CN102435729A - One-step enzyme-linked immunoassay method for quickly detecting melamine and kit thereof - Google Patents
One-step enzyme-linked immunoassay method for quickly detecting melamine and kit thereof Download PDFInfo
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- CN102435729A CN102435729A CN2011103967130A CN201110396713A CN102435729A CN 102435729 A CN102435729 A CN 102435729A CN 2011103967130 A CN2011103967130 A CN 2011103967130A CN 201110396713 A CN201110396713 A CN 201110396713A CN 102435729 A CN102435729 A CN 102435729A
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Abstract
The invention provides a one-step enzyme-linked immunoassay method for quickly detecting melamine and a kit thereof. The kit comprises a melamine specific antibody, melamine standard product solution and a melamine enzyme marker, wherein the antibody is a rabbit anti-melamine polyclonal antibody; and the melamine enzyme marker is a melamine horse-radish oxidase marker. The invention also provides the one-step enzyme-linked immunoassay method for quickly detecting the melamine by using the kit. Main reagents in the kit are provided as working liquid, so that the operating steps of the kit can be reduced, time can be saved for a user, and errors due to the fuzzy operating steps can be reduced. The method has the advantages of high sensitivity, high specificity, high precision, high accuracy, low requirement on apparatuses and equipment, long reagent storage time, high automation degree, no radioactive isotope pollution and the like, and can achieve important functions in the detection of feeds and animal derived products.
Description
Invention field
The present invention relates to the illegal adjuvant residue detection of enzyme linked immunological and food field.Particularly, the present invention relates to a kind of single stage method enzyme-linked immune detection method and kit thereof that is used for the fast detecting melamine.
Background technology
Melamine (Melamine), molecular formula C
3N
6H
6, being called for short triamine, chemical name is 2,4; 6-triamido-1,3,5-triazines is commonly called as melamine, extract of protein; Be a kind of important triazines nitrogen heterocyclic ring Organic Chemicals, white monoclinic crystal, almost tasteless, be slightly soluble in water; Dissolve in methyl alcohol, formaldehyde, acetate, hot monoethylene glycol, glycerine, pyridine etc., insoluble what acetone, ethers are widely used in industries such as timber, plastics, coating, papermaking, weaving, leather, electric and medicine.The MEL nitrogen content is high, and illegal retailer is added into cheap melamine in the food, improves the protein content in the food with falseness.In March, 2007, more than 4000 the cat and dog incident of being poisoned to death takes place in the U.S.; In September, 2008; The contaminated incident of China's outburst Sanlu baby milk powder; The infant who has caused eating contaminated milk powder produces kidney stone illness even death, and the problem milk powder case that breaks out once again at the beginning of 2010, and its reason all is to contain melamine in feed or the milk powder.European Union, the U.S., Canada, Japan and China forbid that all the people is for being added into melamine in food and feed at present; Consider that melamine also might move in the food from wrappage; For guaranteeing health and food security; On April 6th, 2011, five ministries and commissions such as the Ministry of Public Health unite 2011 No. 10 bulletin of issue, and the value of limiting the quantity of of melamine is 1mg/kg in the regulation infant formula (comprising milk and milk products); The value of limiting the quantity of of melamine is 2.5mg/kg in other food, is higher than above-mentioned food of limiting the quantity of and must not sells without exception.
Therefore it is significant to guarantee food and feed safety to set up a kind of melamine rapid detection method quick, sensitive, that be prone to row.The melamine detection method mainly contains high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry chromatogram method (GC-MS) and liquid-matter coupling chromatography etc. at present, and detection sensitivity is high, accuracy good; But these methods need perfect laboratory, experienced technician, and complicated instrument and equipment, and testing process is loaded down with trivial details, the examination of incompatible on-the-spot great amount of samples.For the analyzing and testing of micromolecule residue a new approach is provided based on the immunoreactive immunology detection technology of antigen-antibody.The single stage method direct competitive enzyme linked immunosorbent detection technology of setting up based on this principle has shortened detection time greatly than the indirect competition method.The key of this technology is the preparation of the synthetic and antibody of comlete antigen and enzyme-labelled antigen.
At present, do not see that homemade melamine single stage method enzyme-linked immunologic detecting kit is arranged.
Summary of the invention
The object of the present invention is to provide a kind of single stage method enzyme-linked immunologic detecting kit of fast detecting melamine.
The invention provides a kind of single stage method enzyme-linked immunologic detecting kit of fast detecting melamine, it comprises melamine specific antibody, melamine standard solution and melamine enzyme labeling thing.Wherein said melamine antibody is a polyclonal antibody, is preferably the polyclonal antibody of rabbit anti-melamine, its be with melamine and carrier protein couplet thing as immunogene through immune animal for example rabbit make; Described melamine standard solution is that melamine standard items storing solution (accurately takes by weighing 100mg melamine standard items; With ultrapure water dissolving and to be settled to 100mL formulated) be mixed with phosphate buffer (0.01mol/L, pH 7.4) that concentration is respectively 0,1,3,9,27,81ng/mL; Used marker enzyme is a horseradish peroxidase in the described melamine enzyme labeling thing, and described melamine horseradish peroxidase-labeled thing adopts the chemical method coupling to obtain, and said coupling method is the formaldehyde resin method.
The invention provides a kind of single stage method enzyme-linked immunologic detecting kit of fast detecting melamine, this kit comprises: the ELISA Plate and the melamine horseradish peroxidase-labeled thing working fluid that are coated with the melamine specific antibody.Wherein the melamine specific antibody is the polyclonal antibody of rabbit anti-melamine, its be with melamine and carrier protein couplet thing as immunogene through immune animal for example rabbit make; Described melamine horseradish peroxidase-labeled thing adopts the chemical method coupling to obtain, and marker enzyme is a horseradish peroxidase, and said coupling method is the formaldehyde resin method.
Be used to prepare the solid phase material of said ELISA Plate, include but not limited to, for example, polystyrene, tygon, polypropylene.The form of carrier is the micro-reaction plate shrinkage pool.
The present invention also provides a kind of single stage method enzyme-linked immunologic detecting kit of fast detecting melamine; It also further comprises melamine standard solution, developer, enzyme mark thing dilution, cleansing solution, stop buffer and concentrating sample dilution except comprising ELISA Plate and the melamine enzyme labeling beyond the region of objective existence that is coated with the melamine specific antibody.With convenient on-the-spot the detection and the great amount of samples examination.Described cleansing solution is a 0.05%-0.5% Tween-20 phosphate buffer.Described developer is mixed by 5: 5: 1 (volume ratio) by hydrogen peroxide or urea peroxide, o-phenylenediamine or tetramethyl benzidine and stabilizing agent K, under 4 ℃, can preserve nondiscolouring in 2 years.Described concentrating sample dilution is the phosphate buffer that contains 0.1% Tween-20.Described enzyme mark thing dilution is the phosphate buffer that contains enzyme stabilizers AH and enzymatic protective reagent KEN.Described stabilizing agent K, contain enzyme stabilizers AH and enzymatic protective reagent available from American I nternationnal Diagostica System company.Described melamine enzyme labeling thing is a melamine horseradish peroxidase-labeled thing freeze-dried powder, and being dissolved as working fluid with enzyme mark thing dilution during use can directly can use.Described melamine standard solution is that melamine standard items storing solution (accurately takes by weighing 100mg melamine standard items; With ultrapure water dissolving and to be settled to 100mL formulated) be mixed with phosphate buffer (0.01mol/L, pH 7.4) that concentration is respectively 0,1,3,9,27,81ng/mL.Described stop buffer is the hydrochloric acid solution of 2mol/L.Described concentrating sample dilution is 10 times of concentrated phosphoric acid salt buffers that contain 1% BSA.
The single stage method enzyme-linked immunologic detecting kit of fast detecting melamine of the present invention also comprises the instructions that instructs the single stage method enzyme-linked immune detection method that carries out fast detecting melamine of the present invention.
On the other hand, the present invention also provides the single stage method enzyme-linked immune detection method of the content of melamine in a kind of fast detecting liquid milk, milk powder, dairy produce and the feed sample, comprises step:
(1) sample pre-treatments: the liquid milk sample, get 1.00mL in the Eppendorf centrifuge tube, get middle clear liquid behind 4 ℃ of centrifugal 5min of 12000rpm, directly be used for subsequent experimental, extension rate is 1; The milk powder sample thief accurately takes by weighing 2.00g in the 50mL centrifuge tube, gets 10mL PBS and joins in the milk powder, and 40 ℃ of water-baths are heated to milk powder and dissolve fully, gets middle clear liquid behind 4 ℃ of centrifugal 10min of 4000rpm, directly is used for subsequent experimental, and extension rate is 5; Dairy produce and feed sample accurately take by weighing 2.00g in the 50mL centrifuge tube after the pulverizing, add 10mL 0.1M HCl; Ultrasonic degradation sample 25min after the violent vortex oscillation, the centrifugal 10min of room temperature 5000rpm is with about 1M NaOH adjustment pH value to 8.0; Centrifugal 5min of room temperature 5000rpm or 0.4 μ m filtrator are filtered in the clean pipe; Supernatant or filtered fluid are used for subsequent experimental after further diluting by 1: 1 with PBS, and extension rate is 10.
(2) the single stage method enzyme-linked immunologic detecting kit with fast detecting melamine of the present invention detects; In the ELISA Plate hole that is coated with the melamine specific antibody, add standard items or sample solution; Add melamine horseradish peroxidase-labeled thing working fluid again; After the time of explanation, temperature and the operation notice operation, colour developing, termination are measured absorbance with ELIASA to specifications.The time of these reactions, temperature and operation steps and operation notice are well known in the art.Preferably, the melamine antibody that coating buffer is diluted to 1.8 μ g/mL concentration is by every hole 100 μ L coated elisa plates, and 4 ℃ encapsulate and spend the night, and cleansing solution washing 3 times is clapped and done; Spend the night by 4 ℃ of following sealings of every hole 200 μ L confining liquids, wash 3 times, clap dried subsequent use.Every hole, standard items hole adds the melamine standard items (0,1,3,9,27,81ng/mL) of 50 μ L series concentration; The every hole of sample well adds the good sample of 50 μ L dilution to be measured; Every hole adds the melamine horseradish peroxidase mark thing of 50 μ L dilution in 1: 16000; Room temperature effect 30min washs 3 times, claps and does; Every hole adds 100 μ l developers, room temperature effect 10min, and every hole adds 100 μ L stop buffer cessation reactions, and ELIASA detects A value (450nm).The logarithm that with the combination rate is ordinate, melamine standard items concentration is that horizontal ordinate is set up typical curve, according to typical curve calculate and multiply by its corresponding extension rate get final product the content of melamine in the sample.The single stage method enzyme-linked immunologic detecting kit of wherein said fast detecting melamine comprises ELISA Plate and the melamine horseradish peroxidase-labeled thing working fluid that is coated with the melamine specific antibody; Perhaps further comprise melamine standard solution, developer, enzyme mark thing dilution, cleansing solution, stop buffer and concentrating sample dilution, wherein said cleansing solution is a 0.05%-0.5% Tween-20 phosphate buffer.Described developer is mixed by 5: 5: 1 (volume ratio) by hydrogen peroxide or urea peroxide, o-phenylenediamine or tetramethyl benzidine and stabilizing agent K, under 4 ℃, can preserve nondiscolouring in 2 years.Described concentrating sample dilution is the phosphate buffer that contains 0.1% Tween-20.Described enzyme mark thing dilution is the phosphate buffer that contains 10% enzyme stabilizers AH and 5% enzymatic protective reagent KEN.Described melamine enzyme labeling thing is a melamine horseradish peroxidase-labeled thing freeze-dried powder, and being dissolved as working fluid with enzyme mark thing dilution during use can directly can use.Described melamine standard solution is that melamine standard items storing solution (accurately takes by weighing 100mg melamine standard items; With ultrapure water dissolving and to be settled to 100mL formulated) be mixed with phosphate buffer (0.01mol/L, pH 7.4) that concentration is respectively 0,1,3,9,27,81ng/mL.Described stop buffer is the hydrochloric acid solution of 2mol/L.Described concentrating sample dilution is 10 times of concentrated phosphoric acid salt buffers that contain 1% BSA.Described confining liquid is the phosphate buffer that contains 1% OVA.
(3) analyzing and testing result.Analyzing and testing result's method is well known in the art, and the detection of use and analytical instrument are well known in the art, includes but not limited to ELIASA such as Thermo MK3 etc.
The detection principle of kit of the present invention is:
Melamine antibody is encapsulated on solid phase carrier; Add sample or melamine standard solution and add enzyme labeling melamine horseradish peroxidase-labeled thing working fluid, melamine in the testing sample and enzyme labeling melamine are competed immobilised melamine antibody, remove unconjugated enzyme labeling thing through washing; The colour developing back stops; The absorbance of working sample, the amount of melamine is negative correlation in this value and the sample, relatively can draw the melamine concentration scope with typical curve.
Beneficial effect:
The kit that the present invention detects melamine mainly adopts content of melamine in the qualitative or detection by quantitative sample of direct competitive enzymoimmunoassay; Further having shortened detection time is in 20 minutes, and low to the pre-treatment requirement of sample, sample pretreatment process is simple, simultaneously the fast detecting gross sample.
This kit of the present invention adopts the melamine polyclonal antibody of single step reaction method and high specific; Main agents provides with the working fluid form; Can reduce the operation steps of kit; For the user saves time and reduces the error that causes because of operation steps is miscellaneous; That the present invention has is highly sensitive, high specificity, pinpoint accuracy, pin-point accuracy, low to the instrument and equipment requirement, the reagent holding time is long, automaticity is high, "dead" isotopic contamination etc. advantage, can in feed and animal derived product detect, play a significant role.
Description of drawings
Fig. 1 is the melamine typical curve;
Fig. 2 is melamine kit actual detected structural drawing (before stopping), and 1,2 two is the standard control curve, and all the other holes are the sample detection result;
Fig. 3 is the correlativity that ELISA and HPLC method detect content of melamine in the milk sample;
Fig. 4 is the correlativity that ELISA and HPLC method detect content of melamine in the powdered milk sample;
Fig. 5 is the correlativity that ELISA and HPLC method detect content of melamine in the feed sample.
Embodiment
It is in order further to understand the present invention better that following embodiment is provided, and never content of the present invention and protection domain is constituted any restriction.
The immunogenic synthetic preparation that reaches immune serum of embodiment 1 melamine
1.1 reagent and instrument
Melamine (Melamine, purity 99.5%), horseradish peroxidase (HRP), bovine serum albumin(BSA) (BSA) and ovalbumin (OVA) are the Sigma product; SephacrylTM S-200High Resolution is available from GE HealthcareBio-Sciences AB company; Other reagent are homemade analysis pure chemistry reagent.
Gel imaging analysis system (GeneGenius), UV, visible light sub-ray spectrometer (Shimadzu UV-2501PC), FTIS (Bruker Tensor 27), ultrapure water system (Millipore), vertical electrophoresis system (Bio-Rad), full-automatic protein chromatographic system (Amersham) and sheeter (Shimadzu) etc.
1.2 artificial antigen of melamine is synthetic
Taking by weighing the 12mg melamine is dissolved in the pyridine of 2mL and prepares melamine solution; Taking by weighing 20mg BSA is dissolved in the PBS damping fluid of 2mL and with it and adds in the above-mentioned melamine solution; In solution, slowly splash into 1mL 0.3% glutaraldehyde solution again; Stir 4h down in room temperature condition, the glycocoll 750 μ L that add 1mol/L then are with cessation reaction.The melamine formaldehyde resin method is synthesized coating antigen (MEL-OVA): 37% the formaldehyde of melamine (MEL), 0.082mL that takes by weighing 0.0126g is in covering test tube; The PBS damping fluid that adds 5mL; Insert slowly heating in 65 ℃ water-bath in the three-neck flask again; When treating that MEL all dissolves, using triethylamine to transfer pH is 8.5～9.0.Behind the 30min, add the sodium chloroacetate (being dissolved in 1mL PBS damping fluid in advance) of 0.012g, continue reaction 9h in 65 ℃ of water-baths.After reaction finishes, add the NHS (N-hydroxyl succinamide) of 48mg and the DDC (N, N-dicyclohexylcarbodiimide) of 62mg, this mixed liquor is designated as A liquid.The A drop is added pre-configured B liquid (OVA of 30mg is dissolved in the PBS of 3mL), and room temperature (RT) stirs 3h down.Above product is respectively charged in the bag filter, and to PBS damping fluid 1000mL dialysis 72h ,-20 ℃ frozen subsequent use under 4 ℃ of conditions.Select Sephacryl TMS-200 High Resolution filler dress post, balance, get synthetic immunogene and join chromatographic purifying in the good chromatographic column of balance, eluent is pH 7.4 0.01M PBS, and it is 0.5mL/min that flow velocity is set, 280nm place ultraviolet detection.
The preparation of immunity and specific polyclonal antibody
Above-mentioned immunogene melamine-BSA conjugate is diluted to the 1mg/ml solution for standby with physiological saline.
Choose 6 body weight 2～2.5Kg healthy male new zealand white rabbits.Immunogene melamine-BSA conjugate and equivalent Freund's complete adjuvant are mixed into water in oil emulsion through syringe to the method for taking out, carry out first immunisation, take the subcutaneous multi-point injection in back by the amount of 1mg/Kg body weight.Whenever once, replace Freund's complete adjuvant with incomplete Freund, the same first immunisation of dosage and method at a distance from two all booster immunizations.From immunity beginning for the third time, back 10 days of each immunity, auricular vein is got blood 1ml, carries out antibody titer and detects; When antibody titer no longer raises, do not add adjuvant and carry out for the last time (the 7th time) immunity, leg muscle injection, rear neck artery bloodletting in 7 days; Room temperature is solidified behind the 2h 4 ℃ and is spent the night, and centrifugal 10 minutes of 8000r/min removes clot, and serum partly precipitates with 50% saturated ammonium sulfate solution; The centrifugal supernatant that goes, precipitate resuspended with phosphate buffer, again with twice of 33% saturated ammonium sulfate solution deposition; Sediment is used the SephadexG-25M chromatography with the least possible phosphate buffer dissolving after dialysis, promptly get the melamine polyclonal antibody.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of melamine
Set up the enzyme linked immunological kit that detects melamine, make it comprise following component:
(1) encapsulates the ELISA Plate of the melamine antibody described in the embodiment 1;
(2) melamine horseradish peroxidase-labeled thing freeze-dried powder;
(3) the melamine standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L, 81 μ g/L;
(4) developer, described developer is mixed by 5: 5: 1 (volume ratio) by hydrogen peroxide or urea peroxide, o-phenylenediamine or tetramethyl benzidine and stabilizing agent K;
(5) enzyme mark thing dilution is the phosphate buffer that contains 10% enzyme stabilizers AH and 5% enzymatic protective reagent KEN;
(6) cleansing solution is the phosphate buffer that contains 0.05% polysorbas20;
(7) sample diluting liquid is the phosphate buffer of 0.1% Tween-20;
(8) stop buffer is the hydrochloric acid solution of 2mol/L.
This kit reaches wherein, and each component is used for following embodiment.
The foundation of embodiment 3 immunologic detection methods
2.1ELISA the square formation method is confirmed optimum response concentration
The melamine antibody of 3.0 μ g/ml, 2.4 μ g/ml, 1.8 μ g/ml, 1.2 μ g/ml, 0.6 μ g/ml series concentration is pressed every hole 100 μ l coated elisa plates; 4 ℃ encapsulate and spend the night; Wash 3 times, clap and do, seal down for 4 ℃ by every hole 200 μ l confining liquids (present embodiment is with the phosphate buffer that contains 1%OVA) and spend the night; Wash 3 times, clap and do.Adding is since the melamine horseradish peroxidase mark thing 50 μ L/ holes of 1: 4000 doubling dilution, and room temperature effect 30 minutes is washed three times, adds 100 μ l developers, room temperature effect 10min, and 100 μ l stop buffer cessation reactions, ELIASA detects A value (450nm).Be provided with simultaneously 6 parallel, getting the encapsulate concentration of OD value when being 2.0 left and right sides is optium concentration.Test figure is listed in table 1.
Definite (n=6) of table 1 melamine polyclonal antibody and enzyme mark thing best effort concentration
By confirming in the data of table 1 that it is 1.8 μ g/ml that optimum antibody encapsulates concentration, the enzyme-labelled antigen extension rate reaches optimum response concentration at 1: 16000.
2.2 ELISA detects antibody I C
50Value
The ELISA method of operating is with 2.1, and it is 1.8 μ g/ml that optimum antibody encapsulates concentration, melamine horseradish peroxidase mark thing extension rate 1: 16000; The free melamine standard items of preparation 0,1,3,9,27,81ng/mL series concentration, every hole adds 50 μ l standard items, adds 50 μ L melamine horseradish peroxidase-labeled thing working fluids subsequently; Room temperature reaction 30 minutes is washed plate 3 times, adds the developer incubation 10 minutes; Cessation reaction, the ELIASA reading.The logarithm that with the combination rate is ordinate, melamine concentration is a horizontal ordinate, and the typical curve of melamine series standard article concentration 0,1,3,9,27,81ng/mL is typical S type, and is as shown in Figure 1.In the 1-81ng/mL concentration range, combination rate and concentration are linear, and equation of linear regression is y=-0.5092x+0.8822, R
2=0.9922.Calculate definite through inhibiting rate: 50% inhibiting rate IC
50=10.15ng/mL, the detection sensitivity IC of method
10=1.23ng/mL shows that the antiserum that the present invention prepares is to have specific preferably anti-melamine polyclonal antibody, can satisfy melamine and detect requirement.
The test of experimental example 4 kit precision
This experimental example is the repeatable test of standard.Concrete operations are: through the kit to same production batch and three production batch carry out in the plate with plate between coefficient of variation analysis confirm: mark 96 orifice plates, same batch of different enzyme at same enzyme and mark the different enzymes of 96 orifice plates and mark and do 6 groups of parallel typical curves on 96 orifice plates with different batches; The OD value of under 450nm, surveying is according to the data computation coefficient of variation of being measured.The result shows that plate within variance coefficient (CV%) is between 2.7-7.9%, and average coefficient of variation is 5.4%, and the coefficient of variation between plate (CV%) is between 5.7-10.2%, and average coefficient of variation is 7.9% (table 2).The accuracy that this method is described is higher, can satisfy content of melamine analysis requirement in the food.
Interior and the interassay coefficient of variation of table 2 plate
The recovery test of experimental example 5 kits
Getting concentration is the melamine standard specimen of three concentration of 50-500 μ g/kg (μ g/L), and liquid towards milk, milk powder and feed sample add recovery test, each concentration do 6 parallel, calculate recovery rate respectively.Identical among detection method and the embodiment 3.The result shows (table 3), and the sample of melamine in milk, milk powder and feed adds the recovery 83%～105%, and the recovery is good, and the coefficient of variation is 4%～9.5%, and method is reliable.The kit that this research development is described can be used for detecting the melamine residual in milk, milk powder and the feed.
The sample of table 3 melamine in milk, milk powder and feed adds recovery test (n=6)
The test of experimental example 6 kit storage lives
Kit accelerated test (table 4) shows, 4 ℃ with-20 ℃ of conditions under preserved the B that melamine ELISA indirect competition method detects 210 days
0Be worth constant basically; 37 ℃ of holding times prolong, and influence becomes big, slope of standard curve, IC to typical curve
50Variation.Current test findings shows under 4 ℃ of conditions can preserve 6 months at least.
Table 4 kit stability test (n=3)
Embodiment 7 HPLC compare test
Through the negative sample (milk, milk powder and feed) that detects through HPLC is carried out the artificial contamination, detect comparison with melamine single stage method enzyme linked immunological kit of the present invention and GB HPLC method again.
With reference to national standard " melamine detection method in raw milk and the dairy products " (GB/T22388-2008), " melamine fast detecting liquid phase chromatography in the raw milk " (GB/T 22400-2008) and agricultural industry criteria " mensuration of melamine in the feed " (NY/T 1372-2007) set up the HPLC detection method of melamine; In 0.016-2.56 μ g/mL scope, measure; With the sample concentration is horizontal ordinate; Front cover is long-pending to be ordinate, and the drawing standard curve has good linear relationship; Linear equation is Y=85.107X-0.9714, R
2=0.99998, retention time t
RBe 6.6662 ± 0.0188min, lowest detection is limited to 50.2 μ g/kg.On this basis; The milk, milk powder and the feed sample that have added 50,100,200,400 μ g/kg melamine standard items adopt ELISA and HPLC method to detect respectively; Result (Fig. 3-5) shows, the related coefficient (R of 2 kinds of methods when being used for milk, milk powder and the detection of feed sample melamine
2) be respectively 0.869,0.895 and 0.820, show that ELISA method and HPLC method that this research is set up have good compatibility, can be used for the examination of a large amount of samples that melamine is analyzed in milk, milk powder and the feed sample.
Claims (10)
1. the single stage method enzyme-linked immunologic detecting kit of a fast detecting melamine, comprising: melamine specific antibody, melamine standard items and melamine enzyme labeling thing.
2. the single stage method enzyme-linked immunologic detecting kit of fast detecting melamine according to claim 1 is characterized in that: said kit also comprises ELISA Plate, melamine horseradish peroxidase-labeled thing working fluid, melamine standard solution, developer, enzyme mark thing dilution, cleansing solution, stop buffer, the concentrating sample dilution that is coated with the melamine specific antibody.
3. the single stage method enzyme-linked immunologic detecting kit of fast detecting melamine according to claim 1 and 2 is characterized in that: said melamine specific antibody is a polyclonal antibody, the polyclonal antibody of preferred rabbit anti-melamine.
4. the single stage method enzyme-linked immunologic detecting kit of fast detecting melamine according to claim 1 and 2 is characterized in that: the used enzyme of described melamine enzyme labeling thing is a horseradish peroxidase.
5. the single stage method enzyme-linked immunologic detecting kit of fast detecting melamine according to claim 2 is characterized in that: described cleansing solution is for containing 0.05%-0.5% Tween-20 phosphate buffer, and described stop buffer is the hydrochloric acid solution of 2mol/L.
6. the single stage method enzyme-linked immunologic detecting kit of fast detecting melamine according to claim 2; It is characterized in that: described developer is mixed by 5: 5: 1 volume ratio by hydrogen peroxide or urea peroxide, o-phenylenediamine or tetramethyl benzidine and stabilizing agent K, can under 4 ℃, can preserve nondiscolouring in 2 years.
7. the single stage method enzyme-linked immunologic detecting kit of fast detecting melamine according to claim 2 is characterized in that: described concentrating sample dilution is the phosphate buffer that contains 0.1% Tween-20.
8. the single stage method enzyme-linked immunologic detecting kit of fast detecting melamine according to claim 2 is characterized in that: described enzyme mark thing dilution is the phosphate buffer that contains enzyme stabilizers AH and enzymatic protective reagent KEN; Described melamine enzyme labeling thing is a melamine horseradish peroxidase-labeled thing freeze-dried powder, and being dissolved as working fluid with enzyme mark thing dilution during use can directly can use.
9. the single stage method enzyme-linked immune detection method of content of melamine in the fast detecting sample comprises step:
(1) sample pre-treatments: the liquid milk sample, get 1.00mL in the Eppendorf centrifuge tube, get middle clear liquid behind 4 ℃ of centrifugal 5min of 12000rpm, directly be used for subsequent experimental, extension rate is 1; The milk powder sample thief accurately takes by weighing 2.00g in the 50mL centrifuge tube, gets 10mL PBS and joins in the milk powder, and 40 ℃ of water-baths are heated to milk powder and dissolve fully, gets middle clear liquid behind 4 ℃ of centrifugal 10min of 4000rpm, directly is used for subsequent experimental, and extension rate is 5; Dairy produce and feed sample accurately take by weighing 2.00g in the 50mL centrifuge tube after the pulverizing, add 10mL 0.1M HCl; Ultrasonic degradation sample 25min after the violent vortex oscillation, the centrifugal 10min of room temperature 5000rpm is with about 1M NaOH adjustment pH value to 8.0; Centrifugal 5min of room temperature 5000rpm or 0.4 μ m filtrator are filtered in the clean pipe; Supernatant or filtered fluid are used for subsequent experimental after further diluting by 1: 1 with PBS, and extension rate is 10;
(2) the single stage method enzyme-linked immunologic detecting kit with the arbitrary described fast detecting melamine of claim 1-9 detects; In the ELISA Plate hole that is coated with melamine antibody, add standard items or sample solution; Add melamine horseradish peroxidase-labeled thing working fluid again, after the time of middle explanation, temperature and the operation notice operation, develop the color, stop to specifications; Measure absorbance with ELIASA, (3) analyzing and testing result.
10. the single stage method enzyme-linked immune detection method of content of melamine in the fast detecting sample as claimed in claim 9; Wherein (2) for melamine antibody that coating buffer is diluted to 1.8 μ g/mL concentration by every hole 100 μ L coated elisa plates; 4 ℃ encapsulate and spend the night, and cleansing solution washing 3 times is clapped and done; Spend the night by 4 ℃ of following sealings of every hole 200 μ L confining liquids, wash 3 times, clap dried subsequent use.Every hole, standard items hole adds the melamine standard items (0,1,3,9,27,81ng/mL) of 50 μ L series concentration; The every hole of sample well adds the good sample of 50 μ L dilution to be measured; Every hole adds the melamine horseradish peroxidase mark thing of 50 μ L dilution in 1: 16000; Room temperature effect 30min washs 3 times, claps and does; Every hole adds 100 μ l developers, room temperature effect 10min, and every hole adds 100 μ L stop buffer cessation reactions, and ELIASA detects A value (450nm).The logarithm that with the combination rate is ordinate, melamine standard items concentration is that horizontal ordinate is set up typical curve, according to typical curve calculate and multiply by its corresponding extension rate get final product the content of melamine in the sample.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103145633A (en) * | 2013-02-06 | 2013-06-12 | 天津科技大学 | Novel melamine antigens and antibody as well as application |
| CN103197058A (en) * | 2013-03-01 | 2013-07-10 | 杭州迪恩科技有限公司 | Kit of rapid detection of nitrofurazone by using one-step enzyme-linked immunosorbent assay technique |
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| CN113447646A (en) * | 2021-06-22 | 2021-09-28 | 浙江理工大学 | By using C-SiO2Method for accelerating ELISA detection process by using adsorbing material |
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| CN103145633B (en) * | 2013-02-06 | 2015-12-02 | 天津科技大学 | Novel melamine antigen and antibody and application |
| CN103197058A (en) * | 2013-03-01 | 2013-07-10 | 杭州迪恩科技有限公司 | Kit of rapid detection of nitrofurazone by using one-step enzyme-linked immunosorbent assay technique |
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| CN113495152A (en) * | 2020-03-20 | 2021-10-12 | 郑州达诺生物技术有限公司 | Inhibin B quantitative detection kit and use method thereof |
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| CN113447646A (en) * | 2021-06-22 | 2021-09-28 | 浙江理工大学 | By using C-SiO2Method for accelerating ELISA detection process by using adsorbing material |
| CN113447646B (en) * | 2021-06-22 | 2024-03-19 | 浙江理工大学 | By using C-SiO 2 Method for accelerating ELISA detection process by adsorption material |
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