CN101013131B - Furantoin metabolite enzyme linked immunosorbent analytical reagent casing and uses thereof - Google Patents
Furantoin metabolite enzyme linked immunosorbent analytical reagent casing and uses thereof Download PDFInfo
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- CN101013131B CN101013131B CN2007100638737A CN200710063873A CN101013131B CN 101013131 B CN101013131 B CN 101013131B CN 2007100638737 A CN2007100638737 A CN 2007100638737A CN 200710063873 A CN200710063873 A CN 200710063873A CN 101013131 B CN101013131 B CN 101013131B
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Abstract
The invention provides a detecting nitrofurantoin metabolite ELISA kit, which contains: an ELISA plate covered by the original coated, enzyme marker, nitrofurantoin metabolite specific antibody or nitrofurantoin metabolite derivative antibody, nitrofurantoin metabolite standard solution or nitrofurantoin metabolite derivative standard solution, the substrate color solution, the termination solution, the condensed washing solution, the condensed complex solution. The invention also provides a method to apply the above ELISA kit detecting nitrofurantoin metabolite, which includes steps: firstly, sample pre-treatment, and then using the kit for testing, and finally analyzing testing results. The invention is to provide the nitrofurantoin metabolite residues in the ELISA kit for detection of animal derived foods, such as chicken, pork, fish, shrimp, milk, honey, egg, and other samples, and the detection method is simple, low cost, high sensitivity, and it can monitor on the scene and suitable for screening large number of samples.
Description
Technical field
The present invention relates to field of immunodetection, be specifically related to a kind of enzyme linked immunological kit and application thereof that detects Cistofuran metabolite residual quantity in the animal derived food.
Background technology
The itrofurans medicine once was widely used because of the dynamic (dynamical) characteristic of extraordinary antibacterial action and medicine is arranged, and as the growth-promoting additive of bird, aquatic products and pig.But finding in the process of experimental that for a long time itrofurans medicine and metabolin all can make animal used as test that canceration and gene mutation take place, so this type of drug withdrawal uses in treatment and feed.Furantoin is disabled in nineteen ninety-five.
Because the itrofurans medicine in vivo soon can be by metabolism, the metabolic product of combination then can retain long period of time in tissue, so the product often will analyze its metabolism when analyzing this type of medicine residual after, administrative authority is that means reach the residual purpose of detection itrofurans to detect metabolic product just.
The most popular method that is used for detecting Cistofuran metabolite is LC-UV, and LC-MS and LC-MS/MS are 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
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 Cistofuran metabolite, 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 has reduced the pattern detection lower limit, and the detection sensitivity of raising is higher than the pattern detection sensitivity of this area instrument detecting method.
The invention provides a kind of enzyme linked immunological kit that is used to detect Cistofuran metabolite, it contains:
(1) is coated with the ELISA Plate of coating antigen;
(2) enzyme labeling thing;
(3) Cistofuran metabolite specific antibody or Cistofuran metabolite derivant specific antibody;
(4) Cistofuran metabolite standard items or Cistofuran metabolite derivant standard solution;
(5) substrate colour developing liquid;
(6) stop buffer;
(7) concentrated cleaning solution;
(8) concentrate redissolution liquid.
Above described enzyme labeling thing be enzyme labeling antiantibody, enzyme labeling Cistofuran metabolite antigen or enzyme labeling Cistofuran metabolite derivatives antigens, enzyme labeling Cistofuran metabolite specific antibody or enzyme labeling Cistofuran metabolite derivant specific antibody; Described antiantibody is sheep anti mouse antiantibody or goat-anti rabbit antiantibody.And when coating antigen was Cistofuran metabolite antigen or Cistofuran metabolite derivatives antigens, the enzyme labeling thing was an enzyme mark antiantibody; When coating antigen was antiantibody, the enzyme labeling thing was an antigen.
The marker enzyme of described enzyme labeling thing is that horseradish peroxidase or bacterium are extracted alkaline phosphatase, wherein preferred horseradish peroxidase; Enzyme labeling sheep anti mouse antiantibody or enzyme labeling goat-anti rabbit antiantibody adopt sodium periodate method or carbonization two imido methods 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 carbonization two imido methods, 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 and antiantibody, saved starting material.
Described Cistofuran metabolite specific antibody can be Cistofuran metabolite monoclonal antibody or Cistofuran metabolite polyclonal antibody, and Cistofuran metabolite derivant specific antibody can be Cistofuran metabolite derivant monoclonal antibody or Cistofuran metabolite derivant polyclonal antibody; The Cistofuran metabolite polyclonal antibody is to carry out immunity with the conjugate that Cistofuran metabolite haptens and carrier protein adopt mixed anhydride method to obtain as immunogene to obtain, and Cistofuran metabolite derivant polyclonal antibody is to carry out immunity with the conjugate that Cistofuran metabolite derivative hapten and carrier protein adopt mixed anhydride method to obtain as immunogene to obtain; The Cistofuran metabolite monoclonal antibody is to carry out the splenocyte that immunity obtains with the conjugate that Cistofuran metabolite haptens and carrier protein adopt mixed anhydride method to obtain as immunogene, obtain monoclonal antibody through Fusion of Cells and clone cell again, Cistofuran metabolite derivant monoclonal antibody is to carry out the splenocyte that immunity obtains with the conjugate that Cistofuran metabolite derivative hapten and carrier protein adopt mixed anhydride method to obtain as immunogene, obtains monoclonal antibody through Fusion of Cells and clone cell again; Described Cistofuran metabolite polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody, and Cistofuran metabolite derivant polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody; Described Cistofuran metabolite monoclonal antibody is preferably the Cistofuran metabolite mouse monoclonal antibody, and Cistofuran metabolite derivant monoclonal antibody is preferably Cistofuran metabolite derivant mouse monoclonal antibody; Described Cistofuran metabolite polyclonal antibody is preferably the Cistofuran metabolite rabbit polyclonal antibody, and Cistofuran metabolite derivant polyclonal antibody is preferably Cistofuran metabolite derivant rabbit polyclonal antibody.
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 Cistofuran metabolite haptens and carrier protein can carry out coupling with Cistofuran metabolite haptens and carrier protein by mixed anhydride method and obtain; The conjugate of described Cistofuran metabolite derivative hapten and carrier protein can obtain by Cistofuran metabolite derivative hapten and carrier protein are carried out coupling with mixed anhydride method.
For more convenient on-site supervision and great amount of samples examination, described kit also comprises Cistofuran metabolite standard solution, developer, stop buffer, concentrated cleaning solution, concentrates redissolution liquid.
Described concentrated cleaning solution is 0.02M, and pH7.4 contains the phosphate buffer of 0.8%～1.2% polysorbas20 and 0.05% thimerosal antiseptic.
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 bacterium extraction 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 that 0.2M-0.5M contains 0.1% song and draws logical phosphate buffer.
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 5% calf serum, 1% caseic phosphate buffer.
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 Cistofuran metabolite derivative hapten
React in water with Cistofuran metabolite with to carboxyl benzaldehyde and to carry out derivatization and obtain the Cistofuran metabolite derivative hapten.
2. the preparation of Cistofuran metabolite derivatives antigens
Furantoin 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 mixed anhydride method to carry out coupling Cistofuran metabolite derivative hapten and carrier protein and obtain immunogene.
Adopt mixed anhydride method to carry out coupling Cistofuran metabolite derivative hapten and carrier protein and obtain envelope antigen.
3. the preparation of Cistofuran metabolite antigen
Furantoin 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 mixed anhydride method to carry out coupling Cistofuran metabolite haptens and carrier protein and obtain immunogene.
Adopt mixed anhydride method to carry out coupling Cistofuran metabolite haptens and carrier protein and obtain envelope antigen.
4. the preparation of Cistofuran metabolite or metabolin derivant mouse monoclonal antibody
Animal immune program: adopt the Balb/c mouse as immune animal, with Cistofuran metabolite haptens and carrier protein couplet thing or Cistofuran metabolite derivative hapten and carrier protein couplet thing is immunogene, obtain polyclonal antibody preferably, take out liver and carry 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.
5. the preparation of Cistofuran metabolite or metabolin derivant rabbit polyclonal antibody
Adopt new zealand white rabbit as immune animal, with Cistofuran metabolite haptens and carrier protein couplet or Cistofuran metabolite derivative hapten and carrier protein couplet thing is that immunogene is carried out immunity to new zealand white rabbit, adopt short distance tachysynthesis scheme, repeatedly the immunity back is measured serum antibody titer and is 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.
Cistofuran metabolite standard solution in the kit of the present invention: 6 bottles of standard solutions, 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, 3ml/ bottle.
Detection principle of the present invention is:
When on the ELISA Plate capillary strip, wrapping in advance by Cistofuran metabolite coupling envelope antigen or Cistofuran metabolite derivant coupling envelope antigen, after adding sample solution or standard solution, add Cistofuran metabolite specific antibody or Cistofuran metabolite specific antibody solution again, the Cistofuran metabolite coupling envelope antigen of bag quilt or Cistofuran metabolite derivant coupling envelope antigen competition Cistofuran metabolite specific antibody or Cistofuran metabolite derivant specific antibody on residual Cistofuran metabolite or Cistofuran metabolite standard items or Cistofuran metabolite standard items derivant 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 Cistofuran metabolite in sample light absorption value and the sample is negative correlation, relatively can draw the residual content of Cistofuran metabolite 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 Cistofuran metabolite standard items of series concentration or the Cistofuran metabolite derivant standard solution color concentration range of Cistofuran metabolite residual quantity in the judgement sample roughly.
When on capillary strip, wrap by the Cistofuran metabolite specific antibody in advance or bark mutter appropriate during because of metabolin derivant specific antibody, after adding sample solution or standard solution, add enzyme labeling Cistofuran metabolite antigen or enzyme labeling Cistofuran metabolite derivatives antigens solution again, the competition of residual Cistofuran metabolite or Cistofuran metabolite standard items or Cistofuran metabolite derivant standard items and enzyme labeling Cistofuran metabolite antigen or enzyme labeling Cistofuran metabolite derivatives antigens is coated on Cistofuran metabolite specific antibody or the Cistofuran metabolite derivant specific antibody on the ELISA Plate in the sample, with the colour developing of colour developing liquid, Cistofuran metabolite content in sample light absorption value and the sample is negative correlation, relatively can draw the residual content of Cistofuran metabolite 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 Cistofuran metabolite standard items of series concentration or the Cistofuran metabolite derivant standard solution color concentration range of Cistofuran metabolite residual quantity in the judgement sample roughly.
When on capillary strip, wrapping in advance by Cistofuran metabolite coupling envelope antigen or Cistofuran metabolite derivant coupling envelope antigen, after adding sample solution or standard solution, add enzyme labeling Cistofuran metabolite specific antibody or enzyme labeling Cistofuran metabolite derivant specific antibody solution again, the Cistofuran metabolite envelope antigen of bag quilt or Cistofuran metabolite derivant envelope antigen competition Cistofuran metabolite specific antibody or Cistofuran metabolite derivant specific antibody on residual Cistofuran metabolite or Cistofuran metabolite standard items or Cistofuran metabolite derivant standard items and the ELISA Plate in the sample, with the colour developing of colour developing liquid, the content of the Cistofuran metabolite in sample light absorption value and the sample is negative correlation, relatively can draw the residual content of Cistofuran metabolite 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 Cistofuran metabolite standard items of series concentration or the Cistofuran metabolite derivant standard solution color concentration range of Cistofuran metabolite residual quantity in the judgement sample roughly.
When on capillary strip, wrapping in advance by antiantibody, after adding the furantoin antibody incubation, add sample solution or standard solution, add enzyme labeling Cistofuran metabolite coupled antigen or enzyme labeling Cistofuran metabolite derivant coupled antigen solution again, residual Cistofuran metabolite or Cistofuran metabolite standard items or Cistofuran metabolite derivant standard items and enzyme labeling Cistofuran metabolite coupled antigen or enzyme labeling Cistofuran metabolite derivant coupled antigen competition Cistofuran metabolite specific antibody or Cistofuran metabolite derivant specific antibody in the sample, with the colour developing of colour developing liquid, the content of sample light absorption value and Cistofuran metabolite is negative correlation, relatively can draw the residual content of Cistofuran metabolite 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 Cistofuran metabolite standard items of series concentration or the Cistofuran metabolite derivant standard solution color concentration range of Cistofuran metabolite residual quantity in the judgement sample roughly.
The present invention also provides a kind of method that above-mentioned enzyme linked immunological kit detects Cistofuran metabolite 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 in Cistofuran metabolite animal derived food such as chicken, pork, fishes and shrimps, milk, honey and the egg equal samples.
Tissue sample is thawed, shred, place tissue refiner's high speed homogenate; The centrifugal fat deposit of removing of milk sample; The dissolving of honey sample adding distil water evenly; The egg sample is smashed, stirred evenly the generation that prevents foam with glass bar.
Among the present invention be: when coating antigen is Cistofuran metabolite coupling envelope antigen or Cistofuran metabolite derivant coupling envelope 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 is Cistofuran metabolite coupling envelope antigen or Cistofuran metabolite derivant coupling envelope antigen, adding standard solution or sample solution add enzymic-labelled antibody again in the ELISA Plate micropore, washing pats dry behind the incubation, and colour developing, termination are measured absorbance with microplate reader.
When coating antigen is Cistofuran metabolite specific antibody or Cistofuran metabolite derivant specific antibody, adding standard solution or sample solution add enzyme labeling Cistofuran metabolite antigen or enzyme labeling Cistofuran metabolite derivatives antigens again in the ELISA Plate micropore, washing pats dry behind the incubation, colour developing, termination are measured absorbance with microplate reader; When coating antigen is antiantibody, in the ELISA Plate micropore, add Cistofuran metabolite antibody or Cistofuran metabolite derivant antibody, washing pats dry behind the incubation, add enzyme mark Cistofuran metabolite antigen or enzyme mark Cistofuran metabolite derivatives antigens after adding standard solution or sample solution again, washing pats dry behind the incubation, colour developing, termination are measured 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 Cistofuran metabolite standard items or Cistofuran metabolite derivant 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 Cistofuran metabolite 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 1.5 hours, and lowest detection is limited to 0.1 μ g/L.
The enzyme linked immunological kit that the present invention detects Cistofuran metabolite mainly adopts the residual quantity of Cistofuran metabolite 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 Cistofuran metabolite monoclonal antibody or the Cistofuran metabolite derivant monoclonal antibody of high specific, main agents provides with the form of working fluid, the method of inspection is convenient and easy, has specificity height, highly sensitive, characteristics such as degree of accuracy is high, accuracy height.
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 has reduced the pattern detection lower limit.The detection sensitivity that improves is higher than the pattern detection sensitivity of this area instrument detecting method.
Description of drawings
Fig. 1: the examination criteria curve map of Cistofuran metabolite.
Embodiment
Following embodiment is used for further specifying of the present invention, but is not used for limiting the scope of the invention.
The preparation of embodiment 1 reagent constituents
1. antigen is synthetic
A. the Cistofuran metabolite derivative hapten is synthetic
React in water with Cistofuran metabolite with to carboxyl benzaldehyde and to carry out derivatization and obtain the Cistofuran metabolite derivative hapten.
The preparation process of Cistofuran metabolite derivative hapten:
Getting Cistofuran metabolite 1000mg is dissolved in the 12ml pure water.1000mg is dissolved in the 26ml water carboxyl benzaldehyde, joined in the Cistofuran metabolite solution room temperature reaction 48 hours, obtain faint yellow precipitation and be the Cistofuran metabolite derivative hapten.Clean repeatedly 5～6 times with 50ml water, drying obtains the Cistofuran metabolite derivative hapten.
B. immunogene is synthetic
Adopt mixed anhydride method to carry out coupling Cistofuran metabolite derivative hapten and hemocyanin and obtain immunogene.
Immunogenic preparation process: get Cistofuran metabolite derivative hapten 200mg and be dissolved in the 10ml water, add 4 ℃ of reactions of 1.5ml isobutyl chlorocarbonate 12 hours, getting hemocyanin 500mg is dissolved in the 20ml pure water, join in the Cistofuran metabolite derivative hapten of activation 4 ℃ of reactions spend the night (16 hours), again with pure water dialysis 5 days, concentrate and obtain Cistofuran metabolite derivant immunogene, packing is frozen.
C. the preparation of coating antigen Cistofuran metabolite derivant coupled antigen
Adopt mixed anhydride method to carry out coupling Cistofuran metabolite derivative hapten and thyroprotein and obtain coating antigen.
The preparation process of coating antigen: get Cistofuran metabolite derivative hapten 200mg and be dissolved in the 10ml water, add 4 ℃ of reactions of 1.5ml isobutyl chlorocarbonate 12 hours, getting thyroprotein 500mg is dissolved in the 20ml pure water, join in the Cistofuran metabolite derivative hapten of activation 4 ℃ of reactions spend the night (16 hours), again with pure water dialysis 5 days, concentrate and obtain Cistofuran metabolite derivant coating antigen, packing is frozen.
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 Cistofuran metabolite derivative hapten 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 Cistofuran metabolite coupling envelope 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 Cistofuran metabolite
Set up the enzyme linked immunological kit that detects Cistofuran metabolite, make it comprise following component:
(1) bag is by the ELISA Plate of anti-Cistofuran metabolite coupled antigen;
(2) the sheep anti mouse antiantibody of usefulness horseradish peroxidase-labeled;
(3) Cistofuran metabolite monoclonal antibody working fluid;
(4) the furantoin standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 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 0.02M, and pH7.4 contains the phosphate buffer of 1.1% polysorbas20 and 0.05% thimerosal antiseptic;
(8) concentrate redissolving liquid is that 0.3M contains 0.1% song and draws logical phosphate buffer.
The residual detection of Cistofuran metabolite in embodiment 3 samples
1. sample pre-treatments
Animal tissue's (chicken, pork, fishes and shrimps)
Get the equal pledge of the tissue samples of 1 ± 0.05g, add the distilled water of 4ml, the 2-nitrobenzaldehyde of 0.5ml1MHCl and 100 μ l10mM, fully vibration; At 37 ℃ of night incubation (approximately 16h); Add 5ml0.1M K
2HPO
4, 0.4ml 1M NaOH and 5ml ethyl acetate, thermal agitation 30s; At room temperature (20-25 ℃/68-77F) the above centrifugal 10min of 3000g; Taking out 2.5ml ethyl acetate 50 ℃ of following nitrogen in another container dries up or the Rotary Evaporators evaporate to dryness; With the dry thing of 1ml n-hexane dissolution, diluted good redissolution liquid with 1ml and fully mixed; The above centrifugal 10min of (20-25 ℃/68-77 ℉) 3000g at room temperature; Be used for analyzing with 50 μ l subnatant bodies.
2. detect with kit
In the ELISA Plate micropore that is coated with anti-Cistofuran metabolite coupled antigen, add series standard product solution or sample solution 50 μ l, add Cistofuran metabolite monoclonal antibody working fluid 50 μ l again, 37 ℃ 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.Add 100 μ l enzyme labeling antiantibodys in each micropore, react 30min in the rearmounted 37 ℃ of environment of cover plate membrane cover plate.Take out ELISA Plate, liquid in the hole is dried, the cleansing solution 250 μ l after the adding dilution wash plate 4-5 time in each plate hole, each 10 seconds at interval, pat dry with thieving paper.Every hole adds substrate colour developing liquid A liquid urea peroxide 50 μ l, substrate colour developing liquid B liquid tetramethyl benzidine (TMB) 50 μ l, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge 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 Cistofuran metabolite 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 Cistofuran metabolite from typical curve.
The residual detection of Cistofuran metabolite in embodiment 4 samples
1. sample pre-treatments (milk)
The milk sample that takes out 5ml is in the glass centrifuge tube; Add each 150 μ l of 0.36M sodium nitroprusside damping fluid and 1M zinc sulfate damping fluid respectively; Behind the abundant mixing sample of oscillator, with the above centrifugal 10min of constant temperature hydro-extractor 3000g, 4-12 ℃ (39-54 ℉).Get the centrifuged supernatant 1.1ml of milk, add the distilled water of 5ml, the 2-nitrobenzaldehyde of 1ml 1M HCl and 100 μ l 10mM, fully vibration; At 37 ℃ of night incubation (approximately 16h); Add 5ml0.1M K
2HPO
4, 0.4ml 1M NaOH and 5ml ethyl acetate, thermal agitation 30s; At room temperature (20-25 ℃/68-77F) the above centrifugal 10min of 3000g; Taking out 2.5ml ethyl acetate 50 ℃ of following nitrogen in another container dries up; With the dry thing of 1ml n-hexane dissolution, diluted good redissolution liquid with 1ml and fully mixed; The above centrifugal 10min of (20-25 ℃/68-77 ℉) 3000g at room temperature; Be used for analyzing with 50 μ l subnatant bodies.
2. detect with kit
In the ELISA Plate micropore that is coated with anti-Cistofuran metabolite coupled antigen, add series standard product solution or sample solution 50 μ l, add Cistofuran metabolite monoclonal antibody working fluid 50 μ l again, 37 ℃ 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.Add 100 μ l enzymes mark antiantibody in each micropore, react 30min in the rearmounted 37 ℃ of environment of cover plate membrane cover plate.Take out ELISA Plate, liquid in the hole is dried, the cleansing solution 250 μ l after the adding dilution wash plate 4-5 time in each plate hole, each 10 seconds at interval, pat dry with thieving paper.Every hole adds substrate colour developing liquid A liquid urea peroxide 50 μ l, substrate colour developing liquid B liquid tetramethyl benzidine (TMB) 50 μ l, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge 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 Cistofuran metabolite 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 Cistofuran metabolite from typical curve.
The residual detection of Cistofuran metabolite in embodiment 5 samples
1. sample pre-treatments (honey)
Get 1g honey sample in centrifuge tube; The distilled water vibration dissolving that adds 5ml, the 2-nitrobenzaldehyde of 1ml1MHCl and 100 μ l10mM, fully vibration; At 37 ℃ of night incubation (approximately 16h); Add 5ml0.1M K respectively
2HPO
4, 0.6ml 1M NaOH and 5ml ethyl acetate, thermal agitation 30s; At room temperature (20-25 ℃/68-77F) the above centrifugal 10min of 3000g; Taking out 2ml ethyl acetate 50 ℃ of following nitrogen in another container dries up or the Rotary Evaporators evaporate to dryness; With the dry thing of 1ml n-hexane dissolution, diluted good redissolution liquid with 0.5ml and fully mixed; At room temperature (20-25 ℃/68-77F) the above centrifugal 10min of 3000g; Be used for analyzing with 50 μ l subnatant bodies.
2. detect with kit
In the ELISA Plate micropore that is coated with anti-Cistofuran metabolite coupled antigen, add series standard product solution or sample solution 50 μ l, Cistofuran metabolite monoclonal antibody working fluid 50 μ l, 37 ℃ 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.Add 100 μ l enzyme labeling antiantibodys in each micropore, react 30min in the rearmounted 37 ℃ of environment of cover plate membrane cover plate.Take out ELISA Plate, liquid in the hole is dried, the cleansing solution 250 μ l after the adding dilution wash plate 4-5 time in each plate hole, each 10 seconds at interval, pat dry with thieving paper.Every hole adds substrate colour developing liquid A liquid urea peroxide 50 μ l, substrate colour developing liquid B liquid tetramethyl benzidine (TMB) 50 μ l, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge 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 Cistofuran metabolite 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 Cistofuran metabolite from typical curve.
The residual detection of Cistofuran metabolite in embodiment 6 samples
1. sample pre-treatments (egg)
Take by weighing the egg sample 2g for preparing, join in the 50ml centrifuge tube, add 6ml water respectively, 1ml 1M HCl, 200 μ l0.36M sodium nitroprusside damping fluids, vibration mixing; It is slow to add 200 μ l1M zinc sulfate, the 5min that fully vibrates, the above centrifugal 10min of room temperature (20～25 ℃) 3000g; Get whole supernatants, add the 2-nitrobenzaldehyde of 200 μ l10mM, fully vibration, 50 ℃ of water-bath 2h (middle per half an hour thermal agitation 1-2 minute); Add 5ml0.1M K respectively
2HPO
4, 0.5ml1M NaOH4ml ethyl acetate, thermal agitation 30s, the above centrifugal 10min of room temperature (20～25 ℃) 3000g; Getting the 2.5ml upper organic phase dries up in 50 ℃ of following nitrogen; The dry thing of 1ml n-hexane dissolution adds the redissolution liquid after 2ml dilutes, vibration 10s, the above centrifugal 10min of 3000g; Remove upper organic phase; Taking off layer water 50 μ l is used for analyzing.
2. detect with kit
In the ELISA Plate micropore that is coated with anti-Cistofuran metabolite coupled antigen, add series standard product solution or sample solution 50 μ l, Cistofuran metabolite monoclonal antibody working fluid 50 μ l, 37 ℃ 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.Add 100 μ l enzyme labeling antiantibodys in each micropore, react 30min in the rearmounted 37 ℃ of environment of cover plate membrane cover plate.Take out ELISA Plate, liquid in the hole is dried, the cleansing solution 250 μ l after the adding dilution wash plate 4-5 time in each plate hole, each 10 seconds at interval, pat dry with thieving paper.Every hole adds substrate colour developing liquid A liquid urea peroxide 50 μ l, substrate colour developing liquid B liquid tetramethyl benzidine (TMB) 50 μ l, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge 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 Cistofuran metabolite 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 Cistofuran metabolite 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 0.9 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 6.8%-19.4%.
Experimental example 2
Sample precision and accuracy test
A. sample precision test:
, add in the sample animal tissue, milk, honey and egg with the Cistofuran metabolite of 1.0 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 milk sample
The repeatable test of table 4 honey sample
The repeatable test of table 5 egg sample
The result shows that animal tissue, milk, honey and egg sample coefficient of variation all are lower than 25%.
B. sample accuracy test
The Cistofuran metabolite standard solution of getting two concentration is respectively 0.5 μ g/kg (L) and 1.0 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 accurate seat of table 6 kit
The result shows that muscle, fishes and shrimps, milk, honey and egg sample add accuracy between 62.5%-96.7%
Experimental example 3
The cross reacting rate test:
Select to have 3 kinds of drug monitoring cross reacting rates of similar structures and similar functions with Cistofuran metabolite.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 Cistofuran metabolite so.
Cross reacting rate (%)=(cause 50% concentration that suppresses Cistofuran metabolite/cause
The 50% analog concentration that suppresses) * 100%
The specificity of table 7 kit
| Medicine name | Cross reacting rate (%) |
| |
100 |
| Furaxone metabolite | <0.01 |
| AMOZ | <0.01 |
| Nitrofurazone (nitrofurazone) metabolin | <0.01 |
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, Cistofuran metabolite 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 Cistofuran metabolite is characterized in that it contains:
(1) be coated with the ELISA Plate of coating antigen, wherein coating antigen is Cistofuran metabolite envelope antigen or Cistofuran metabolite derivant envelope antigen;
(2) the sheep anti mouse antiantibody of usefulness horseradish peroxidase-labeled;
(3) Cistofuran metabolite monoclonal antibody working fluid;
(4) the furantoin standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 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 0.02M, and pH 7.4, contains the phosphate buffer of 1.1% polysorbas20 and 0.05% thimerosal antiseptic;
(8) concentrate redissolving liquid is that 0.3M contains 0.1% song and draws logical phosphate buffer;
Wherein, the Cistofuran metabolite envelope antigen of bag quilt is the conjugate that Cistofuran metabolite haptens and carrier protein obtain by the mixed anhydride method coupling in the described ELISA Plate, and Cistofuran metabolite derivant envelope antigen is the conjugate that Cistofuran metabolite derivative hapten and carrier protein obtain by the mixed anhydride method coupling; Described carrier protein is mouse haemocyanin, thyroprotein, bovine serum albumin(BSA), rabbit anteserum albumen, human serum albumins, ovalbumin or hemocyanin; The needed immunizing antigen of preparation Cistofuran metabolite monoclonal antibody is described Cistofuran metabolite immunizing antigen or described Cistofuran metabolite derivant immunizing antigen, and described Cistofuran metabolite immunizing antigen is by mixed anhydride method Cistofuran metabolite haptens and carrier protein couplet to be obtained; Described Cistofuran metabolite derivant immunizing antigen is by mixed anhydride method Cistofuran metabolite derivative hapten and carrier protein couplet to be obtained.
2. method that detects Cistofuran metabolite residual quantity 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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| US11275084B2 (en) * | 2010-01-15 | 2022-03-15 | Stanford University | Successive sampling device and associated method |
| CN102565032A (en) * | 2010-12-15 | 2012-07-11 | 北京勤邦生物技术有限公司 | Magnetic granule chemiluminescence kit for detecting nitrofurantoin metabolite and application of magnetic granule chemiluminescence kit |
| CN103288961A (en) * | 2012-02-29 | 2013-09-11 | 华中农业大学 | Monoclonal antibody of furantoin residue marker aminohydantoin, and preparation method and application thereof |
| CN103364556B (en) * | 2012-04-05 | 2016-02-24 | 北京勤邦生物技术有限公司 | A kind of kit and method detecting Cistofuran metabolite |
| CN105601730A (en) * | 2014-10-30 | 2016-05-25 | 北京维德维康生物技术有限公司 | Preparation methods of nitrofurantoin hapten and nitrofurantoin antigen, and applications of nitrofurantoin hapten and nitrofurantoin antigen in chemiluminescence immunoassay kit |
| CN107315091A (en) * | 2017-06-29 | 2017-11-03 | 重庆师范大学 | One kind detection Cistofuran metabolite quantum dot immune chromatographic test paper, preparation method and applications |
| CN115286580B (en) * | 2022-08-01 | 2024-04-16 | 上海源本食品质量检验有限公司 | Preparation method of nitrofurantoin metabolite derivatization hapten |
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| 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 |
| 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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| 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 |
| 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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