CN1996022B - Enzyme-linked immunologic kit for detecting neomycin - Google Patents
Enzyme-linked immunologic kit for detecting neomycin Download PDFInfo
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- CN1996022B CN1996022B CN 200610171540 CN200610171540A CN1996022B CN 1996022 B CN1996022 B CN 1996022B CN 200610171540 CN200610171540 CN 200610171540 CN 200610171540 A CN200610171540 A CN 200610171540A CN 1996022 B CN1996022 B CN 1996022B
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Abstract
The enzyme immune agent box for inspecting mycifradin comprises wrapped mycifradin and carrier protein coupling, specific antibody of the mycifradin and enzyme label, with the said antibody being immune acquired monoclonal antibody or monoclonal antibody. It has low requirement for the pretreatment of the sample, with simple process, with milk and pork meat lowest inspecting limit being 7 mug/L and 68 mug/kg, directly deciding whether the sample overproof, with high specificity, high sensitivity and accuracy, playing a very important role in inspecting the residue of novomycin of animal foodstuff.
Description
Technical field
The present invention relates to a kind of enzyme linked immunological kit that detects neomycin in enzyme linked immunological and the detection of veterinary drugs in food analysis technical field.
Background technology
Neomycin is the aminoglycoside medicaments with powerful bactericidal action, and stable in properties, and good water solubility is widely used in herding is produced.But existing research proof, in several kinds of aminoglycoside medicaments neomycins, kanamycins, western plain mycin, TOB, streptomysin, the ototoxicity of neomycin is the strongest, also is that cochleotoxicity is the strongest in all microbiotic.Neomycin is also toxic to kidney simultaneously, and aminoglycoside medicaments to the toxicity of kidney is in proper order under routine dose: neomycin>kanamycins>TOB>streptomysin.And neomycin can cause the cobalamin malabsorption, thereby causes megaloblastic anemia, and neomycin also can impel the eliminating of elements such as fat and potassium in the enteron aisle, sodium, tungsten, phosphorus.And the infant is to this type of medicaments insensitive, and neomycin can see through placenta infringement Fetal Hearing Monitored.Therefore China, the international food code council and European Union all to formulate the MRL of neomycin in animal tissue be 500～1000 μ g/kg (L).
U.S. FSIS adopts neomycin content in the high performance liquid chromatogram mass spectrometric determination at present, detects to be limited to 100～400ug/L; Domestic have the liquid phase fluorescence method (SN0646-1997) of employing and microbial method (agriculture and animal husbandry is sent out [2001] No. 38) to detect neomycin.But high performance liquid chromatogram mass spectroscopy and fluorescence method are high to instrument requirement, complicated operation, and the derivant that generates in the liquid phase fluorescence method is unstable; The microbial method poor specificity, the cycle is long.Therefore,, can cause damage to fetus, in the residual monitoring of China, neomycin not listed in the Supervisory Surveillance Program always through placental barrier although neomycin cochleotoxicity in all microbiotic is the strongest.
Summary of the invention
The purpose of this invention is to provide a kind of enzyme linked immunological kit that detects neomycin.
The enzyme linked immunological kit of detection neomycin provided by the present invention comprises the neomycin that encapsulates and the conjugate of carrier protein, the specific antibody of neomycin and ELIAS secondary antibody; The specific antibody of said neomycin is polyclonal antibody or the monoclonal antibody that immunogene obtains for the conjugate with neomycin and carrier protein.
Said polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, pig source, rabbit source or cavy source antibody.
Said polyclonal antibody is preferably rabbit polyclonal antibody.
Said monoclonal antibody can be mouse monoclonal antibody, like the monoclonal antibody of monoclonal hybridoma strain secretion.
Said 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).
Carrier protein in the conjugate of said neomycin that encapsulates and carrier protein is preferably albumin rabbit serum; Carrier protein in the said immunogene is preferably human serum albumins.
For more convenient on-site supervision and great amount of samples examination, said 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.
Said A, B, C, D, E, F reagent solution are neomycin series concentration standard solution.
Said G reagent solution is the ELIAS secondary antibody of 0.1-10mg/L.
Said H reagent solution is an antibody concentrated solution, is neomycin monoclonal antibody or the polyclonal antibody of 0.1-10mg/L.。
Said I reagent solution is a tetramethyl benzidine.
Said J reagent solution is the phosphate buffer that contains 0.5% tween.
Said K reagent solution is a citrate buffer solution.
Said L reagent solution is a stop buffer.
Marker enzyme in the said ELIAS secondary antibody can be horseradish peroxidase or alkaline phosphatase, is preferably horseradish peroxidase, and horseradish peroxidase can be crosslinked on two antibody through glutaraldehyde method or sodium periodate method; Said 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 neomycin and carrier protein is a lot, like 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.
Detection principle of the present invention is adsorbed on the solid phase carrier as coating antigen for the conjugate with neomycin and carrier protein; Add sample and neomycin specific antibody, add ELIAS secondary antibody again, the neomycin that encapsulates on residual neomycin and the solid phase carrier in the testing sample and the conjugate of carrier protein are competed binding specificity antibody; The colour developing back stops; The working sample light absorption value, the neomycin residuals content is negative correlation in this value and the sample, relatively can draw the content of neomycin 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 neomycin thing standard solution color of series concentration.
The enzyme linked immunological kit of detection neomycin of the present invention is single residue detection kit; The main residual quantity that adopts neomycin drug in the qualitative or samples such as detection by quantitative animal tissue (musculature of pig, chicken, ox, sheep and liver, kidney, the flesh of fish, feed etc.), milk of ELISA competing method; Pre-treatment to sample requires low; Sample pretreatment process is simple; Milk sample can directly be measured after containing the dilution of polysorbas20 phosphate buffer, and pork and kidney can be gone up appearance after containing the extracting of polysorbas20 phosphate buffer, and ability while fast detecting gross sample; Assay method is simple, saves time, and sample can draw the mensuration result within 2 hours; LDL in milk and the pork is respectively 7 μ g/L and 68 μ g/kg, has characteristics such as high specific, high sensitivity, pinpoint accuracy, pin-point accuracy, can in animal food neomycin drug residue detection, play a significant role.
Embodiment
Experimental technique among the following embodiment if no special instructions, is conventional method.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
The preparation of the enzyme linked immunological kit of embodiment 1, detection neomycin drug
One, the preparation of antigen and antibody
1, neomycin antigen is synthetic
With the albumin rabbit serum is carrier, and neomycin is a haptens, with ethyloic azanol-carbodlimide method neomycin is coupled on the albumin rabbit serum, obtains neomycin antigen.
Concrete operations are following: get 20-30mg neomycin and 30-40mg ethyloic azanol half hydrochloride in the round bottom cucurbit; Add 10-15ml ethanol, backflow 2-4 hour, stirring at room 12-18 hour again; Decompression volatilizes solvent, and the solid that obtains is neomycin-ethyloic oxime.Get 10-30mg neomycin-ethyloic oxime and be dissolved in the 5-10ml water, mixing adds 100-200mg1-ethyl-3-(3-dimethylaminopropyl)-carbodiimides successively; Slowly stirred 10-20 minute; The albumin rabbit serum that adds 20-50mg, mixing, 2-8 ℃ stir 12-18 hour after; The PBS dialysis obtains neomycin antigen.-20 ℃, preservation.
What 2, immunity (resisting) was former synthesizes
Adopt the EDC method to carry out coupling neomycin and human serum albumins and obtain neomycin and human serum albumin conjugate, former as immunity (resisting).
Concrete operations are following: get the 20-30mg neomycin; Add 200-300mg1-ethyl-3-(3-dimethylaminopropyl)-carbodiimides successively, slowly stirred 10-20 minute, add the human serum albumins of 20-50mg; Mixing; After 12-18 hour, the PBS dialysis obtains neomycin and human serum albumin conjugate 2-8 ℃ of stirring.-20 ℃, preservation.
3, the preparation of neomycin rabbit polyclonal antibody
Adopting new zealand rabbit as immune animal, is immunogene with neomycin and human serum albumin conjugate synthetic in the step 2, and first immunisation is prepared according to following method with emulsion: the immunogene WS 2mL of 2mg/mL adds complete freund adjuvant 2mL emulsification; The booster immunization of band adjuvant is prepared according to following method with emulsion: the immunizing antigen WS 2mL of 2mg/mL adds the incomplete freund adjuvant emulsification of 2mL.First immunisation dosage is 1mg/, and booster immunization dosage is 0.5mg/, and each immunity is 2 weeks at interval; Booster immunization is 5-10 time altogether, and for the last time not with the direct intramuscular injection of immunologic adjuvant, blood sampling detects after 7 days; After measuring serum antibody titer; Serum is extracted in the arteria carotis bloodletting, obtains the polyclonal antibody of purifying through ammonium sulfate precipitation.
4, neomycin mouse monoclonal antibody preparation
Adopt BALB/C mice as immune animal; Neomycin and human serum albumin conjugate with synthetic in the step 2 are immunogene; Dosage is that 50 μ g/ (volume is 0.1mL, and solvent is a water) immunogenes add the complete freund adjuvant emulsification of 0.1mL, carries out subcutaneous first multi-point injection immunity.After January, get same amount immunizing antigen and add incomplete freund adjuvant, booster immunization is carried out in emulsification, and immunizing antigen does not add the adjuvant lumbar injection and carries out booster immunization after January, and antibody titer is measured in blood sampling in 5 days afterwards.Extracting spleen cell carries out Fusion of Cells in 4:1 ratio and myeloma cell SP2/0.Adopt limiting dilution or soft agar flat band method screening hybridoma, obtain the monoclonal antibody of complete homogeneity and stable monoclonal hybridoma strain.
Cell cryopreservation is got the monoclonal hybridoma strain that is in exponential phase with recovery and is processed 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.Make regular check on the stability of cell activity and secretory antibody.
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 paraffinum liquidum 0.5ml/, 7-14 days pneumoretroperitoneum injection monoclonal hybridoma strain cells 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, the preparation of ELIAS secondary antibody
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.
Two, detect the enzyme linked immunological kit of neomycin drug
1, detects the structure of the enzyme linked immunological kit of neomycin drug
This kit is mainly by box body, ELISA Plate, A reagent bottle (standard solution 1), B reagent bottle (standard solution 2); C reagent bottle (standard solution 3), D reagent bottle (standard solution 4), E reagent bottle (standard solution 5), F reagent bottle (standard solution 6); G reagent bottle (enzyme combination two is anti-), H reagent bottle (antibody concentrated solution), I reagent bottle (tetramethyl benzidine), J reagent bottle (containing tween-20 phosphate buffer); K reagent bottle (citrate buffer solution), L reagent bottle (stop buffer), the cover plate film, carriage is formed.Above-mentioned A is housed, B, C, D, E, F, G, H, I reagent bottle in the carriage.The foam carriage, ELISA Plate, J reagent bottle (containing tween-20 phosphate buffer), K reagent bottle (citrate buffer solution), L reagent bottle (stop buffer) 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, the preparation of agents useful for same
A, B, C, D, E, F reagent solution are neomycin series concentration standard solution, its concentration is respectively 0,0.2,1,5,25,125 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 neomycin 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 (0.5M pH7.2) that contains 0.5% polysorbas20.
K reagent solution: citrate buffer solution (0.1M pH5.0).
L reagent solution: stop buffer, 1-2mol/L sulfuric acid or hydrochloric acid.
3, the preparation of ELISA Plate
With encapsulating damping fluid (0.05M pH9.6 carbonate buffer solution) the neomycin envelope antigen is diluted to 0.1-1 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h; 4 ℃ are spent the night, and inclining encapsulates damping fluid, with the J reagent solution of 10 times of dilutions washing 3 times; Each 30 seconds, clap and do, 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.
Embodiment 2, kit sensitivity, specificity, precision, accuracy and critical value (cut-off) test
One, the pre-treatment of sample and detection
1, milk appearance: get the milk of fresh or freeze thawing, direct with the J reagent solution dilution back of 10 times of dilutions as supplying test agent;
2, tissue sample: get pork, each 1g of renal tissue, add the J reagent solution 5mL of 10 times of dilutions, behind the mixing, vibration 15min, the centrifugal 5min of 5000g gets the J reagent solution 0.95mL dilution of supernatant 0.05mL with 10 times of dilutions, as supplying test agent.
3, detect
Before detecting kit is recovered room temperature (18 ℃-30 ℃); The J reagent solution adds water and is mixed with washing lotion (phosphate buffer of 0.05% polysorbas20); With H reagent solution and washing lotion preparation antibody-solutions (0.1mg/L when the protein concentration of H reagent solution is 0.1mg/L, does not need to dilute with washing lotion)).In the ELISA Plate aperture, add 50 μ l sample solutions or neomycin series concentration standard solution, 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, clap with thieving paper and do, afterwards with G reagent and washing lotion configuration enzyme labelled antibody solution (0.1mg/L; When the protein concentration of G reagent solution is 0.1mg/L, do not need to dilute with washing lotion) and add in the ELISA Plate aperture, 30min placed for 18 ℃-30 ℃; Wash ELISA Plate 3 times with washing lotion again, clap and do, with I reagent solution and K reagent solution preparation zymolyte mixed liquor (0.1%; When the I reagent solution was 1mg/ml tetramethyl biphenyl amine aqueous solution, the I reagent solution need not used K reagent solution dilution), 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 ELIASA
450Value.Be calculated as follows the percentage absorbance:
(B-be the mean light absorbency value of standard solution or sample; B
0-be the standard solution mean light absorbency value of 0 μ g/L)
Logarithm with neomycin concentration in the standard solution is the X axle, and the percentage absorbance is the Y axle, and the drawing standard curve calculates neomycin concentration the sample solution from typical curve.
Two, kit sensitivity, specificity, precision, accuracy and critical value (cut-off) test
According to the relevant regulations of animal-derived food veterinary drug residue examination criteria establishment rule, respectively sensitivity, specificity, accuracy, precision and the critical value (cut-off) of this kit are measured.
1, kit sensitivity test
50% inhibition concentration (both IC
50, referring to the corresponding drug concentration in 50% place of the absorbance of neomycin 0 μ g/L standard solution) and Chang Zuowei estimates the index of competitive ELISA kit sensitivity.Measure 50% inhibition concentration of 10 substandard curves respectively, confirm this kit standard curve I C
50Should be within the scope of 1.5-7.0 μ g/L.Measure the blank sample of 20 portions of milk and the blank sample of 20 portions of porks respectively, and will measure mean value and add 3 times of standard deviations as LDL.
The kit that the result shows an anti-embodiment 1 for rabbit polyclonal antibody is limited to 7 μ g/L to the lowest detection of milk blank sample; The kit of one anti-embodiment 1 for rabbit polyclonal antibody is limited to 68 μ g/kg to the lowest detection of pork blank sample.
2, specific assay
Select and the similar 7 kinds of medicines of neomycin 26S Proteasome Structure and Function; Neomycin 50% inhibition concentration is compared with each medicine 50% inhibition concentration; Draw a series of cross reacting rates, the result is as shown in table 1, and test shows that 7 kinds of medicines of test and neomycin almost do not have cross reaction.
The cross reaction of table 1. neomycin ELISA detection method
3, accuracy, precision are measured
In blank milk, pork sample, adding neomycin titer to final concentration respectively is 250 μ g/L (kg), 500 μ g/L (kg) and 1000 μ g/L (kg).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 (recovery=measured value/interpolation value), plate within variance coefficient and plate.The result shows that the kit of an anti-embodiment 1 for rabbit polyclonal antibody is in milk; The recovery that 250 μ g/L neomycins add concentration is 80%-105%; The recovery that 500 μ g/L neomycins add concentration is 80%-95%, and the recovery that 1000 μ g/L neomycins add concentration is 60%-80%; Variation within batch coefficient CV≤15%, interassay coefficient of variation CV≤20%.The kit of one anti-embodiment 1 for rabbit polyclonal antibody is in pork; The recovery that 250 μ g/L neomycins add concentration is 50-80%; The recovery that 500 μ g/L neomycins add concentration is 50-90%; The recovery that 1000 μ g/L neomycins add concentration is 60-110%, variation within batch coefficient CV≤25%, interassay coefficient of variation CV≤25%.
4, the mensuration of critical value (cut-off value)
Through in milk and pork, adding neomycin to MRL 500 μ g/L, measure 20 times, repeat 3 times, calculate the cut-off value.The kit that the result shows an anti-embodiment 1 for rabbit polyclonal antibody is respectively 345 μ g/L and 310 μ g/kg at milk and cut-off mean value in the pork detection.
5, kit storage life test
The kit preservation condition of embodiment 1 is 2-8 ℃, and through 6 months mensuration, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, neomycin added the practical measurement value all within normal range.Consideration has improper preservation condition and occurs in transportation and use, and kit the condition held of 37 ℃ of preservations 4 days, is carried out accelerated deterioration and tests, and the result shows that this kit each item index meets the requirements fully.Can draw kit from above result can preserve more than 6 months at 2-8 ℃ at least.
Claims (4)
1. enzyme linked immunological kit that detects neomycin comprises the neomycin that encapsulates and the conjugate of albumin rabbit serum, the specific antibody of neomycin and ELIAS secondary antibody; The specific antibody of said neomycin is the polyclonal antibody that immunogene obtains for the conjugate with neomycin and human serum albumins; The conjugate of said neomycin and albumin rabbit serum is to be carrier with the albumin rabbit serum, and neomycin is a haptens, neomycin is coupled at the neomycin antigen that obtains on the albumin rabbit serum with ethyloic azanol-carbodlimide method; The conjugate of said neomycin and human serum albumins adopts the EDC method to carry out coupling neomycin and human serum albumins and obtains.
2. enzyme linked immunological kit according to claim 1 is characterized in that: said polyclonal antibody is a rabbit polyclonal antibody.
3. enzyme linked immunological kit according to claim 1 is characterized in that: said 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 and L reagent solution; Said A, B, C, D, E, F reagent solution are neomycin series concentration standard solution;
Said G reagent solution is the said ELIAS secondary antibody of 0.1-10mg/L;
Said H reagent solution is an antibody concentrated solution, is neomycin monoclonal antibody or the polyclonal antibody of 0.1-10mg/L;
Said I reagent solution is a tetramethyl benzidine;
Said J reagent solution is the phosphate buffer that contains 0.5% tween;
Said K reagent solution is a citrate buffer solution;
Said L reagent solution is a stop buffer.
4. enzyme linked immunological kit according to claim 1 is characterized in that: the marker enzyme in the said ELIAS secondary antibody is horseradish peroxidase or alkaline phosphatase.
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| CN 200610171540 CN1996022B (en) | 2006-12-30 | 2006-12-30 | Enzyme-linked immunologic kit for detecting neomycin |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102128923B (en) * | 2010-12-24 | 2013-05-15 | 江南大学 | One-step ELISA (Enzyme Linked Immunosorbent Assay) method for neomycin (NEO) residues in milk |
| CN102585006B (en) * | 2012-02-27 | 2013-12-04 | 华中农业大学 | Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting neomycin, amikacin and paromomycin |
| CN103513041A (en) * | 2012-06-29 | 2014-01-15 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting beta-agonist in raw milk |
| CN103575888B (en) * | 2012-08-02 | 2016-04-20 | 北京勤邦生物技术有限公司 | A kind of test strips and method detecting neomycin |
| CN105572370A (en) * | 2014-10-16 | 2016-05-11 | 镇江先创生物科技有限公司 | Time-resolved fluorescent immunoassay kit for detecting neomycin and detecting method of kit |
| CN106520705B (en) * | 2016-12-06 | 2019-04-30 | 得利斯集团有限公司 | The anti-paromomycin monoclonal antibody hybridoma cell strain C1 of one plant of secretion and its application |
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| CN1563994A (en) * | 2004-03-25 | 2005-01-12 | 中国农业大学 | Enzyme-linked immune kit for detecting corn bakanae alcohol |
| CN2694277Y (en) * | 2003-07-25 | 2005-04-20 | 北京望尔生物技术有限公司 | Streptomycin ELISA checking reagent case |
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| CN2694277Y (en) * | 2003-07-25 | 2005-04-20 | 北京望尔生物技术有限公司 | Streptomycin ELISA checking reagent case |
| CN1563994A (en) * | 2004-03-25 | 2005-01-12 | 中国农业大学 | Enzyme-linked immune kit for detecting corn bakanae alcohol |
| CN1707265A (en) * | 2004-06-11 | 2005-12-14 | 中国兽医药品监察所 | Enzyme-linked immunological kit for detecting tetracycline drug |
| CN1766629A (en) * | 2005-11-03 | 2006-05-03 | 北京望尔生物技术有限公司 | ELISA kit for detecting chloramphenicols in animal derived food |
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| LOOMANS等.Neamin as an immunogen for the development of a generic ELISA detecting gentamicin, kanamycin, and neomycin in milk.《Journal of Agricultural and Food Chemmistry》.2003,第51卷(第3期), * |
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