CN101021536B - Enzyme-linked immunological kit for detecting neomycin drug and method - Google Patents
Enzyme-linked immunological kit for detecting neomycin drug and method Download PDFInfo
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- CN101021536B CN101021536B CN2007100643472A CN200710064347A CN101021536B CN 101021536 B CN101021536 B CN 101021536B CN 2007100643472 A CN2007100643472 A CN 2007100643472A CN 200710064347 A CN200710064347 A CN 200710064347A CN 101021536 B CN101021536 B CN 101021536B
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Abstract
The invention provides an enzyme linked immune regent box to detect neomycin medicine. It includes: the enzyme labeled board which encrusts the encrusting substance, the enzyme marker, the special antibody for the neomycin, the neomycin standard solution, the substrate color solution, the stop solution, the condense washing solution, the condense recovery solution. The invention also discloses the method which includes the process: first to pre-treat the sample, then to detect by the regent box and last to analyze the detecting result. The invention is to detect the neomycin residue weight in the animal food such as the chicken, chicken liver, pork, pork liver, milk powder, egg and feedstuff. The method is simple, low cost and has high sensitivity, also it is proper for detect the mass sample.
Description
Technical field
The present invention relates to the immunology detection field, specifically, relate to a kind of enzyme linked immunological kit and detection method thereof that detects in the animal derived food as chicken, chicken gizzard, pork, pork liver, milk powder, egg and feed neomycin class medicine.
Background technology
The neomycin has a broad antifungal spectrum all has good inhibition effect to gram-positive bacteria and tubercle bacillus.Be used widely in animal husbandry is produced as a kind of veterinary drug commonly used.On the veterinary clinic, neomycin is mainly used in treatment fowl bacterial enteritis.On livestock and poultry cultivation, can improve efficiency of feed utilization, promote growth of animal, but because of its toxicity is bigger, residual in animal food can produce bigger potential hazard to the consumer, produce threat to food hygiene and publilc health.
The chemical method that detects the neomycin residual quantity mainly contains thin-layered chromatography (TLC), high performance liquid chromatography (HPLC), Capillary Electrophoresis (CE) etc., because complicated instrument and equipment and loaded down with trivial details process are not suitable for on-site supervision and great amount of samples examination.
Summary of the invention
(1) technical matters that will solve
The object of the present invention is to provide a kind of simple in structure, easy to use, low price, the portable enzyme linked immunological kit that is used for the animal derived food neomycin drug, and a kind of efficient, accurate, easy, qualitative and quantitative analysis method of being suitable for the batch samples screening is provided.
The working fluid form that the main contents thing of this detection kit has adopted convenience to use, working fluid keeping quality and good stability have overcome the technical matters of most of this areas product; The detection method that this kit adopts provides the recovery method of multiple sample, can be used for the detection of multiple sample.
(2) technical scheme
Detection principle of the present invention is:
When on the ELISA Plate capillary strip, wrapping in advance by neomycin coupling envelope antigen, after adding sample solution or standard solution, add neomycin specific antibody solution again, the neomycin coupled antigen competition neomycin specific antibody of bag quilt on residual neomycin drug or neomycin standard items and the ELISA Plate in the sample, add the enzyme labeling antiantibody and carry out amplification, with the colour developing of colour developing liquid, the content of the neomycin drug in sample light absorption value and the sample is negative correlation, relatively can draw the residual content of neomycin in the sample with typical curve.Simultaneously also can be according to the depth of the color on the ELISA Plate, with the comparison of the neomycin standard solution color of the series concentration concentration range of neomycin residual quantity in the judgement sample roughly.
When on capillary strip, wrapping in advance by the neomycin specific antibody, after adding sample solution or standard solution, add enzyme labeling neomycin antigenic solution again, the competition of residual neomycin drug or neomycin standard items and enzyme-labelled antigen is coated on the neomycin specific antibody on the ELISA Plate in the sample, with the colour developing of colour developing liquid, the content of the neomycin drug in sample light absorption value and the sample is negative correlation, relatively can draw the residual content of neomycin in the sample with typical curve.Simultaneously also can be according to the depth of the color on the ELISA Plate, with the comparison of the neomycin standard solution color of the series concentration concentration range of neomycin residual quantity in the judgement sample roughly.
When on capillary strip, wrapping in advance by the neomycin coupled antigen, after adding sample solution or standard solution, add enzyme labeling neomycin specific antibody solution again, the neomycin antigenic competition neomycin specific antibody of bag quilt on residual neomycin drug or neomycin standard items and the ELISA Plate in the sample, with the colour developing of colour developing liquid, the content of the neomycin drug in sample light absorption value and the sample is negative correlation, relatively can draw the residual content of neomycin in the sample with typical curve.Simultaneously also can be according to the depth of the color on the ELISA Plate, with the comparison of the neomycin standard solution color of the series concentration concentration range of neomycin residual quantity in the judgement sample roughly.
When on capillary strip, wrapping in advance by antiantibody, after adding the neomycin antibody incubation, add sample solution or standard solution, add enzyme labeling neomycin antigenic solution again, residual neomycin drug or neomycin standard items and enzyme labeling neomycin antigenic competition neomycin specific antibody in the sample, with colour developing liquid colour developing, the content of sample light absorption value and neomycin drug is negative correlation, relatively can draw the residual content of neomycin in the sample with typical curve.Simultaneously also can be according to the depth of the color on the ELISA Plate, with the comparison of the neomycin standard solution color of the series concentration concentration range of neomycin residual quantity in the judgement sample roughly.
The invention provides a kind of enzyme linked immunological kit that is used to detect neomycin drug, it contains:
(1) is coated with the ELISA Plate (the enzyme labeling thing is an enzyme-labelled antigen for envelope antigen on the ELISA Plate, the antiantibody of bag quilt when the enzyme labeling thing is the enzyme labeling antiantibody or on the ELISA Plate) of coating antigen;
(2) enzyme labeling thing (being enzyme-labelled antigen, enzymic-labelled antibody or enzyme labeling antiantibody);
(3) neomycin specific antibody (the enzyme labeling thing is an enzyme-labelled antigen for envelope antigen on the ELISA Plate, the antiantibody of bag quilt when the enzyme labeling thing is the enzyme labeling antiantibody or on the ELISA Plate);
(4) neomycin standard solution;
(5) substrate colour developing liquid;
(6) stop buffer;
(7) concentrated cleaning solution;
(8) concentrate redissolution liquid.
The enzyme linked immunological kit of detection neomycin provided by the present invention comprises the neomycin specific antibody and is coated with the elisa plate and the enzyme labeling thing of coating antigen; Described enzyme labeling thing is the antiantibody of enzyme labeling, enzyme labeling neomycin antigen or enzyme labeling neomycin specific antibody; Described antiantibody is sheep anti mouse antiantibody or goat-anti rabbit antiantibody.
The marker enzyme of described enzyme labeling thing is horseradish peroxidase or alkaline phosphatase, wherein preferred horseradish peroxidase; The sheep anti mouse antiantibody of enzyme labeling or goat-anti rabbit antiantibody adopt glutaraldehyde method or sodium periodate method that marker enzyme and antiantibody are carried out coupling and obtain, horseradish peroxidase can adopt several different methods of the prior art that itself and antiantibody are carried out coupling, as glutaraldehyde method, sodium periodate method etc., the present invention creates through long-term work the sodium periodate method is improved, make it save time, reduce the concentration rate of horseradish peroxidase (HRP) and antiantibody, saved starting material.
Described neomycin specific antibody can be neomycin monoclonal antibody or neomycin polyclonal antibody; They all are to obtain as immunogene with the conjugate that neomycin and carrier protein adopt the direct activation protein method to obtain; Described neomycin polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody, and described neomycin monoclonal antibody is preferably the neomycin mouse monoclonal antibody, and described neomycin polyclonal antibody is preferably the neomycin rabbit polyclonal antibody.
Above antibody all can prepare by the direct activation protein method as immunogene with the conjugate of neomycin and carrier protein.Described carrier protein can be common carrier albumen such as mouse haemocyanin, thyroprotein (BCG), bovine serum albumin(BSA), rabbit anteserum albumen, human serum albumins, ovalbumin or hemocyanin; The conjugate of described neomycin and carrier protein can obtain by neomycin and carrier protein are carried out coupling with the direct activation protein method.
For more convenient on-site supervision and great amount of samples examination, described kit also comprises neomycin standard solution, developer, stop buffer, concentrated cleaning solution, concentrates redissolution liquid.
Described concentrated cleaning solution is 0.01M, and pH7.4 contains the phosphate buffer of 0.8～1.2% polysorbas20 and 0.5 ‰ sodium azide antiseptics.
Described when marker enzyme is horseradish peroxidase, developer is made up of colour developing liquid A liquid and colour developing liquid B liquid, and colour developing liquid A liquid is hydrogen peroxide or urea peroxide, and described colour developing liquid B liquid is o-phenylenediamine or tetramethyl benzidine, and stop buffer is 1～2mol/L sulfuric acid; When marker enzyme is alkaline phosphatase, show that liquid is p-nitrophenyl phosphate damping fluid, stop buffer is the 2mol/L sodium hydroxide solution.
Described concentrated redissolution liquid is the phosphate buffer that contains 0.01～0.05mol/L, contains 1% methyl alcohol.
Wherein to be cushioned liquid be pH9.6 to ELISA Plate used bag in preparation process, the carbonate buffer solution of 0.05mol/L; Used confining liquid contains the solution of 5% hyclone.
The preparation process of ELISA Plate is among the present invention: be cushioned liquid with bag coating antigen is diluted to 0.1～1 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h, and 4 ℃ are spent the night again, coating buffer inclines, with cleansing solution washing 2 times, each 30 seconds, pat dry, in every hole, add 150-200 μ l confining liquid then, 37 ℃ of incubation 1-2h, liquid pats dry in the hole of inclining, and preserve with the vacuum seal of aluminium film dry back.
The building-up process of antigen is among the present invention:
1. the preparation of neomycin antibody
Neomycin is a small-molecule substance, has only immunoreactivity, does not have immunogenicity, can not bring out body and produce immune response, must with the macromolecular carrier albumen coupling after just have immunogenicity.
Adopt the direct activation protein method to carry out coupling neomycin and carrier protein and obtain immunogene.
Adopt the direct activation protein method to carry out coupling neomycin and carrier protein and obtain coating antigen.
2. the preparation of neomycin mouse monoclonal antibody
The animal immune program: adopting the Balb/c mouse as immune animal, is immunogene with neomycin and carrier protein couplet thing, obtains polyclonal antibody preferably, takes out liver and carries out Fusion of Cells.
Fusion of Cells and cloning: get immune BALB/c mouse splenocyte and SP2/0 myeloma cell and merge, the screening cell line is up to the hybridoma cell strain that obtains the stably excreting monoclonal antibody.
3. the preparation of neomycin rabbit polyclonal antibody
Adopting new zealand white rabbit as immune animal, is that immunogene is carried out immunity to new zealand white rabbit with neomycin and carrier protein couplet thing, and repeatedly the immunity back is measured serum antibody titer and obtained polyclonal antibody.
The preparation process of antiantibody of the present invention: the sheep anti mouse antiantibody be with sheep as immune animal, be that immunogene is carried out immunity to anosis thalline sheep with mouse source antibody, obtain the sheep anti mouse antiantibody; Goat-anti rabbit antiantibody be with sheep as immune animal, be that immunogene is carried out immunity to anosis thalline sheep with rabbit source antibody, obtain goat-anti rabbit antiantibody.
Neomycin standard solution in the kit of the present invention: 6 bottles of standard solutions, 0 μ g/L, 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L and 3ml/ bottle.
The present invention also provides a kind of method that above-mentioned enzyme linked immunological kit detects neomycin drug of using, and it comprises step:
(1) sample pre-treatments;
(2) detect with kit;
(3) analyzing and testing result.
The invention provides the disposal route of neomycin in animal derived food such as chicken, chicken gizzard, pork, pork liver, milk powder, egg and feed equal samples.
Take by weighing and remove fatty pulverizing animal tissue sample in centrifuge tube, after adding PB damping fluid mixes up and down, put in the water-bath and heat, room temperature is centrifugal, gets supernatant and redissolves sample analysis; The milk powder sample is placed centrifuge tube, add PB and normal hexane, taking off layer liquid after centrifugal redissolves sample analysis; The egg sample is smashed, stirred evenly the generation that prevents foam with glass bar; The feed sample is placed centrifuge tube, add PBS
1, sample analysis is redissolved in centrifugal back.
Among the present invention be: when coating antigen is the neomycin coupled antigen with the kit detection, adding standard solution or sample solution add antibody again in the ELISA Plate micropore, washing pats dry behind the incubation, add enzyme mark antiantibody again, washing pats dry behind the incubation, colour developing, termination are measured absorbance with microplate reader; When coating antigen was the neomycin coupled antigen, adding standard solution or sample solution added enzymic-labelled antibody again in the ELISA Plate micropore, and washing pats dry behind the incubation, and colour developing, termination are measured absorbance with microplate reader.
When coating antigen was the neomycin specific antibody, adding standard solution or sample solution added enzyme labeling neomycin antigen again in the ELISA Plate micropore, and washing pats dry behind the incubation, and colour developing, termination are measured absorbance with microplate reader; When coating antigen is antiantibody, in the ELISA Plate micropore, add neomycin antibody, washing pats dry behind the incubation, adds enzyme mark neomycin antigen after adding standard solution or sample solution again, and washing pats dry behind the incubation, colour developing, stops, and measures absorbance with microplate reader.
The testing result analytic process is among the present invention: the absorbance mean value (B) of standard solution of using each concentration that is obtained is divided by the absorbance (B of first standard solution (0 standard)
0) multiply by 100% again, i.e. percentage absorbance.Computing formula is:
Percentage absorbance (%)=(B/B
0) * 100%
Semilog value with the concentration (μ g/L) of neomycin standard solution is an X-axis, and the percentage absorbance is a Y-axis, the drawing standard curve map.With the percentage absorbance of same way calculation sample solution, the concentration of corresponding each sample then can be read the residual quantity of neomycin the sample from typical curve.
The analysis of testing result also can be adopted regression equation method among the present invention, calculates sample solution concentration.
The analysis of testing result can also utilize computer professional software among the present invention, the be more convenient for express-analysis of a large amount of samples of this method, and whole testing process only needed to finish in 1 hour, and lowest detection is limited to 0.5 μ g/L.
(3) beneficial effect
The enzyme linked immunological kit that the present invention detects neomycin mainly adopts the residual quantity of neomycin in the qualitative or detection by quantitative sample of indirect competitive ELISA method; Pre-treatment requirement to sample is low, and sample pretreatment process is simple, simultaneously the fast detecting gross sample; Adopt the neomycin monoclonal antibody of high specific, main agents provides with the form of working fluid, and the method for inspection is convenient and easy, has specificity height, highly sensitive, characteristics such as degree of accuracy is high, accuracy height.Enzyme linked immunological kit of the present invention, simple in structure, easy to use, low price, carrying convenience, detection method is efficient, accurate, easy, be suitable for the qualitative, quantitative of batch samples screening.Kit of the present invention will play a significant role in the detection of neomycin.Method of the present invention is efficient, accurate, easy, be suitable for the qualitative and quantitative analysis of batch samples screening.
Description of drawings
Fig. 1: the examination criteria curve map of neomycin.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only are used to illustrate the present invention, and be not used for limiting the scope of the invention.
The preparation of embodiment 1 reagent constituents
1. antigen is synthetic
A. the neomycin immunizing antigen is synthetic
Adopt the direct activation protein method to carry out coupling neomycin and bovine serum albumin(BSA) and obtain immunogene.
Immunogenic preparation process: get bovine serum albumin(BSA) 18mg and separate (I liquid) with 1ml is water-soluble; Get EDC20mg and add in the solution (I liquid) stirring reaction 30min with the water-soluble back of separating of 0.5ml; Get neomycinsulphate 5mg and add in the solution (II liquid) with the water-soluble back of separating of 1.5ml, stirring reaction spends the night, and obtains immunogene.
B. the preparation of coating antigen neomycin coupled antigen
Adopt the direct activation protein method to carry out coupling neomycin and thyroprotein and obtain coating antigen.
The preparation process of coating antigen: get BCG protein 18 mg and separate (I liquid) with 1ml is water-soluble; Get EDC20mg and add in the solution (I liquid) stirring reaction 30min with the water-soluble back of separating of 0.5ml; Get neomycinsulphate 5mg and add in the solution (II liquid) with the water-soluble back of separating of 1.5ml, stirring reaction spends the night, and obtains coating antigen.
2. MONOCLONAL ANTIBODIES SPECIFIC FOR
A. animal immune
Immunogene is injected in the Balb/c mouse body, and immunizing dose is 100 μ g/, makes it produce polyclonal antibody.
B. Fusion of Cells and cloning
Get immune Balb/c mouse boosting cell, merge, obtain the hybridoma cell strain of monoclonal antibody in 5:1 ratio and SP2/0 myeloma cell.
C. cell cryopreservation and recovery
Hybridoma is made 1 * 10 with cryopreserving liquid
6The cell suspension of individual/ml is 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.
D. MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
The Balb/c mouse peritoneal is only injected sterilization paraffin oil 0.4ml/, 7 days pneumoretroperitoneum injection hybridomas 5 * 10
5Individual/as only, to gather ascites after 7 days.Carry out the ascites purifying with sad-saturated ammonium sulfate method ,-20 ℃ of preservations.
3. Polyclonal Antibody Preparation
Adopt new zealand white rabbit as immune animal, with neomycin and ovalbumin conjugate is immunogene, immunizing dose is 1.5mg/kg, when head exempts from the Fu Shi of immunogene and equivalent helped fully and be mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, getting the same dose immunogene 3～4 weeks adds equivalent incomplete Freund mixing and emulsifying at interval, and booster immunization is once, immunity is 5 times altogether, does not add adjuvant for the last time.Serum antibody titer is measured in last immunity blood sampling after 10 days, and heart is taken a blood sample, and obtains the polyclonal antibody of purifying with ammonium sulfate precipitation.
4. the preparation process of sheep anti mouse antiantibody: as immune animal, is that immunogene to pathogen-free domestic sheep carry out immunity with mouse source antibody with sheep, obtains the sheep anti mouse antiantibody; The preparation of goat-anti rabbit antiantibody: as immune animal, is that immunogene to pathogen-free domestic sheep carry out immunity with rabbit source antibody with sheep, obtains goat-anti rabbit antiantibody.
5. the preparation of ELISA Plate
Be cushioned liquid with bag neomycin coupled antigen, antibody or antiantibody are diluted to 0.1-1 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h or 4 ℃ spend the night, and the coating buffer that inclines is with the concentrated cleaning solution washing of 20 times of dilutions 2 times, each 30 seconds, pat dry, in every hole, add 200 μ l confining liquids, 37 ℃ of incubation 2h then, the liquid in the hole that inclines, preserve with the vacuum seal of aluminium film dry back.
6. enzyme labeling sheep anti mouse antiantibody purchases
Adopt the sodium periodate method after improveing to carry out coupling antiantibody and horseradish peroxidase (HRP).The molar concentration rate of enzyme and antiantibody is 4:1 in traditional sodium periodate method requirement reflection system; Because horseradish peroxidase produces many sites that combine with antiantibody under the effect of strong oxidation, Huo Hua horseradish peroxidase molecule has served as the bridge that connects each molecule like this, reduced the enzymatic activity of enzyme labeling thing, make in the conjugate of preparation and be mixed with many condensates, in order to address this problem, we improve traditional method, that is:
1) saved amino closed process, because can produce self amino amino reality that connects seldom.
2) reduced horseradish peroxidase: the volumetric molar concentration ratio of antiantibody is to 2:1, and the method after the improvement is easier than traditional method, and the loss of the activity of enzyme is reduced.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of neomycin
Set up the enzyme linked immunological kit that detects neomycin, make it comprise following component:
(1) bag is by the ELISA Plate of anti-neomycin antibody;
(2) the sheep anti mouse antiantibody of usefulness horseradish peroxidase-labeled;
(3) neomycin monoclonal antibody body running liquid;
(4) the neomycin standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 0.5 μ g/L, 1.5 μ μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L;
(5) substrate colour developing liquid is made up of A liquid and B liquid, and substrate colour developing liquid A liquid is urea peroxide, and substrate colour developing liquid B liquid is tetramethyl benzidine;
(6) stop buffer is a 2mol/L hydrochloric acid;
(7) concentrated cleaning solution is 0.01M, and pH7.4 contains the phosphate buffer of 0.8～1.2% polysorbas20 and 0.5 ‰ sodium azide antiseptics;
(8) concentrate redissolving liquid is 0.01～0.05mol/L, contains the phosphate buffer of 1% methyl alcohol.
The residual detection of neomycin in embodiment 3 samples
1. sample pre-treatments
Animal tissue's (chicken, chicken gizzard, pork, pork liver)
Take by weighing tissue samples that the 2.0g homogeneous crosses in the 50ml centrifuge tube, add 6ml0.1MPBS (PH=10～11) damping fluid, mix 10min fully up and down, put into 60 ℃ of water-bath environment 60min, taking-up is cooled to room temperature and shakes up, more than the 6000g, the centrifugal 10min of room temperature, get supernatant, dilute, get 20 μ l and analyze with the volume ratio of 1:9.
2. detect with kit
In the ELISA Plate micropore that is coated with anti-neomycin antibody, add series standard product solution or sample solution 20 μ l, add enzyme-labelled antigen 80 μ l again, 25 ℃ of reaction 30min.Pour out liquid in the hole, every hole adds the cleansing solution after the dilution, pours out liquid in the hole behind the 30s, and so repetitive operation is washed plate 5 times altogether, pats dry with thieving paper.Every hole adds substrate colour developing liquid A liquid urea peroxide, substrate colour developing liquid B liquid tetramethyl benzidine (TMB), each the 50 μ l mixing that vibrates gently, 25 ℃ of constant temperature oven lucifuges colour developing 15～30min.Every hole adds stop buffer 50 μ l, and the mixing that vibrates gently at the 450nm place, is measured every hole absorbance (OD value) with the microplate reader wavelength set.
3. testing result analysis
With the absorbance mean value (B) of the standard solution of each concentration that is obtained absorbance (B divided by first standard solution (0 standard)
0) multiply by 100% again, obtain the percentage absorbance.Semilog value with neomycin standard items concentration (μ g/L) is an X-axis, and the percentage absorbance is a Y-axis, the drawing standard curve map.With the percentage absorbance of same way calculation sample solution, the concentration of corresponding each sample, the residual quantity that then can read neomycin from typical curve.
The residual detection of neomycin in embodiment 4 samples
1. sample pre-treatments
The egg sample
Take by weighing the even good egg sample of matter of 1g ± 0.05g, add 8ml0.1M PBS
1(pH=10) hatched 30 minutes for 80 ℃, 3000g is above centrifugal, draws the redissolution liquid after supernatant 100 μ l add 900 μ l dilution, mixes, and gets 50 μ l and is used for analyzing.
2. detect with kit
In the ELISA Plate micropore that is coated with anti-neomycin antibody, add series standard product solution or sample solution 20 μ l, add enzyme-labelled antigen 80 μ l again, 25 ℃ of reaction 30min.Pour out liquid in the hole, every hole adds the cleansing solution after the dilution, pours out liquid in the hole behind the 30s, and so repetitive operation is washed plate 5 times altogether, pats dry with thieving paper.Every hole adds substrate colour developing liquid A liquid urea peroxide, substrate colour developing liquid B liquid tetramethyl benzidine (TMB), each the 50 μ l mixing that vibrates gently, 25 ℃ of constant temperature oven lucifuges colour developing 15～30min.Every hole adds stop buffer 50 μ l, and the mixing that vibrates gently at the 450nm place, is measured every hole absorbance (OD value) with the microplate reader wavelength set.
3. testing result analysis
With the absorbance mean value (B) of the standard solution of each concentration that is obtained absorbance (B divided by first standard solution (0 standard)
0) multiply by 100% again, obtain the percentage absorbance.Semilog value with neomycin standard items concentration (μ g/L) is an X-axis, and the percentage absorbance is a Y-axis, the drawing standard curve map.With the percentage absorbance of same way calculation sample solution, the concentration of corresponding each sample, the residual quantity that then can read neomycin from typical curve.
The detection of gentamicin residue in embodiment 5 samples
1. sample pre-treatments
The milk powder sample
Take by weighing 1g ± 0.05g milk powder sample, add 4ml0.02M PB, add the 5ml normal hexane, 3000g is above centrifugal, draws the redissolution liquid after the liquid 40 μ l of lower floor add 960 μ l dilution, mixes, and gets 50 μ l and is used for analyzing.
2. detect with kit
In the ELISA Plate micropore that is coated with anti-neomycin antibody, add series standard product solution or sample solution 20 μ l, add enzyme-labelled antigen 80 μ l again, 25 ℃ of reaction 30min.Pour out liquid in the hole, every hole adds the cleansing solution after the dilution, pours out liquid in the hole behind the 30s, and so repetitive operation is washed plate 5 times altogether, pats dry with thieving paper.Every hole adds substrate colour developing liquid A liquid urea peroxide, substrate colour developing liquid B liquid tetramethyl benzidine (TMB), each the 50 μ l mixing that vibrates gently, 25 ℃ of constant temperature oven lucifuges colour developing 15～30min.Every hole adds stop buffer 50 μ l, and the mixing that vibrates gently at the 450nm place, is measured every hole absorbance (OD value) with the microplate reader wavelength set.
3. testing result analysis
With the absorbance mean value (B) of the standard solution of each concentration that is obtained absorbance (B divided by first standard solution (0 standard)
0) multiply by 100% again, obtain the percentage absorbance.Semilog value with neomycin standard items concentration (μ g/L) is an X-axis, and the percentage absorbance is a Y-axis, the drawing standard curve map.With the percentage absorbance of same way calculation sample solution, the concentration of corresponding each sample, the residual quantity that then can read neomycin from typical curve.
The detection of gentamicin residue in embodiment 6 samples
1. sample pre-treatments
The feed sample
Take by weighing 1g ± 0.05g feed sample, add 10ml0.02M PBS
1Abundant vibration mixing, the above centrifugal 5min of 3000g draws the redissolution liquid after 100 μ l add 900 μ l dilution, mixes, and gets 50 μ l and is used for analyzing.
2. detect with kit
In the ELISA Plate micropore that is coated with anti-neomycin antibody, add series standard product solution or sample solution 20 μ l, add enzyme-labelled antigen 80 μ l again, 25 ℃ of reaction 30min.Pour out liquid in the hole, every hole adds the cleansing solution after the dilution, pours out liquid in the hole behind the 30s, and so repetitive operation is washed plate 5 times altogether, pats dry with thieving paper.Every hole adds substrate colour developing liquid A liquid urea peroxide, substrate colour developing liquid B liquid tetramethyl benzidine (TMB), each the 50 μ l mixing that vibrates gently, 25 ℃ of constant temperature oven lucifuges colour developing 15～30min.Every hole adds stop buffer 50 μ l, and the mixing that vibrates gently at the 450nm place, is measured every hole absorbance (OD value) with the microplate reader wavelength set.
3. testing result analysis
With the absorbance mean value (B) of the standard solution of each concentration that is obtained absorbance (B divided by first standard solution (0 standard)
0) multiply by 100% again, obtain the percentage absorbance.Semilog value with neomycin standard items concentration (μ g/L) is an X-axis, and the percentage absorbance is a Y-axis, the drawing standard curve map.With the percentage absorbance of same way calculation sample solution, the concentration of corresponding each sample, the residual quantity that then can read neomycin from typical curve.
Experimental example 1
The test of standard items precision:
Respectively extract a collection of ELISA Plate out respectively from the ELISA Plate of three different time period preparations, every batch is extracted 10 kits, and every plate is extracted 20 micropores out, measures the absorbance of 4.5 μ g/L standard solution, calculates the coefficient of variation.
The repeatable test of table 1 standard (CV%)
Can draw by above-mentioned test findings, every batch of each 10 standard items coefficient of variation of kit meet precision and are less than or equal to 25% regulation between 4.6%-16.8%.
Experimental example 2
Sample precision and accuracy test
A. sample precision test:
, add in the sample animal tissue, milk powder, egg and feed with the neomycin of 50 μ g/L concentration, get each five of the kits of three different batches respectively, each concentration repeats 5 times, calculates the coefficient of variation respectively, the results are shown in Table 2～5.
The repeatable test of table 2 animal tissue sample
The repeatable test of table 3 powdered milk sample
The repeatable test of table 4 egg sample
The repeatable test of table 5 feed sample
The result shows that the Variation Lines number average in animal tissue, milk powder, egg and the feed sample is lower than 25%.
B. sample accuracy test
The neomycin standard solution of getting two concentration is respectively 20 μ g/kg (L), 50 μ g/kg (L), respectively sample is added recovery test, each concentration do 4 parallel, sample pre-treatments step among concrete grammar such as the embodiment 1, respectively accuracy in computation.
The accuracy of table 6 kit
The result shows that muscle, fishes and shrimps, milk powder, egg and feed sample add accuracy between 64.2%-98.5%.
Experimental example 3
The cross reacting rate test:
Select to have 5 kinds of drug monitoring cross reacting rates of similar structures and similar functions with neomycin.The typical curve of logical various medicines obtains its 50% inhibition concentration respectively.Calculate the cross reacting rate of kit with following formula to other medicines.Cross reaction is big more, and this kit is just good more to the specificity of the detection of neomycin so.
Cross reacting rate (%)=(cause 50% concentration that suppresses neomycin/cause, 50% analog that suppresses
Concentration) * 100%
The specificity of table 7 kit
| Medicine name | Cross reacting rate (%) |
| |
100 |
| Streptomysin | <0.1 |
| Kanamycins | <0.1 |
| Apramycin | <0.1 |
| Gentamicin | <0.7 |
| Tobramycin | <0.1 |
Experimental example 4
The kit preservation condition 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 was placed 6 days under the condition of 37 ℃ of preservations, carries out the accelerated deterioration experiment, and the result shows that the every index of this kit meets the requirements fully.Consider that the freezing situation of kit takes place, kit was put into-20 ℃ of refrigerators freezing 5 days, measurement result shows that also the every index of kit is normal fully.Can draw kit from above result can preserve more than 6 months at least at 2-8 ℃.
Claims (2)
1. enzyme linked immunological kit that detects neomycin drug is characterized in that it contains:
(1) bag is by the ELISA Plate of anti-neomycin antibody;
(2) the sheep anti mouse antiantibody of usefulness horseradish peroxidase-labeled;
(3) neomycin monoclonal antibody body running liquid;
(4) neomycin standard solution, concentration are respectively 0 μ g/L, 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L;
(5) substrate colour developing liquid is made up of A liquid and B liquid, and substrate colour developing liquid A liquid is urea peroxide, and substrate colour developing liquid B liquid is tetramethyl benzidine;
(6) stop buffer: 2mol/L hydrochloric acid;
(7) concentrated cleaning solution: 0.01M, pH 7.4, contain the phosphate buffer of 0.8～1.2% polysorbas20 and 0.5 ‰ sodium azide antiseptics;
(8) concentrate the phosphate buffer that redissolves liquid: 0.01～0.05mol/L, contains 1% methyl alcohol;
Wherein, the preparation method of ELISA Plate is as follows: be cushioned liquid the neomycin antibody dilution is become 0.1-1 μ g/ml with bag, every hole adds 100 μ l, and 37 ℃ of incubation 2h or 4 ℃ spend the night, coating buffer inclines, concentrated cleaning solution with 20 times of dilutions washs 2 times, each 30 seconds, pats dry, in every hole, add 200 μ l confining liquids then, 37 ℃ of incubation 2h, liquid in the hole of inclining, preserve with the vacuum seal of aluminium film dry back;
The preparation method of above-mentioned enzyme labeling sheep anti mouse antiantibody is as follows: adopt the sodium periodate method after improveing to carry out coupling antiantibody and horseradish peroxidase, traditional sodium periodate method is improved, that is:
1) saved amino closed process, because can produce self amino amino reality that connects seldom;
2) reduced horseradish peroxidase: the volumetric molar concentration ratio to 2 of antiantibody: 1, the method after the improvement is easier than traditional method, and the loss of the activity of enzyme is reduced.
2. one kind is detected the residual method of neomycin drug in the animal derived food, comprises step:
(1) sample pre-treatments;
(2) detect with the described kit of claim 1;
(3) analyzing and testing result.
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Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN102353704A (en) * | 2011-06-27 | 2012-02-15 | 江南大学 | Preparation method and use of universal antibiotic detection sensor |
| 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 |
| 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 |
| CN103575914B (en) * | 2012-08-02 | 2016-08-03 | 北京勤邦生物技术有限公司 | The enzyme linked immunological kit of detection vitamin B12 and application thereof |
| CN105572345A (en) * | 2014-10-16 | 2016-05-11 | 镇江亿特生物科技发展有限公司 | Fluorescence assay method for detecting neomycin in chicken meat tissue |
| 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 |
| CN110470831B (en) * | 2019-07-09 | 2021-03-12 | 华南农业大学 | Chemiluminescence immunoassay method and kit for directly detecting tenuazonic acid based on camel source antibody |
| CN111848792B (en) * | 2020-07-16 | 2023-02-24 | 浙江省食品药品检验研究院 | Preparation method and detection method of antibody of bovine serum albumin and other impurities in vaccine |
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| CN1029232C (en) * | 1991-07-11 | 1995-07-05 | 宜昌市三峡制药厂 | Extraction process of neomycin sulfate by use of static method |
| CN1766624A (en) * | 2005-11-03 | 2006-05-03 | 北京望尔生物技术有限公司 | ELISA kit for detecting furazolidone metabolites and detection method thereof |
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|---|---|---|---|---|
| CN1029232C (en) * | 1991-07-11 | 1995-07-05 | 宜昌市三峡制药厂 | Extraction process of neomycin sulfate by use of static method |
| CN1766624A (en) * | 2005-11-03 | 2006-05-03 | 北京望尔生物技术有限公司 | ELISA kit for detecting furazolidone metabolites and detection method thereof |
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| Yong Jin等.Development of ELISA and immunochromatographic assay for the detection of neomycin.《Clinica Chimica Acta》.2005,第364卷(第1-2期),260-266. * |
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