti-second kind animal protein of 0.01-30 μ g/mL, regulate the label concentration of the 2nd kind animal protein of reference line encrusting substance concentration and colloid gold label, the color that makes 2 reference lines respectively with standard model in determining enrofloxacin content when being B, A μ g/kg the color of detection line identical, determine reference line encrusting substance concentration according to this; Interval 2-6mm between each bar line; When the detection line color is deeper than the 2nd reference line color, show that sample is negative; Be shallower than the 1st when detecting when the detection line color is deeper than the 2nd reference line color, show that Enrofloxacin concentration is greater than A μ g/kg, less than B μ g/kg in the sample; When the detection line color is deeper than the 1st reference line color and is shallower than the 1st reference line, show that Enrofloxacin concentration is greater than B μ g/kg in the sample.
Enrofloxacin coupling antigen is synthetic
Enrofloxacin coupling antigen is divided into immunity and uses antigen with antigen and detection, and the former is used for immune animal, and the latter is used for colloid gold label or the detection line bag quilt in screening of Antibody Preparation detection of antibodies and the test paper preparation.
Antigen preparation use in immunity: 20mg Enrofloxacin and 70mg water-soluble carbodiimide and 25mgN-N-Hydroxysuccinimide are dissolved in the DMF aqueous solution of 6mL 50% room temperature reaction 8h, adding 80mg bovine serum albumin(BSA), add 1mL PBS (0.5mol/L again, pH7.8), stirred overnight at room temperature, reactant liquor PBS (0.1mol/L, pH 7.4) dialysis 48h, centrifugal (10000r/min, 15min) mL, supernatant adjust concentration to 2mg/mL, packing promptly gets Enrofloxacin immunity antigen.
Detect and use antigen: replace bovine serum albumin(BSA) BSA with oralbumin OVA, all the other steps are with the immunity antigen preparation.
The enrofloxacin monoclonal antibody preparation
Get 6 of health female secondary Balb/c mouse in 5 age in week and carry out immunity.Fundamental immunity uses antigen and equivalent complete Freund's adjuvant with the abundant mixing emulsification of stirrer the immunity of 0.1mL Enrofloxacin for the first time, carries out lumbar injection, and injection volume 0.2mL/ only.Begin all around to carry out booster immunization, adjuvant is changed to incomplete Freund's adjuvant, every two all booster immunizations once, gets spleen behind the last immunity 3d and merges.
With HAT nutrient solution, the incomplete nutrient solution of IMDM, IMDM complete culture solution and 50%PEG-6000 solution pre-temperature in 37 ℃ of water-baths, the beaker that another is filled with water is put into the pre-temperature of 37 ℃ of water-baths simultaneously earlier.The above-mentioned splenocyte for preparing and myeloma cell and splenocyte are mixed in 1: 5 ratio, and the full nutrient solution that toos many or too much for use in the 50mL plastic centrifuge tube is washed 1 time, and the centrifugal 8min of 1200r/min abandons supernatant, with the dropper residual liquid that exhausts.Put in 37 ℃ of water-baths, at the bottom of the suction pipe tubular stinger, in 90s, add 1mL, the 50%PEG-6000 of preheating while stirring.After leaving standstill 90s, in 37 ℃ of water-baths, add the complete culture solution of 10mL preheating in the 60s.Suspend once gently, with the centrifugal 6min of 1000r/min, supernatant discarded suspends gently with 6mL left and right sides complete culture solution earlier.According to the quantity of used 96 well culture plates, add the HAT nutrient solution to 76.8mL, mixing gently, in 96 orifice plates that feeder cells are arranged, every hole 0.1mL once inoculates 8 blocks of plates with the mixing suspension inoculation.Culture plate is placed 37 ℃, 5%CO
2Cultivate in the incubator.Merge back 7d, change liquid 1 time, behind 13d, use the complete culture solution of 20%NBS instead according to the propagation situation with HT nutrient solution half amount.Occur the hybridoma colony behind about 7d, cell is big, round and bright.Treat that colony grows at the bottom of the hole at 1/3 o'clock, draw the mark of clonal growth on the cover plate of culture plate, be that desirable supernatant detects corresponding specific antibody this moment.After the complete cloning of cell, cell is injected mouse peritoneal, when ascites such as certain interval of time are abundant, get ascites, behind ascites usefulness albumin A immunoaffinity chromatography purifying, promptly obtain enrofloxacin monoclonal antibody.
Sample liquid absorption portion is handled
Filter paper or all-glass paper etc. are immersed 2min among the PBS of 0.1mol/L pH7.4, take out, 80 ℃ of oven dry or other mode dryings, sample liquid absorption portion.
The preparation and the processing of colloid gold label part
The preparation of colloid gold label part and processing comprise the preparation of collaurum colloidal sol, collaurum mark Enrofloxacin antibody, colloid gold label section processes.
Collaurum colloidal sol preparation: with gold chloride (HAuCL
4) be configured to 1% mother liquor with ultrapure water, get the mother liquor of 1mL, use the ultrapure water constant volume to 100mL, be made into 0.01% solution, be heated to boiling, add a certain amount of 1% trisodium citrate aqueous solution, continue to be heated to occur transparent orange red till, be collaurum colloidal sol.
Colloid gold label enrofloxacin monoclonal antibody preparation: enrofloxacin monoclonal antibody and porcine hemoglobin are used PBS (0.01mol/L respectively, pH7.0～7.5) dissolved dilution is to 3mg/mL, every 100mL collaurum colloidal sol adds enrofloxacin monoclonal antibody and the 1mL 2mg/mL porcine hemoglobin of 1mL 4mg/mL, concussion 2min is with the K of 0.2mol/L
2CO
3Regulate pH to 8.4, vibration 5min adds 11%PEG-100002mL, vibration 5min, the centrifugal 15min of 15000r/min removes supernatant, with PBS (0.01mol/L, pH7.4) redissolve, with the centrifugal 15min of 6000～13000r/min, remove supernatant, will precipitate (0.01mol/L with PBS, pH7.5) dilution is 1000 times, and product is marked enrofloxacin monoclonal antibody as gold.
The colloid gold label section processes: gold is marked enrofloxacin monoclonal antibody pour in the groove, all-glass paper or filter paper are immersed 1min, take out, drying at room temperature or vacuum freeze drying promptly become the colloid gold label part.
Article 1, the detection paper reactive moieties of detection line+2 a reference line array configuration is handled
Glutaraldehyde solution with 0.8% or 0.2% carbodiimide solution soak nitrocellulose membrane 30min, takes out, and 37 ℃ of oven dry, the top is detected with the antigen line as detection line by 1 Enrofloxacin with flush coater spraying bag, and concentration is 3 μ g/mL; As the reference line, the concentration of close lower end is 2 μ g/mL to bag by the IgG line of 2 goat-anti porcine hemoglobin, and another is 1 μ g/mL.This is the detection reaction part, and detection line and reference line array configuration are 1 detection line+2 reference line.
Suction section processes and test paper assembling
After the thieving paper drying at room temperature, promptly as the suction part.
On backing, paste sample liquid absorption portion, colloid gold label part, detection reaction part and suction part successively, array configuration be that the Enrofloxacin rapid semi-quantitative of 1 detection line+2 reference line detects test paper.
Detect
The milk sample: directly drip 2 milk for the detection paper part, about 100 μ L behind the 2min, observe color.If 2 detection lines all present color, show that sample is negative; If the 1st detection line colour generation, the 2nd colour generation not shows that then determining enrofloxacin content is more than the 80 μ g/kg in the sample; If the 1st detection line and the 2nd detection line do not develop the color, show that then determining enrofloxacin content is more than the 190 μ g/kg in the sample.Regardless of determining enrofloxacin content in the sample, reference line should develop the color, if reference line does not develop the color, shows that test paper lost efficacy.
Tissue samples such as animal's liver, kidney, muscle: the 10g that removes fat organizes sample to smash to pieces, adds among the 30mL PBS (0.01mol/L pH7.4), hatches 30min at 80 ℃, place ice bath 10min then, centrifugal, the careful fat deposit of removing the surface, supernatant is as detecting liquid.Remaining operation is with the milk sample.
The test paper preparation of other array configurations, detection and preceding similar substantially repeat no more.
Good effect of the present invention:
1. high specificity. This test paper specificity is extremely strong, with penicillin, kanamycins, sarafloxacin, native mould The medicines such as element, aureomycin, sulfamido, chloramphenicol, erythromycin do not have cross reaction substantially, have avoided other The interference of medicine to detecting met the requirement of Ministry of Agriculture's detection Enrofloxacin composite medicine.
2. highly sensitive. Although Enrofloxacin maximum residue limit in the milk of European Union and China Ministry of Agriculture regulation Be 100 μ g/kg, if but need, also can when preparing test paper, improve according to testing requirement and detect lower limit, this Be limited to 3ug/kg under the test paper lowest detection.
3. can realize half-quantitative detection. This test paper not only can qualitative detection, and prior is according to inspection Survey line color and reference line color contrast or reach the half-quantitative detection purpose according to detection line colour generation number.
4. simple to operation. But to milk, animals urine direct-detection, the tissue samples such as meat are simply located Can detect after the reason. This test paper does not need any professional training, does not need any instrument and equipment, common amateur people The member can operate, as long as test paper is inserted in the detection liquid with the naked eye interpretation. Therefore, this test paper is suitable for face width, Sanitary inspection superintendent office, animal-breeding unit and consumer are individual can be used per capita.
5. speed is fast, and flux is big, colour stable. Test paper inserts in the sample liquid, and 3min is just observable result later on, People can be simultaneously, continuous detecting, detects flux and be higher than the HPLC method far away, than Enrofloxacin ELISA The kit detection speed is then fast more than 40-160 times, but and its color persistence, and can not resemble ELISA Substrate is variable color like that soon.
6. the detected temperatures optimum range is wide. All can use at 4-40 ℃, the result is normal, does not need to advance at low temperatures OK, need not take Insulation, indoor field all can be used.
7. test paper long shelf-life. According to the accelerated aging test result, the test paper shelf-life can reach 2 years under the drying condition.
8. with low cost. This test paper method testing cost is well below Enrofloxacin ELISA kit detection method, After large-scale production, its cost also can decrease.
Claims (5)
1. method for detecting Enrofloxacin through semiquantitative test paper of side stream immunity chromatography, on the backing of test paper, be pasted with sample liquid absorption portion successively, the colloid gold label part, detection reaction part and suction part, it is characterized in that: the material that colloid gold label partly is labeled is the potpourri of the second kind animal protein and Enrofloxacin antibody, or second kind animal protein and Enrofloxacin detect potpourri with antigen, being coated with Enrofloxacin above the detection reaction part detects with the 1-3 bar as detection line, or be coated with Enrofloxacin antibody 1-3 bar as detection line, the IgG 1-3 bar that is coated with the anti-second kind animal protein simultaneously is as reference line, can not be when detection line and reference line combination simultaneously more than 1, the combination line number is the 2-4 bar; The test paper of various array configurations, when preparing, passes through by test paper to regulate detection line and reference line encrusting substance concentration, the colour developing depth of detection line and reference line during control detection, and its colour developing depth or colour developing bar number and standard substance concentration be mapped, reach half-quantitative detection.
2,, it is characterized in that it is the conjugates that Enrofloxacin and carrier mass form that Enrofloxacin detects with antigen according to the said detection method of claim 1.
3,, be protein or protein fragments or synthetic polypeptide or semi-synthetic polypeptide or polysaccharide according to the said carrier mass of claim 2.
4,, it is characterized in that the second kind animal protein is the albumen that the non-antibody source belongs to animal according to the said detection method of claim 1.
5, according to the said detection method of claim 1, the array mode that it is characterized in that detection line and reference line is 1 detection line and 1 reference line, 1 detection line and 2 reference lines, 1 detection line and 3 reference lines, 2 detection lines and 1 reference line, 3 detection lines and 5 kinds of forms of 1 reference line.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101498726B (en) * | 2008-01-30 | 2013-04-24 | 北京望尔生物技术有限公司 | Colloidal gold test paper card for detecting enrofloxacin medicament residue |
| CN104880552A (en) * | 2015-05-20 | 2015-09-02 | 集美大学 | Colloidal gold immunochromatography test strip for detecting enrofloxacin and preparation method of colloidal gold immunochromatography test strip |
| CN105277665A (en) * | 2014-07-24 | 2016-01-27 | 江苏维赛科技生物发展有限公司 | Test paper strip for rapidly detecting overstandard preservative benzoic acid |
| CN110412260A (en) * | 2019-04-26 | 2019-11-05 | 山东省食品药品检验研究院 | Fluoroquinolones direct competitive chemiluminescence qualitative, quantitative immunoassay method in cosmetics |
-
2005
- 2005-06-20 CN CN 200510035242 patent/CN1727899A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101498726B (en) * | 2008-01-30 | 2013-04-24 | 北京望尔生物技术有限公司 | Colloidal gold test paper card for detecting enrofloxacin medicament residue |
| CN105277665A (en) * | 2014-07-24 | 2016-01-27 | 江苏维赛科技生物发展有限公司 | Test paper strip for rapidly detecting overstandard preservative benzoic acid |
| CN104880552A (en) * | 2015-05-20 | 2015-09-02 | 集美大学 | Colloidal gold immunochromatography test strip for detecting enrofloxacin and preparation method of colloidal gold immunochromatography test strip |
| CN110412260A (en) * | 2019-04-26 | 2019-11-05 | 山东省食品药品检验研究院 | Fluoroquinolones direct competitive chemiluminescence qualitative, quantitative immunoassay method in cosmetics |
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