CN100338467C - Enzyme-linked immunological kit for detecting tetracycline drug - Google Patents
Enzyme-linked immunological kit for detecting tetracycline drug Download PDFInfo
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- CN100338467C CN100338467C CNB2004100465664A CN200410046566A CN100338467C CN 100338467 C CN100338467 C CN 100338467C CN B2004100465664 A CNB2004100465664 A CN B2004100465664A CN 200410046566 A CN200410046566 A CN 200410046566A CN 100338467 C CN100338467 C CN 100338467C
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- tetracycline
- linked immunological
- immunological kit
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Abstract
The present invention discloses an enzyme-linked immunological kit for detecting tetracycline drugs. The enzyme-linked immunological kit comprises conjugate of coated tetracycline drugs and carrier protein, specific antibodies of tetracycline drugs, and enzyme-linked diantibodies. The enzyme-linked immunological kit is a multiple residue detecting kit which can simultaneously detect residues of tetracycline drugs, such as tetracycline, minocycline, chlorotetracycline, doxycycline, etc. The present invention has the advantages of low sample pretreatment requirement and simple sample pretreatment process, and can simultaneously and rapidly detect large numbers of samples; the minimum detection limit can be below 10 mu g/kg, and the present invention can directly judge whether samples exceed standards; the present invention has the characteristics of specificity, high sensitivity, high precision, high accuracy, etc. The present invention has the important function for detecting tetracycline drug residues in animal foods.
Description
Technical field
The present invention relates to a kind of enzyme linked immunological kit that detects tetracycline medication in enzyme linked immunological and the detection of veterinary drugs in food analysis technical field.
Background technology
Tetracycline medication (Tetracyclines TCs) is to be a family antibiotic of parent nucleus with four acenes, wherein tetracycline, terramycin, aureomycin and Doxycycline are because of its good antibiotic property, the stable property of medicine and cheap price, in the Production of Livestock and Poultry of being everlasting as medicine and medicated premix and be extensive use of, as commodity, tetracycline medication is to use one of microbiotic the most widely.Tetracycline medication is used to prevent and treat enteric infection and growth promotion, and TCs is used for treating diseases such as mastitis in dairy.But the easy inducible resistance strain growth of the excessive use of tetracycline medication, and cause high medicament residue, it is healthy directly or indirectly to influence the consumer.Tetracycline, terramycin and aureomycin monomer or compound maximum residue limit(MRL) are 100 μ g/L in the middle regulation milk of China's " animal food herbal medicine maximum residue limit(MRL) ", and the maximum residue limit(MRL) in muscle, liver and the kidney is respectively 100,300 and 600 μ g/kg.
The analytical approach of the tetracycline medication residual quantity of now having reported has microbial method, high performance liquid chromatography and high performance liquid chromatography-tandem mass method etc.The tetracycline medication poor stability, strict to sample treatment, the recovery is lower.Microbial method is a screening technique, can reach the detection requirement of maximum residue limit(MRL) substantially, but this method complex operation is time-consuming, just can obtain a result in the fastest 2 days, and poor specificity, there is cross reaction with other class microbiotic.High performance liquid chromatography is to analyze the main method of tetracycline medication residual quantity at present, but needs a series of loaded down with trivial details extractions and purified treatment step, and exists the recovery low, and sensitivity is low, and impurity disturbs problems such as big.The high performance liquid chromatography-tandem mass method is the method for newly developing, and needs expensive instrument and equipment, is difficult to popularize.
The innovation and creation content
The purpose of this invention is to provide a kind of enzyme linked immunological kit that detects tetracycline medication.
The enzyme linked immunological kit of detection tetracycline medication provided by the present invention comprises the tetracycline medication of quilt and the conjugate of carrier protein, the specific antibody of described tetracycline medication and ELIAS secondary antibody.
For more convenient on-site supervision and great amount of samples examination, described kit also comprises A, B, C, D, E, F reagent solution, G reagent solution, H reagent solution, I reagent solution, J reagent solution, K reagent solution, L reagent solution and M reagent solution.
Described A, B, C, D, E, F reagent solution are tetracycline medication series concentration standard solution.
Described G reagent solution is that protein concentration is the ELIAS secondary antibody of 0.1-10mg/L.
Described H reagent solution is that protein concentration is tetracycline medication monoclonal antibody or the polyclonal antibody of 0.1-10mg/L.
Described I reagent solution is a tetramethyl benzidine.
Described J reagent solution is the phosphate buffer that contains 0.1-10%BSA.
Described K reagent solution is the phosphate buffer that contains 0.05% tween.
Described L reagent solution is a citrate buffer solution.
Described M reagent solution is a stop buffer.
Wherein, described tetracycline medication is tetracycline, terramycin, aureomycin, fortimicin, minocycline, bristacin or demethylchlortetra cylinum.
The specific antibody of described tetracycline medication can be tetracycline medication monoclonal antibody or tetracycline medication polyclonal antibody; Described tetracycline medication monoclonal antibody or polyclonal antibody can be mouse source, Ma Yuan, pig source, Yang Yuan, rabbit source or cavy source antibody, described tetracycline medication monoclonal antibody is preferably the tetracycline medication mouse monoclonal antibody, and described tetracycline medication polyclonal antibody is preferably the tetracycline medication rabbit polyclonal antibody.
The conjugate of all available tetracycline medication of above antibody and carrier protein prepares according to a conventional method as immunogene.
Described carrier protein can be bovine serum albumin(BSA) (BSA), human serum albumins (HSA), albumin rabbit serum (RSA), thyroglobulin (TG), ovalbumin (OVA) or hemocyanin common carrier albumen such as (KLH); The conjugate of described tetracycline medication and carrier protein can adopt formaldehyde method, hydrazine method, diazotising method or EDC method synthetic.
Described marker enzyme can be horseradish peroxidase or alkaline phosphatase, is preferably horseradish peroxidase, and horseradish peroxidase can be crosslinked on two antibody by glutaraldehyde method or sodium periodate method; Described two anti-anti-mouse or the anti-rabbit antiantibodys of can be.
The material that can be used as the fixing carrier of the conjugate of tetracycline medication and carrier protein is a lot, as polystyrene, cellulose, polyacrylamide, tygon, polypropylene, cross-link dextran, glass, silicon rubber, Ago-Gel etc.The form of this carrier can be test tube, micro-reaction plate shrinkage pool, globule, sequin etc.
Under preferred condition, the enzyme linked immunological kit of detection tetracycline medication of the present invention comprises the tetracycline of quilt and the conjugate of carrier protein, the specific antibody of tetracycline and ELIAS secondary antibody.
Detection principle of the present invention is adsorbed on the solid phase carrier as coating antigen for the conjugate with tetracycline medication and carrier protein, add sample and tetracycline medication specific antibody, add ELIAS secondary antibody again, in the testing sample on residual tetracycline medication and the solid phase carrier tetracycline medication of bag quilt compete binding specificity antibody with the conjugate of carrier protein, the colour developing back stops, the working sample light absorption value, the tetracycline medication residuals content is negative correlation in this value and the sample, relatively can draw the content of tetracycline medication with typical curve.Simultaneously according to the depth of the color sample on the ELISA Plate, but with the concentration range of the comparison judgement sample of the tetracycline medication standard solution color of series concentration.
The enzyme linked immunological kit of detection tetracycline medication of the present invention is many residue detection kit, can measure tetracycline simultaneously, terramycin, and tetracycline medications such as aureomycin and fortimicin are residual; The main residual quantity that adopts tetracycline medication in the samples such as the qualitative or detection by quantitative animal tissue (musculature of pig, chicken, ox, sheep, fishes and shrimps and liver, kidney etc.) of indirect competitive ELISA method, honey, milk; Pre-treatment requirement to sample is low, and sample pretreatment process is simple, simultaneously the fast detecting gross sample; Assay method is simple, and same curve can be measured milk and muscle samples simultaneously, saves time, and milk sample can draw measurement result within two hours; Lowest detectable limit reaches below the 10 μ g/kg, and directly whether judgement sample exceeds standard, and has characteristics such as high specific, high sensitivity, pinpoint accuracy, pin-point accuracy, can play a significant role in animal food tetracycline medication residue detection.
Embodiment
The preparation of embodiment 1, antigen and antibody
(1) envelope antigen is synthetic
Adopt formaldehyde method, hydrazine method, EDC method or diazotising method to carry out coupling tetracycline medication and albumin rabbit serum (RSA) and obtain envelope antigen.
What (2) immunity (resisting) was former synthesizes
Adopting formaldehyde method, hydrazine method, EDC method or diazotising method to carry out coupling tetracycline medication and human serum albumins, to obtain immunity (resisting) former.
(3) preparation of tetracycline medication rabbit polyclonal antibody
Adopt new zealand white rabbit as immune animal, with tetracycline medication and human serum albumin conjugate is immunogene, first immunisation adds complete freund adjuvant 2mL emulsification with 2mg/mL immunizing antigen 2mL, and booster immunization adds the incomplete freund adjuvant emulsification of 1mL with immunizing antigen 1mL.Respectively get the 5-10 point in both sides, white rabbit back, every some hypodermic injection antigen 0.2-0.3mL, booster immunization all around, booster immunization is 5-10 time altogether, and each booster immunization is 2 weeks at interval, last immunity blood sampling after 7 days, measure serum antibody titer, serum is extracted in the arteria carotis bloodletting, obtains the polyclonal antibody of purifying through ammonium sulfate precipitation.
(4) preparation of tetracycline medication mouse monoclonal antibody
The animal immune program adopts BALB/c mouse as immune animal, with tetracycline medication and human serum albumin conjugate is immunogene, immunizing dose is that 50 μ g (0.1mL) immunogenes add the complete freund adjuvant emulsification of equal-volume, carries out hypodermic injection immunity first.After January, get same amount immunizing antigen and add the incomplete freund adjuvant of equivalent, booster immunization is carried out in emulsification, after January, gets same amount immunizing antigen and does not add the adjuvant lumbar injection and carry out booster immunization, and blood sampling in 5 days is afterwards measured antibody titer, extracting spleen cell.
Immune BALB/c mouse splenocyte is got in Fusion of Cells and cloning, merges in 4: 1 ratios and SP2/0 myeloma cell, adopts indirect competitive ELISA to measure cell conditioned medium liquid, screens positive hole.Utilize limiting dilution or soft agar flat band method that cloning is carried out in positive hole, obtain the monoclonal antibody of complete homogeneity and stable monoclonal hybridoma strain.
Cell cryopreservation and recovery are got the hybridoma that is in exponential phase and are made 1 * 10 with cryopreserving liquid
6-5 * 10
6The cell suspension of individual/mL is sub-packed in frozen pipe, in the medium-term and long-term preservation of liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
Adopt in the body and induce method, BALB/c mouse (8 age in week) abdominal cavity is only injected sterilization paraffin oil 0.5ml/, 7-14 days pneumoretroperitoneum injection hybridomas 5 * 10
5-10
6Individual/as only, to gather ascites after 7-10 days.Carry out the ascites purifying through sad-ammonium sulfate precipitation method, bottle packing ,-20 ℃ of preservations.
(5) two anti-preparations
As immune animal, is that immunogene carry out immunity with mouse or rabbit igg with sheep, obtains sheep anti mouse or goat-anti rabbit antiantibody.
With sodium periodate method or the synthetic horseradish peroxidase-goat anti-rabbit igg of glutaraldehyde method or horseradish peroxidase-sheep anti-mouse igg label.
The enzyme linked immunological kit of embodiment 2, detection tetracycline medication
(1) structure of the enzyme linked immunological kit of detection tetracycline medication
This kit is mainly by box body, vacuum-packed 96 holes of aluminium film/40 hole ELISA Plate, A, B, C, D, E, F reagent solution (tetracycline series concentration standard solution), G reagent solution (enzyme is anti-in conjunction with two), H reagent solution (tetracycline medication antibody), J reagent solution (containing the BSA phosphate buffer); K reagent solution (containing the Tween-20 phosphate buffer); I reagent solution (tetramethyl benzidine); L reagent solution (citrate buffer solution); M reagent solution (stop buffer); Cover plate film and carriage are formed.Mentioned reagent liquid leaves in the reagent bottle.Above-mentioned A is housed, B, C, D, E, F, G, H, I reagent bottle in the carriage.Carriage, ELISA Plate, the J reagent bottle, the K reagent bottle, the L reagent bottle, M reagent bottle and cover plate film are installed in the box body.ELISA Plate is made up of plastic stent and the plastic strip with holes that separates separately.
(2) preparation of agents useful for same
A, B, C, D, E, the F reagent solution: tetracycline medication series concentration standard solution, its concentration are respectively 0,3,10,30,100,300 μ g/L.
The G reagent solution: protein concentration is horseradish peroxidase-goat anti-rabbit igg label or the horseradish peroxidase-sheep anti-mouse igg label of 0.1-10mg/L.
The H reagent solution: protein concentration is tetracycline medication mouse monoclonal antibody or the rabbit polyclonal antibody of 0.1-10mg/L.
I reagent solution: 1-10mg/ml tetramethyl biphenyl amine aqueous solution.
J reagent solution: the phosphate buffer (0.01M pH7.2) that contains 0.1-10%BSA.
K reagent solution: the phosphate buffer (0.5M pH7.2) that contains 0.5% tween.
L reagent solution: 0.1M pH5.0 citrate buffer solution.
M reagent solution: 1-2mol/L sulfuric acid or hydrochloric acid.
(3) preparation of ELISA Plate
Be cushioned liquid (0.05M pH9.6 carbonate buffer solution) with bag the tetracycline medication envelope antigen is interpreted into 0.1-1 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h, and 4 ℃ are spent the night, the bag that inclines is cushioned liquid, K reagent solution with 10 times of dilutions washs 3 times, each 30 seconds, pats dry, in every hole, add 5% skim milk 0.05M phosphate buffer, 250 μ l then, 37 ℃ of incubation 1-2h, liquid in the hole of inclining, preserve with the vacuum seal of aluminium film dry back.
The pre-treatment of embodiment 3, sample and detection
1, milk sample: directly get the fresh milk or the milk of freeze thawing, supply to measure as sample solution with J reagent solution dilution back.
2, tissue sample: get muscle, the liver of chicken, pig respectively, each tissue sample of kidney respectively takes by weighing 2g, add the MCI damping fluid (the 0.1M citrate buffer solution, 0.1M EDTA, pH3.8) 8mL, the vibration 30min, centrifugal 10min, it is standby to get supernatant.C
18Solid-phase extraction column is used absolute methanol 3mL, water 2mL prewashing successively.Get reserve liquid 5.0mL and cross post, water 2mL drip washing is extracted.With 20mmol/L methyl ethyl oxalate alcoholic solution 1.0mL wash-out, extract, collect eluent.The eluent of collecting is shaken up, dilute the back more than 10 times or 10 times with the J reagent solution and supply to measure as sample solution.
3, detect
Before detecting kit is recovered room temperature (18 ℃-30 ℃), use A again, B, C, D, E, F reagent solution and J reagent solution preparation tetracycline medication series concentration standard solution 0,0.3,1,3,10,30 μ g/L, add water at the K reagent solution and be mixed with washing lotion (phosphate buffer of 0.05% tween), with H reagent solution and washing lotion preparation antibody-solutions (0.1mg/L), the tetracycline series concentration standard solution that in the ELISA Plate aperture, adds 50 μ l sample solutions or prepare, add 50 μ l antibody-solutions, be covered with the cover plate film and place 1h in 18 ℃-30 ℃, wash ELISA Plate 3 times with washing lotion, pat dry with thieving paper, afterwards with G reagent and washing lotion configuration enzyme labelled antibody solution (0.1mg/L) and add in the ELISA Plate aperture, place 30min for 18 ℃-30 ℃, wash ELISA Plate 3 times with washing lotion again, pat dry, with I reagent solution and L reagent solution preparation zymolyte mixed liquor (citrate buffer solution of 0.1% tetramethyl benzidine), get 100 μ l and add mixing behind the ELISA Plate aperture, the about 15min of lucifuge colour developing, last every hole adds M stop buffer (1mol/L sulfuric acid) 100 μ l, measures the 450nm A of place with microplate reader
450Value.Be calculated as follows the percentage absorbance:
(B-is the mean light absorbency value of standard solution or sample; B
0-be the standard solution mean light absorbency value of 0 concentration)
Logarithm with tetracycline concentration in the standard solution is an X-axis, and the percentage absorbance is a Y-axis, and the drawing standard curve calculates tetracycline medication concentration the sample solution from typical curve.
Embodiment 5, kit sensitivity, specificity, precision and accuracy
The antigen of this kit is the conjugate of tetracycline and RSA, and the specific antibody of tetracycline is the tetracycline rabbit polyclonal antibody, and ELIAS secondary antibody is horseradish peroxidase-goat anti-rabbit igg label, and wherein, standard solution is a tetracycline series concentration standard solution.
1, kit sensitivity test
Carried out the kit sensitivity test according to a conventional method, the result shows that the typical curve minimum point of this kit is 0.3 μ g/L, and standard curve range is 0.3 μ g/L-30 μ g/L; In milk and chicken meat sample, detect and be limited to about 7 μ g/L.
2, kit specificity test
Select 7 kinds of tetracyclines commonly used as shown in table 1, measure cross reacting rate respectively according to conventional method, the result is as shown in table 1.
Table 1. cross reaction
| Medicine name | Cross reacting rate % |
| Tetracycline aureomycin terramycin Doxycycline minocycline demethylchlortetra cylinum bristacin | 100 100 54 100 100 100 100 |
3, accuracy, precision are measured
Adding tetracyclines (tetracycline, terramycin, Doxycycline or aureomycin) titer to final concentration respectively in blank milk, chicken meat sample is 50 μ g/L, 100 μ g/L and 200 μ g/L.Each concentration prepares 5 parts in sample respectively, measures its content then respectively, repeats 3 times.The coefficient of variation between difference calculate recovery rate, plate within variance coefficient and plate.The result shows that in milk the recovery that 100 μ g/L add concentration is 45%-120%; In chicken muscle, the recovery that 100 μ g/kg add concentration is 40%-120%; Add in milk and the chicken meat sample variation within batch coefficient CV≤20%, interassay coefficient of variation CV≤25% in blank.
Claims (7)
1, a kind of enzyme linked immunological kit that detects tetracycline medication, comprise the tetracycline medication of quilt and the conjugate of carrier protein, protein concentration is tetracycline medication monoclonal antibody or the polyclonal antibody of 0.1-10mg/L, protein concentration is the horseradish peroxidase of 0.1-10mg/L or the ELIAS secondary antibody of alkaline phosphate ester enzyme labeling, concentration is respectively 0,3,10,30, the tetracycline medication series concentration standard solution of 100 and 300 μ g/L, the tetramethyl biphenyl amine aqueous solution, the phosphate buffer that contains 0.1-10%BSA, the phosphate buffer that contains 0.05% tween, citrate buffer solution and stop buffer.
2, enzyme linked immunological kit according to claim 1 is characterized in that: described tetracycline medication is tetracycline, terramycin, aureomycin, Doxycycline, minocycline, demethylchlortetra cylinum or bristacin.
3, enzyme linked immunological kit according to claim 1 is characterized in that: described tetracycline medication monoclonal antibody or polyclonal antibody are mouse source, Ma Yuan, Yang Yuan, pig source, rabbit source or cavy source antibody.
4, enzyme linked immunological kit according to claim 3 is characterized in that: described tetracycline medication monoclonal antibody is the tetracycline medication mouse monoclonal antibody.
5, enzyme linked immunological kit according to claim 3 is characterized in that: described tetracycline medication polyclonal antibody is the tetracycline medication rabbit polyclonal antibody.
6, according to any described enzyme linked immunological kit of claim 1-5, it is characterized in that: described carrier protein is bovine serum albumin(BSA), human serum albumins, albumin rabbit serum, thyroglobulin, ovalbumin or hemocyanin.
7, enzyme linked immunological kit according to claim 1, it is characterized in that: the enzyme linked immunological kit of described detection tetracycline medication, comprise the tetracycline of quilt and the conjugate of carrier protein, protein concentration is tetracycline monoclonal antibody or the polyclonal antibody of 0.1-10mg/L, protein concentration is the horseradish peroxidase of 0.1-10mg/L or the ELIAS secondary antibody of alkaline phosphate ester enzyme labeling, tetracycline series concentration standard solution, the tetramethyl biphenyl amine aqueous solution, the phosphate buffer that contains 0.1-10%BSA, the phosphate buffer that contains 0.05% tween, citrate buffer solution and stop buffer.
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Families Citing this family (7)
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| CN1996022B (en) * | 2006-12-30 | 2012-07-11 | 中国兽医药品监察所 | Enzyme-linked immunologic kit for detecting neomycin |
| CN101639477B (en) * | 2009-08-20 | 2013-06-26 | 华中农业大学 | ELISA detection method for doxycycline remnant, kit and application |
| CN102288753A (en) * | 2011-05-10 | 2011-12-21 | 重庆市科学技术研究院 | Quadruple colloidal gold immunochromatography testing strip for rapid detection of residual tetracycline, chlortetracycline, oxytetracycline and doxycycline and preparation method of same |
| CN102353769A (en) * | 2011-06-22 | 2012-02-15 | 珠海市英平生物科技有限公司 | Fluoroquinolones labeling method with HRP |
| CN103149167A (en) * | 2013-02-26 | 2013-06-12 | 上海交通大学 | Method for detecting tetracycline residues in milk and drinking water |
| CN106290927B (en) * | 2016-07-26 | 2018-12-14 | 鲁东大学 | Fortimicin quick detection kit and its preparation, application method |
| CN107389667A (en) * | 2017-08-24 | 2017-11-24 | 太原瑞盛生物科技有限公司 | A kind of chemiluminescence detection kit of terramycin and preparation method thereof |
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