A kind of melamine rapid detection method based on latex and kitTechnical field
The present invention relates to the interdisciplinary field of immunology, medical science, biology and food security, more specifically relate to a kind of method detecting melamine based on polystyrene latex microspheres.
Background technology
In food service industry, by triumphant formula nitriding (GB/T5009.5-2010; First method) measure nitrogen atom content thus indirect calculation protein content.In recent years, industrial chemicals melamine adds in food or feed by some illegal retailers, in the hope of improving the apparent protein content measured by triumphant formula nitriding.Melamine can cause human body urinary system to produce calculus, as the disease such as vesical calculus and kidney stone, is therefore strictly prohibited and adds in food.
The detection method of current melamine has high performance liquid chromatography, LC-MS, gas chromatography mass spectrometry method etc., although these method detection sensitivities are high, but the pre-treatment step running time is long, instrument price is expensive, and need sample and send laboratory back to and detect, be not suitable for the on-site quick screening of melamine in sample.Can be used for the method for field quick detection at present as enzyme linked immunosorbent assay, should use more flexible in comparison, but testing cost is still very expensive, also certain technical requirement is had to technical operation human users, although and colloid gold card operates very easy but can only be qualitative analysis, quantitative test accurately can not be realized.
Immunoturbidimetry (J.M.Singeretal., Am.J.Med.21,888-92; 1956) detection and the clinical diagnosis of microorganism and virus infections etc. is mainly used in.In immunoturbidimetry, antibody is coated on microsphere surface.When the sample comprising specific antigen mixes with latex suspension, cause visible aggegation, add the turbidity of sample.By measuring sample and antibody bag by the reacted absorbance of latex suspension, and compare with the typical curve that the antigen of use concentration known is drawn, thus the concentration of antigen in working sample.
The microballoon used in immunoturbidimetry comprises polytype, such as glass, silica, nano metal, polystyrene, polyacrylamide particle etc.By passive adsorption or chemical covalent coupling, antibody is connected on microballoon by the modes such as such as carbodiimides (EDC) method, bromination coupling.Usually need in the method for latex microsphere pan coating antibody the functional group first activating latex microsphere surface, can react with antibody protein.Such as, usually use EDC to activate the carboxyl on latex microsphere surface, make the amino coupled of itself and protein.
It has been generally acknowledged that the interaction by using the activation method of carbodiimides (EDC)/N-hydroxy thiosuccinimide (Sulfo-NHS) to be more conducive between protein and microballoon, is therefore the coupling method commonly used the most.In addition, the activation method of EDC/NHS (N-hydroxy-succinamide) potpourri is used in addition.But successively will use two kinds of reagent in these two kinds of methods, reactive system and method are comparatively complicated, very easily cause microballoon generation aggegation, be unfavorable for that antibody bag is by the suitability for industrialized production of microballoon.
In contrast to this, the step being used alone the activation method (or being called a step EDC method) of EDC is comparatively simple and productive rate is higher.But, a step EDC fado be used for not wrapping carboxylic part (such as oligonucleotides, as DNA) and surface with carboxyl microballoon between coupling.If treat that the part of coupling also comprises carboxylic group (such as protein, as antibody), it may be activated by EDC, causes the polymerization between part, thus cannot obtain the product of expectation.
Therefore, need to develop one and avoid mutual aggegation generation precipitation between microballoon or antibody, thus be applicable to steady production antibody bag by the production technology of microballoon, thus acquisition can utilize quick, the accurate melamine detected in sample of immunoturbidimetry.
Summary of the invention
At present, in EDC activation method, use 2-(N-morpholine) ethyl sulfonic acid (MES) as activation buffering agent, but the present inventor find to use the EDC solution of MES buffering cannot prepare bag by the microspheres solution of melamine antibody through experiment more.The present inventor shows through great many of experiments, in latex system, very easily makes the mutual aggegation of the microballoon of band carboxyl produce precipitation after adding activating solution, keeps latex state then to need to improve the composition of activation buffer.Therefore, the present inventor has carried out large quantity research to EDC activation method, found that the phosphate buffer comprising polysorbas20 can provide the environmental optima being beneficial to coupling between antibody and microballoon, the mutual aggegation of microballoon can be avoided to produce precipitation, be applicable to steady production antibody bag by microballoon, thus complete the present invention.
In first aspect, the invention provides a kind of bag by the preparation method of the latex microsphere solution of melamine antibody, said method comprising the steps of: surface is diluted in activation buffer with the latex microsphere of carboxyl, wherein, described activation buffer is the 10 ~ 40mM phosphate buffer comprising 0.5 ~ 1.0g/L polysorbas20, pH7.4-7.8.
In preferred embodiments, preparation method of the present invention uses a step EDC activation method to activate the carboxyl on described latex microsphere surface.In the present invention, term " a step EDC activation method " refers to the EDC activation method not using other activators except carbodiimides in reactivation process.Described " other activators " comprises the reagent of such as Sulfo-NHS, NHS.
In an embodiment, preparation method of the present invention comprises the following steps:
1) be diluted in activation buffer by surface with the latex microsphere of carboxyl, wherein, described activation buffer is the 10 ~ 40mM phosphate buffer comprising 0.5 ~ 1.0g/L polysorbas20, pH7.4-7.8.
2) add activator mix reaction, wherein said activator is carbodiimides;
3) add melamine monoclonal antibody, carry out wrapping and reacted;
4) cancellation liquid cessation reaction is added;
5) add confining liquid to close free carboxyl, thus obtain bag by the latex microsphere solution of melamine antibody.
In the present invention, described latex microsphere can be any latex microsphere being generally used for immune detection, such as polystyrene microsphere, polyacrylamide microsphere etc.There will be a known the latex microsphere that a lot of manufacturer provides various different model, such as MicroparticlesGmbH, PolyMicrospheresInc. etc.Those skilled in the art can select the surface that is applicable to the latex microsphere of carboxyl according to specific needs.
In the present invention, preferably polystyrene latex microspheres is used.The particle diameter of latex microsphere of the present invention can between 100-200nm, preferred 130-170nm.In the present invention, use surface with the latex microsphere of carboxyl, thus be connected with melamine monoclonal antibody by Carbodiimide reaction.The carboxyl-content of described latex microsphere is 100-200 μ eq/g, preferably 160 ~ 170 μ eq/g.
In the present invention, the addition of carbodiimides can be determined according to specific needs by those skilled in the art.Such as, the carbodiimides of carboxyl quantity 15%-30% can be added.
The melamine monoclonal antibody that the present invention uses can be prepared by methods known in the art, such as hybridoma technology etc.; Also can pass through commercially available, such as, purchased from Beijing Bo Ao biotechnology Ltd (model bsm-2182M) etc.
In the present invention, the ratio between latex microsphere and melamine monoclonal antibody can be determined according to specific needs by those skilled in the art.Ratio between latex microsphere and melamine monoclonal antibody usually between 5:1 to 10:1, preferred 10:1.The antibody added is too much or very fewly all easily cause latex microsphere generation aggegation.
The cancellation liquid that the present invention uses can be any reagent being generally used for stopping carbodiimides coupled reaction, such as 60-80g/L glycocoll, preferred 75g/L glycocoll.
The cancellation liquid that the present invention uses can be any reagent being generally used for the carboxyl that closed latex microsphere dissociates, such as seralbumin, as 180-220g/L bovine serum albumin(BSA), and preferred 200g/L bovine serum albumin(BSA).
The bag prepared by the method for the invention can be now with the current by the latex microsphere solution of melamine antibody.In use, by centrifugal segregation supernatant, use redissolution liquid cleaning latex microsphere, and be settled to the concentration of specifying.Described redissolution liquid can comprise 8-15g/L glycocoll, 0.5-1.5g/L Sodium azide, 8-13g/L sorbierite, 3-8g/L bovine serum albumin(BSA) and 1.5-2.5g/LEDTANa2, pH7.8-8.2.
Preferably, described redissolution liquid comprises 11.26g/L glycocoll, 1.0g/L Sodium azide, 10.0g/L sorbierite, 5.0g/L bovine serum albumin(BSA) and 2.0g/LEDTANa2, pH8.0.
Also the bag prepared according to the method for the invention can be redissolved in described redissolution liquid by the latex microsphere of melamine antibody, and under 2 ~ 8 ° of C Cord blood.
In one embodiment, preparation method of the present invention comprises the following steps:
1) use activation buffer by particle diameter 130-170nm, carboxyl-content is the latex solution of the polystyrene latex microspheres dilution 1% of 160 ~ 170 μ eq/g, wherein said activation buffer comprises 10 ~ 40mM phosphate buffer of 0.5 ~ 1g/L polysorbas20, pH7.4-7.8;
2) add the carbodiimides of carboxyl quantity 15%-30%, after mixing, react 5min;
3) ratio being 10:1 with latex microsphere and melamine monoclonal antibody adds melamine monoclonal antibody, and reacts 2-3 hour at 37 ° of C, 300rpm;
4) add 75g/L glycocoll, stop bag and reacted;
5) add 200g/L bovine serum albumin(BSA), react 30-60min at 37 ° of C, 300rpm, thus close free carboxyl;
6) centrifugal segregation supernatant, use the cleaning of redissolution liquid, wherein said redissolution liquid comprises 11.26g/L glycocoll, 1.0g/L Sodium azide, 10.0g/L sorbierite, 5.0g/L bovine serum albumin(BSA) and 2.0g/LEDTANa2, pH8.0-8.2;
7) repeat step 6) three times, using described redissolution liquid to be settled to latex concentration is subsequently 0.1%, and under 2-8 ° of C Cord blood.
Compared with cushioning activating solution with conventional MES, the activation buffer that the present invention adopts provides coupling is carried out on melamine monoclonal antibody and surface the suitableeest ion concentration and pH with the latex microsphere of carboxyl, avoids, after adding melamine antibody, disadvantageous agglutination phenomenon occurs.
The redissolution liquid that the present invention adopts provides and is conducive to bag by the suitableeest ion concentration of the latex microsphere solution remained stable of melamine antibody and pH.Use described redissolution liquid bag can be kept more than 1 year at normal temperatures by the latex microsphere solution of melamine antibody, avoid antibody to come off simultaneously.
In second aspect, the invention provides described activation buffer for the preparation of bag by the purposes of the latex microsphere solution of melamine antibody.
In the third aspect, the invention provides a kind of kit for detecting melamine in sample, described kit comprises following component:
1) R1 reagent: the phosphate buffer comprising PEG6000 and polysorbas20, pH7.0-7.6;
2) R2 reagent: wrap by the latex microsphere solution of melamine antibody; With
3) melamine standard items.
Described R1 reagent is the buffering agent being generally used for immunoturbidimetry.Those skilled in the art can prepare applicable buffering agent as required.In the present invention, the phosphate buffer comprising PEG6000 and polysorbas20 can be used, such as, comprise the 40mM phosphate buffer of 3%PEG6000 and 0.2% polysorbas20, pH7.0-7.6, preferred pH7.4.When needs are preserved for a long time, also optionally add 0.1% Sodium azide to be used as antiseptic.
Described R2 reagent is that bag is by the latex microsphere solution of melamine antibody.Described melamine antibody and latex microsphere are as described in the first aspect of the invention.Preferably, described R2 reagent be according to a first aspect of the present invention the bag prepared of described preparation method by the latex microsphere solution of melamine antibody.The concentration of described latex microsphere solution can be 1% (10 times of concentrates) or 0.1%.
Described kit also optionally comprises the instructions using described reagent to detect melamine in sample.
Compared with the method for conventional sense melamine, kit of the present invention is simple to operate, cost is low, detection speed is fast, and can quantitatively analyze.
Described kit can be used for the concentration detecting melamine in sample.Therefore, in the third aspect, the invention provides described kit for detecting the application of melamine concentration in sample.Described sample can be food, particularly dairy products, as fresh milk, milk powder, cheese, and cream and Yoghourt.
Fourth aspect, the invention provides a kind of immunoturbidimetry method for detecting melamine concentration in sample, it comprises the following steps:
1) by described sample and the phosphate buffer (pH7.0-7.6) comprising PEG6000 and polysorbas20, mixing incubation;
2) bag is added by the latex microsphere solution of melamine antibody;
3) the gained potpourri absorbance difference Δ A of any two time points between 10s to 5 minute after adding described latex microsphere solution is measured540;
4) according to the melamine concentration in the typical curve determination sample using melamine standard items to draw.
The method of the typical curve using melamine standard items to draw is known in the art.Usually, melamine standard items known for concentration and the buffering agent (such as, R1 reagent of the present invention) be applicable to are mixed incubation, subsequently, add bag by the latex microsphere solution of melamine antibody, incubation 5 minutes after mixing, and according to the change drawing standard curve of absorbance.
The present invention compared with prior art, has the following advantages and effect:
Melamine detection kit of the present invention and method thereof, by adding R1, R2 two kinds of reagent, the concentration of quantitative test sample melamine according to absorbance change before and after reaction.
The all preparation of reagents of the present invention are simple, processing ease, conjugate difficult drop-off in R2 reagent, and stability is very strong, and reagent cost of manufacture is low, consuming time short, can produce in a large number within a short period of time.Not high to the technical requirement of technician detected, detection speed fast, detect overall process only needs 7 minutes, in addition, do not rely on special instrument, can really realize detecting real-time.
Accompanying drawing explanation
Fig. 1 is the response curve figure of melamine standard items absorbance change in course of reaction of display variable concentrations, and wherein horizontal ordinate is the reaction time, and ordinate is absorbance change.
Fig. 2 is the typical curve recorded under different time, and wherein horizontal ordinate is reactant concentration (ppb), and ordinate is absorbance change.
Fig. 3 is the absorbance difference drawing standard curve according to variable concentrations standard items, and wherein horizontal ordinate is reactant concentration (ppb), and ordinate is absorbance change.
Fig. 4 is the comparing result of exhibit stabilization experiment, and wherein, horizontal ordinate is reaction time (min), and ordinate is absorbance change.Detectable concentration is 27ppb, and the time interval is 7 days, preserves under 37 degree of conditions.
Embodiment
The present invention is further illustrated by the following examples, but the present invention is not limited to these embodiments.
Embodiment 1: the preparation of buffering agent R1
The PBS preparation of 40mM, PH7.4: 1. take sodium dihydrogen phosphate 0.0624g, with ultrapure water constant volume 100ml; 2. sodium hydrogen phosphate 7.16g is taken, with ultrapure water constant volume 500ml; 3. take sodium chloride 0.14g, add 1. reagent 3.8ml and 2. reagent 16.2ml.
This is the phosphate buffer (PBS) of 40mM, PH7.4.
The preparation of R1:
PEG60000.3g
Sodium azide 0.01g
Tween-20 20ul
After weighing good various chemicals, be settled to 10ml with the PBS of 40mM, PH7.4.
Because PEG6000 does not dissolve at normal temperatures, therefore this reagent is placed in 60 degree of water-baths and heat, and constantly stir mixing.Reagent after dissolving is colourless transparent liquid.
Preserve in 2-8 ° of C refrigerator after preparing reagent, the pot-life is 12 months.
Embodiment 2: wrap by the reagent of the latex microsphere of melamine antibody for the preparation of with preservation
Activation buffer:
The preparation of the PBS (PH7.4) of 10mM: the PBS of 40mM embodiment 1 prepared dilutes four times, subsequently, adds the Tween-20 of 0.70g/L in gained 10mMPBS.
Redissolution liquid:
Glycocoll 11.3g/L
Sodium azide 1.0g/L
Sorbierite 10.0g/L
BSA5.0g/L
EDTA·Na22.0g/L
First weighing various solid chemical compound, then use ultrapure water constant volume, for increasing dissolution velocity, 37 degree of incubators can be placed in.Finally, hydro-oxidation sodium solution (20%) adjusts PH8.00 ± 0.08.The reagent prepared is the liquid that achromaticity and clarification is transparent.The redissolution liquid prepared can be kept in 2-8 ° of C refrigerator, and the pot-life is 12 months.
Cancellation liquid:
Glycocoll 75g/L
After taking glycocoll, use ultrapure water constant volume.After mixing, this solution is colourless transparent liquid.Preserve in 2-8 degree refrigerator after preparing reagent.Pot-life is 60 days.
Confining liquid:
BSA200g/L
Weigh BSA solid, use ultrapure water constant volume, place in 60 degree of water-baths, limit heating edge shake bottle, accelerate dissolution.BSA solution after dissolving is flaxen liquid.The confining liquid prepared can be kept in 2-8 ° of C refrigerator, and the pot-life is 60 days.
Embodiment 3: wrap by the preparation technology of the latex microsphere of melamine antibody
Reaction system is 100ul.
Activation
Get 1.5ml centrifuge tube, add activation buffer 70ul, polystyrene latex microspheres (the purchased from American PolyMicrospheres company of 10ul is got with pipettor, particle size 130-170nm, carboxyl-content 160-170 μ eq/g) in this centrifuge tube, continuous piping and druming, makes latex particle be distributed in activation buffer uniformly, thus obtains white emulsion.
Weigh solid carbodiimide hydrochloride (EDCA; Purchased from Shanghai covalent chemical Science and Technology Ltd.) be dissolved in activation buffer, concentration is 0.001mg/ul.In above-mentioned latex particle damping fluid, add 10ulEDCA solution, activate 5min at normal temperatures.The latex system activated is still white emulsion, but viscosity declines to some extent.
Coupling
The latex system activated is added melamine monoclonal antibody (purchased from Beijing Bo Aosen biotechnology Ltd, model bsm-2182M), addition is 10ul(10mg/ml), rapidly with the piping and druming of rifle head, should mix fast at short notice during interpolation as far as possible.White emulsion is obtained after mixing.The constant temperature oscillator latex system being added with melamine antibody being placed in 37 ° of C, 300rpm reacts 2 hours.After completing, system is still white emulsion.
Cancellation
Now melamine latex conjugate is formed, adds cancellation liquid 80ul in system, with rifle head piping and druming mixing, in 37 degree, react 30min in 300rpm constant temperature oscillator.After terminating, system answers deposit-free.
Close
Add confining liquid 17ul, with rifle head piping and druming mixing, be positioned over 30 degree, in 300rpm constant temperature oscillation case, off-period is 45min.Close after terminating, this system is white emulsion, and deposit-free.
Purifying
Centrifuge tube is put into high-speed refrigerated centrifuge, 45000g, 8min centrifugal (note: hydro-extractor should be pre-chilled to 10 degree before centrifugation prevents from producing higher temperature during high speed centrifugation), after centrifugal, white latex microsphere major part is all deposited in bottom centrifuge tube, containing minute quantity microballoon in supernatant, but still seem water white transparency, outwell supernatant, or with rifle head, supernatant is taken out, add 100ul and redissolve liquid, with rifle head piping and druming mixing, then be placed in ultrasonic washing instrument microballoon is disperseed.Ultrasonic washing instrument power 99%, time 1min.Repeat this step 3 time.Add redissolution liquid 1ml for the last time.
Now solution is white emulsion, does not have without granular precipitate after testing in system.This is R2 reagent, and absorbance is about 0.2-0.9, as absorbance is too high, can again dilute.
Preserve
This reagent should be placed in 2-8 ° of C refrigerator sealing and preserve, and the pot-life is 12 months.
Embodiment 4: different EDC activation method is on the impact of latex system stability
In the present embodiment, adopt the activation method of conventional EDC+NHS to carry out the activation of latex microsphere, and compare with activation method of the present invention.Concrete steps are as follows:
First, EDC is used to activate latex microsphere according to the description of embodiment 3:
Polystyrene latex microspheres (the purchased from American PolyMicrospheres company of 10ul is got with pipettor, particle size 130-170nm, carboxyl-content 160-170 μ eq/g) in centrifuge tube, continuous piping and druming, latex particle is made to be distributed in activation buffer of the present invention uniformly, to final concentration 1%.
Weighing solid EDCA (purchased from Shanghai covalent chemical Science and Technology Ltd.) is dissolved in activation buffer of the present invention, and concentration is 0.001mg/ul.In above-mentioned latex particle damping fluid, add the above-mentioned EDCA solution of 10ul, vibration mixing, reaction 5min, surveys light absorption value 1.
Subsequently, gained latex microsphere is divided into two parts, and a copy of it does not add NHS, and another part adds NHS, and consumption is about the 2-3 of EDC consumption doubly, after mixing, measures the light absorption value 2 of two increment product.
Table 1: Different Activation Methods is on the impact of latex system stability
| Activator | Absorbance 1 | Absorbance 2 | ΔA |
| EDC | 2.45 | 2.47 | 0.02 |
| EDC+NHS | 2.46 | 3.57 | 1.11 |
Note: Δ A is absorbance 2-absorbance 1.
Use EDC+NHS activation method, latex system before activation rear absorbance changes greatly, and about 1.11, this illustrates this system generation coagulation sedimentation.
And being used alone EDC, latex system before activation rear absorbance is almost unchanged, and this system is more stable, does not produce agglutination phenomenon.
Embodiment 5: different activation buffer system is on the impact of latex system stability
2-(N-morpholine) ethyl sulfonic acid (MES) solution of the most frequently used buffer system of current EDC cross-linking reaction to be ion concentration be 40mM, PH is 5-6.Compared with cushioning activating solution with conventional MES, the activating solution that the present invention adopts provides coupling is carried out on melamine monoclonal antibody and surface the suitableeest ion concentration and pH with the latex microsphere of carboxyl, avoids, after adding melamine antibody, disadvantageous agglutination phenomenon occurs.
use MES activation buffer activation latex microsphere
Get 1.5ml centrifuge tube, add MES activation buffer 70ul.Polystyrene latex microspheres (the purchased from American PolyMicrospheres company of 10ul is got with pipettor, particle size 130-170nm, carboxyl-content 160-170 μ eq/g) in this centrifuge tube, constantly blow and beat, latex particle is distributed in MES activation buffer uniformly.
Weighing solid EDCA (purchased from Shanghai covalent chemical Science and Technology Ltd.) is dissolved in MES activation buffer, and concentration is 0.001mg/ul.In above-mentioned latex particle damping fluid, add the above-mentioned EDCA solution of 10ul, immediately with the piping and druming of rifle head, slightly a moment, observe latex system.
Found that, after the activator adding MES buffering, occur a small amount of flocky precipitate immediately in latex system, after about 30 seconds, occur a large amount of flocculent deposit, placed 5-6 as a child, this system layering.
To this, have detected and use MES to cushion activation buffer described in activating solution and embodiment 2, latex system is adding the change of absorbance before and after activator EDC.Result is as shown in the table:
Table 2: different activation buffer is on the impact of latex system stability
| Activating solution composition | Absorbance 1 | Absorbance 2 | ΔA |
| The activation buffer of embodiment 2 | 2.56 | 2.57 | 0.01 |
| MES | 2.53 | 3.85 | 1.32 |
Note: absorbance 1 is after each activation buffer of latex dilutes, the absorbance recorded under 540nm; ;
Absorbance 2 is after each latex dilution adds activator EDC reaction 10s, in the absorbance that 540nm records;
Δ A is absorbance 2-absorbance 1.
As shown in data in table 1, MES damping fluid latex system is used to change greatly adding absorbance before and after EDCA, about 1.32, this illustrates this system generation coagulation sedimentation, and MES damping fluid is also not suitable for the latex microsphere of activation zone carboxyl.
And using activation buffer of the present invention, latex system is to add absorbance before and after EDCA almost unchanged, and this system is more stable, does not produce agglutination phenomenon.
Embodiment 6: for detecting the kit of melamine concentration in sample
Kit comprises R1 reagent, R2 reagent and melamine standard items.
Composition is as follows:
The PBS:PEG6000(3% of R1:40mM), polysorbas20 (0.2%) and Sodium azide (0.1%);
R2:1% bag, by the latex microsphere solution of melamine antibody, is prepared according to embodiment 3; With
Melamine standard items, purchased from China National Measuring Science Research Inst..
Embodiment 7: the detection of melamine concentration in food samples
Use instrument: twin-beam ultraviolet-visible pectrophotometer, model TU-1901, production firm: Beijing Puxi General Instrument Co., Ltd.Determined wavelength: 540nm.
Detecting step: two-point method.
In cuvette, add the R1 reagent 250ul of preparation in embodiment 1, get 2.5ul standard items or liquid milk sample with pipettor, join in cuvette, incubation 5min under being incorporated in 37 degree with described R1 reagent is mixed; To add in embodiment 3 the R2 reagent 50ul of preparation, mix immediately, start timing after 10S, and to survey absorbance be A1, after 5min reaction, surveys absorbance is A2, calculating absorbance difference A2-A1, i.e. absorbance difference Δ A.
A typical curve is drawn according to the absorbance difference of variable concentrations standard items.The concentration of melamine in described liquid milk sample can be calculated according to typical curve.
Embodiment 8: detection kit recovery detected value
The sample that with the addition of concentration known melamine is detected according to the method described in embodiment 7.The melamine recovery of kit of the present invention is calculated according to detectable concentration and interpolation concentration.
Result is as shown in the table:
Table 3: the concentration of standard items and sample and the absorbance of detection:
According to the absorbance difference drawing standard curve of standard items, as shown in Figure 3.
Table 4: the sample melamine concentration calculated according to typical curve and the kit recovery
From above result, the melamine sample recovery rate of interpolation 5,10ppb, between 83.1%-106.05%, has good degree of accuracy.
Embodiment 8: stability experiment
Experimental procedure:
After preparing R2 reagent according to embodiment 3, carry out stability test to it, method of testing is as follows:
Choose 40ppb melamine standard items, test absorbance with two-point method, and to change the figure that runs a curve according to absorbance difference under different time, to the results are shown in Figure in 3 detection curve first.
After depositing 7 days (simulation accelerated experiment deposits 1 year under being equivalent to 4 ° of C) under kit described in embodiment 4 is placed in 37 ° of C, again detect with same concentrations melamine standard items.The results are shown in Figure Detection of Stability curve in 4.
As shown in Figure 4, result two curves almost overlap, and this confirms, this latex conjugate does not come off, and antibody activity is better, and stabilization of kit set forth in the present invention is good.