CN104034893A - Latex-based melamine rapid-detection method and kit - Google Patents
Latex-based melamine rapid-detection method and kit Download PDFInfo
- Publication number
- CN104034893A CN104034893A CN201310074726.5A CN201310074726A CN104034893A CN 104034893 A CN104034893 A CN 104034893A CN 201310074726 A CN201310074726 A CN 201310074726A CN 104034893 A CN104034893 A CN 104034893A
- Authority
- CN
- China
- Prior art keywords
- melamine
- latex
- latex microsphere
- antibody
- add
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a melamine antibody-coated latex microsphere solution, a preparation method of the melamine antibody-coated latex microsphere solution, a kit containing the melamine antibody-coated latex microsphere solution, and a method for detecting melamine in a sample by the melamine antibody-coated latex microsphere solution.
Description
Technical 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 that detects melamine based on polystyrene latex microballoon.
Background technology
In food service industry, by triumphant formula nitriding (GB/T5009.5-2010; First method) thus nitrogen atom content indirect calculation protein content measured.In recent years, some illegal retailers add industrial chemicals melamine in food or feed to, in the hope of improving the apparent protein content of measuring 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 melamine has high performance liquid chromatography, LC-MS, gas chromatography mass spectrometry method etc. at present, although these method detection sensitivities are high, but the pre-treatment step running time is long, instrument is expensive, and need to sample and send back to laboratory and detect, be not suitable for the on-site quick screening of melamine in sample.Can be used at present the method for field quick detection as enzyme linked immunosorbent assay, should use in comparison more flexible, but testing cost is still very expensive, technical operation personnel operation is also had to certain technical requirement, although and colloid gold card operates very easy but can only be qualitative analysis, can not realize quantitative test accurately.
Immunoturbidimetry (J.M.Singer et al., Am.J.Med.21,888-92; 1956) be mainly used in detection and the clinical diagnosis of microorganism and virus infections etc.In immunoturbidimetry, antibody is coated on to microsphere surface.In the time that the sample that comprises specific antigen mixes with latex suspension, cause visible aggegation, increase the turbidity of sample.By measuring the coated reacted absorbance of latex suspension of sample and antibody, and compare with the typical curve that uses the antigen of concentration known to draw, thus the concentration of antigen in working sample.
The microballoon using in immunoturbidimetry comprises polytype, such as glass, silica, nano metal, polystyrene, polyacrylamide particle etc.Can pass through passive adsorption or chemical covalent coupling, the modes such as such as carbodiimides (EDC) method, bromination coupling are connected to antibody on microballoon.Conventionally need to first activate the functional group on latex microsphere surface in the method for latex microsphere pan coating antibody, can react with antibody protein.For example, conventionally use the carboxyl on EDC activation latex microsphere surface, make the amino coupled of itself and protein.
It has been generally acknowledged that the activation method by using carbodiimides (EDC)/N-hydroxy thiosuccinimide (Sulfo-NHS) is more conducive to the interaction between protein and microballoon, is therefore the coupling method of commonly using the most.In addition use in addition, the activation method of EDC/NHS (N-hydroxy-succinamide) potpourri.But, in these two kinds of methods, to successively use two kinds of reagent, reactive system and method are comparatively complicated, very easily cause microballoon generation aggegation, are unfavorable for the suitability for industrialized production of the coated microballoon of antibody.
In contrast to this, use separately that the step of activation method (or being called a step EDC method) of EDC is comparatively simple and productive rate is higher.But a step EDC fado for example, for not wrapping carboxylic part (oligonucleotides, as DNA) and surface with the coupling between the microballoon of carboxyl.For example, if treat that the part of coupling also comprises carboxylic group (protein, as antibody), it may be activated by EDC, causes the polymerization between part, thereby cannot obtain the product of expectation.
Therefore, need to develop one and avoid mutual aggegation between microballoon or antibody to produce precipitation, thereby be applicable to the production technology of the coated microballoon of steady production antibody, thereby acquisition can utilize immunoturbidimetry fast, accurately to detect the melamine in sample.
Summary of the invention
At present, in EDC activation method, many use 2-(N-morpholine) ethyl sulfonic acids (MES), as activation buffering agent, cannot be prepared the microspheres solution of coated melamine antibody but the inventor finds the EDC solution of use MES buffering through testing.The inventor shows through great many of experiments, in latex system, very easily makes the mutual aggegation of microballoon with carboxyl produce precipitation after adding activating solution, keeps latex state to need to improve the composition of activation damping fluid.Therefore, the inventor has carried out large quantity research to EDC activation method, found that the phosphate buffer that comprises polysorbas20 can provide the environmental optima that is beneficial to coupling between antibody and microballoon, can avoid the mutual aggegation of microballoon to produce precipitation, be applicable to the coated microballoon of steady production antibody, thereby completed the present invention.
In first aspect, the invention provides a kind of preparation method of latex microsphere solution of coated melamine antibody, said method comprising the steps of: surface is diluted in activation damping fluid with the latex microsphere of carboxyl, wherein, described activation damping fluid is the 10～40mM phosphate buffer that comprises 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 that does not use other activators except carbodiimides in reactivation process.Described " other activators " comprises the reagent such as Sulfo-NHS, NHS.
In an embodiment, preparation method of the present invention comprises the following steps:
1) surface is diluted in activation damping fluid with the latex microsphere of carboxyl, wherein, described activation damping fluid is the 10～40mM phosphate buffer that comprises 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, be coated with reaction;
4) add cancellation liquid cessation reaction;
5) add confining liquid to seal free carboxyl, thereby obtain the latex microsphere solution of coated melamine antibody.
In the present invention, described latex microsphere can be any latex microsphere that is generally used for immune detection, such as polystyrene microsphere, polyacrylamide microsphere etc.The known latex microsphere that has a lot of manufacturers that various different models are provided, for example Microparticles GmbH, PolyMicrospheres Inc. etc.Those skilled in the art can select the surface that the is applicable to latex microsphere with carboxyl according to specific needs.
In the present invention, preferably use polystyrene latex microballoon.The particle diameter of latex microsphere of the present invention can be between 100-200nm, preferably 130-170nm.In the present invention, use the latex microsphere of surface with carboxyl, thereby 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.For example, can add the carbodiimides of carboxyl quantity 15%-30%.
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 be by commercially available, for example, 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 conventionally between 5:1 to 10:1, preferably 10:1.The antibody that adds 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 that is generally used for stopping carbodiimides coupled reaction, for example 60-80g/L glycocoll, preferably 75g/L glycocoll.
The cancellation liquid that the present invention uses can be any reagent that is generally used for sealing the free carboxyl of latex microsphere, for example seralbumin, and as 180-220g/L bovine serum albumin(BSA), preferably 200g/L bovine serum albumin(BSA).
The latex microsphere solution of the coated melamine antibody of being prepared by the method for the invention can be now with the current.In use, by centrifugal removal supernatant, use redissolution liquid to clean latex microsphere, and be settled to the concentration of appointment.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/L EDTANa2, 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/L EDTANa2, pH8.0.
Also the latex microsphere of the coated melamine antibody of preparing according to the method for the invention can be redissolved in described redissolution liquid, and low temperature is preserved under 2～8 ° of C.
In one embodiment, preparation method of the present invention comprises the following steps:
1) use activation damping fluid by particle diameter 130-170nm, carboxyl-content is the latex solution of the polystyrene latex microballoon dilution 1% of 160～170 μ eq/g, 10～40mM phosphate buffer that wherein said activation damping fluid comprises 0.5～1g/L polysorbas20, pH7.4-7.8;
2) add the carbodiimides of carboxyl quantity 15%-30%, mix rear reaction 5min;
3) add melamine monoclonal antibody taking latex microsphere and melamine monoclonal antibody as the ratio of 10:1, and at 37 ° of C, 300rpm reaction 2-3 hour;
4) add 75g/L glycocoll, end coated reaction;
5) add 200g/L bovine serum albumin(BSA), at 37 ° of C, 300rpm reaction 30-60min, thereby seal free carboxyl;
6) centrifugal removal supernatant, uses redissolution liquid to clean, and 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/L EDTANa2, pH8.0-8.2;
7) repeating step 6) three times, using subsequently described redissolution liquid to be settled to latex concentration is 0.1%, and low temperature is preserved under 2-8 ° of C.
Compared with cushioning activating solution with conventional MES, the suitableeest ion concentration and pH that the activation damping fluid that the present invention adopts provides melamine monoclonal antibody and surface to carry out coupling with the latex microsphere of carboxyl, avoided disadvantageous agglutination phenomenon occurring adding after melamine antibody.
The redissolution liquid that the present invention adopts provides the latex microsphere solution that is conducive to coated melamine antibody to keep stable the suitableeest ion concentration and pH.Use described redissolution liquid the latex microsphere solution of coated melamine antibody can be kept more than 1 year at normal temperatures, avoid antibody to come off simultaneously.
In second aspect, the invention provides the purposes of described activation damping fluid for the preparation of the latex microsphere solution of coated melamine antibody.
In the third aspect, the invention provides a kind of kit for detection of melamine in sample, described kit comprises following component:
1) R1 reagent: the phosphate buffer that comprises PEG6000 and polysorbas20, pH7.0-7.6;
2) R2 reagent: the latex microsphere solution of coated melamine antibody; With
3) melamine standard items.
Described R1 reagent is the buffering agent that is generally used for immunoturbidimetry.Those skilled in the art can prepare applicable buffering agent as required.In the present invention, can use the phosphate buffer that comprises PEG6000 and polysorbas20, for example, comprise the 40mM phosphate buffer of 3%PEG6000 and 0.2% polysorbas20, pH7.0-7.6, preferably pH7.4.The in the situation that of the long-term preservation of needs, also optionally add 0.1% Sodium azide to be used as antiseptic.
Described R2 reagent is the latex microsphere solution of coated melamine antibody.Described melamine antibody and latex microsphere are as described in first aspect present invention.Preferably, described R2 reagent is the latex microsphere solution of the coated melamine antibody prepared according to preparation method described in first aspect present invention.The concentration of described latex microsphere solution can be 1% (10 times of concentrates) or 0.1%.
Described kit also optionally comprises the instructions that uses 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 detecting the concentration of melamine in sample.Therefore,, in the third aspect, the invention provides the application of described kit for detection 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 detection of melamine concentration in sample, and it comprises the following steps:
1) by described sample and the phosphate buffer (pH7.0-7.6) that comprises PEG6000 and polysorbas20, mix incubation;
2) add the latex microsphere solution that is coated with melamine antibody;
3) measure gained potpourri and adding after described latex microsphere solution the absorbance difference Δ A of any two time points between 10s to 5 minute
540;
4) determine the melamine concentration in sample according to the typical curve that uses melamine standard items to draw.
It is known in the art using the method for the typical curve of melamine standard items drafting.Conventionally, melamine standard items known concentration and applicable buffering agent (for example, R1 reagent of the present invention) are mixed to incubation, subsequently, add the latex microsphere solution of coated melamine antibody, incubation 5 minutes after mixing, and according to the variation 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, two kinds of reagent of R2, change and the concentration of quantitative test sample melamine according to absorbance before and after reaction.
The all reagent preparations 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 technician's the technical requirement detecting, detection speed fast, detect overall process only needs 7 minutes, in addition, do not rely on professional instrument, can really realize real-time and detecting.
Brief description of the drawings
Fig. 1 is the melamine standard items response curve figure that absorbance changes in course of reaction that shows variable concentrations, and wherein horizontal ordinate is the reaction time, and ordinate is that absorbance changes.
Fig. 2 is the typical curve recording under different time, and wherein horizontal ordinate is reactant concentration (ppb), and ordinate is that absorbance changes.
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 that absorbance changes.
Fig. 4 is the comparing result of exhibit stabilization experiment, and wherein, horizontal ordinate is reaction time (min), and ordinate is that absorbance changes.Detectable concentration is 27ppb, and the time interval is 7 days, under 37 degree conditions, preserves.
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. take sodium hydrogen phosphate 7.16g, with ultrapure water constant volume 500ml; 3. take sodium chloride 0.14g, add 1. reagent 3.8ml and 2. reagent 16.2ml.
This is 40mM, the phosphate buffer (PBS) of PH7.4.
The preparation of R1:
PEG6000 0.3g
Sodium azide 0.01g
Tween-20 20ul
Weigh after 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 water-baths and heated, and constantly stir and mix.Reagent after dissolving is colourless transparent liquid.
After preparing reagent, in 2-8 ° of C refrigerator, preserve,, the pot-life is 12 months.
Embodiment 2: for the preparation of with the reagent of latex microsphere of preserving coated melamine antibody
Activation damping fluid:
The preparation of the PBS (PH7.4) of 10mM: four times of the PBS dilutions of 40mM prepared by embodiment 1 subsequently, add the Tween-20 of 0.70g/L in gained 10mM PBS.
Redissolution liquid:
Glycocoll 11.3g/L
Sodium azide 1.0g/L
Sorbierite 10.0g/L
BSA 5.0g/L
EDTA·Na2 2.0g/L
First weigh various solid chemical compounds, then use ultrapure water constant volume, for increasing dissolution velocity, can be placed in 37 degree incubators.Finally, hydro-oxidation sodium solution (20%) is adjusted PH8.00 ± 0.08.The reagent preparing is the liquid that achromaticity and clarification is transparent.The redissolution liquid preparing can be kept in 2-8 ° of C refrigerator, and the pot-life is 12 months.
Cancellation liquid:
Glycocoll 75g/L
Take after glycocoll, use ultrapure water constant volume.After mixing, this solution is colourless transparent liquid.After preparing reagent, in 2-8 degree refrigerator, preserve.Pot-life is 60 days.
Confining liquid:
BSA 200g/L
Weigh BSA solid, use ultrapure water constant volume, place in 60 degree water-baths limit heating edge shake bottle, accelerate dissolution.BSA solution after dissolving is flaxen liquid.The confining liquid preparing can be kept in 2-8 ° of C refrigerator, and the pot-life is 60 days.
Embodiment 3: the preparation technology of the latex microsphere of coated melamine antibody
Reaction system is 100ul.
Activation
Get 1.5ml centrifuge tube, add activation damping fluid 70ul, the polystyrene latex microballoon of getting 10ul with pipettor is (purchased from PolyMicrospheres company of the U.S., particle size 130-170nm, carboxyl-content 160-170 μ eq/g) in this centrifuge tube, constantly piping and druming, is distributed to uniformly in activation damping fluid latex particle, thereby obtains white emulsion.
Weigh solid carbodiimide hydrochloride (EDCA; Purchased from Shanghai covalent chemical Science and Technology Ltd.) be dissolved in activation damping fluid, concentration is 0.001mg/ul.In above-mentioned latex particle damping fluid, add 10ul EDCA solution, activate at normal temperatures 5min.The latex system having activated is still white emulsion, but viscosity declines to some extent.
Coupling
Add melamine monoclonal antibody (purchased from Beijing Bo Aosen biotechnology Ltd the latex system having activated, model bsm-2182M), addition is 10ul(10mg/ml), when interpolation, should, rapidly with the piping and druming of rifle head, mix fast at short notice as far as possible.After mixing, obtain white emulsion.The constant temperature oscillator that the latex system that is added with melamine antibody is placed in to 37 ° of C, 300rpm reacts 2 hours.After completing, system is still white emulsion.
Cancellation
Now melamine latex conjugate forms, adds cancellation liquid 80ul in system, blows and beats and mixes with rifle head, in 37 degree, 300rpm constant temperature oscillator, reacts 30min.After finishing, system is answered deposit-free.
Sealing
Add confining liquid 17ul, blow and beat and mix with rifle head, be positioned in 30 degree, 300rpm constant temperature oscillation case, be 45min off-period.After sealing finishes, 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 centrifugal, produce higher temperature while preventing high speed centrifugation), centrifugal after, white latex microsphere major part is all deposited in centrifuge tube bottom, in supernatant, contain minute quantity microballoon, but still seem water white transparency, outwell supernatant, or supernatant is taken out with rifle head, add 100ul to redissolve liquid, blow and beat and mix with rifle head, then be placed in ultrasonic washing instrument microballoon is disperseed.Ultrasonic washing instrument power 99%, time 1min.Repeat this step 3 time.Add for the last time redissolution liquid 1ml.
Now solution is white emulsion, does not precipitate without graininess after testing in system.This is R2 reagent, and absorbance is about 0.2-0.9, as too high in absorbance, 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: the impact of different EDC activation methods on 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, use EDC activation latex microsphere according to the description of embodiment 3:
The polystyrene latex microballoon of getting 10ul with pipettor is (purchased from PolyMicrospheres company of the U.S., particle size 130-170nm, carboxyl-content 160-170 μ eq/g) in centrifuge tube, constantly piping and druming, latex particle is distributed in activation damping fluid of the present invention, to final concentration 1% uniformly.
Weigh solid EDCA (purchased from Shanghai covalent chemical Science and Technology Ltd.) and be dissolved in activation damping fluid of the present invention, concentration is 0.001mg/ul.In above-mentioned latex particle damping fluid, add the above-mentioned EDCA solution of 10ul, vibration mixes, and reaction 5min, surveys light absorption value 1.
Subsequently, gained latex microsphere is divided into two parts, a copy of it does not add NHS, and another part adds 2-3 that NHS, consumption be about EDC consumption doubly, after mixing, measures the light absorption value 2 of two duplicate samples.
Table 1: the impact of Different Activation Methods on 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 absorbance before and after activation changes greatly, and approximately 1.11, this illustrates this system generation coagulation sedimentation.
And use separately EDC, and latex system absorbance before and after activation is almost unchanged, and this system is more stable, does not produce agglutination phenomenon.
Embodiment 5: the impact of different activation buffer systems on latex system stability
The most frequently used buffer system of EDC cross-linking reaction is that ion concentration is 2-(N-morpholine) ethyl sulfonic acid (MES) solution of 40mM at present, and PH is 5-6.Compared with cushioning activating solution with conventional MES, the suitableeest ion concentration and pH that the activating solution that the present invention adopts provides melamine monoclonal antibody and surface to carry out coupling with the latex microsphere of carboxyl, avoided disadvantageous agglutination phenomenon occurring adding after melamine antibody.
use MES activation damping fluid activation latex microsphere
Get 1.5ml centrifuge tube, add MES activation damping fluid 70ul.The polystyrene latex microballoon of getting 10ul with pipettor is (purchased from PolyMicrospheres company of the U.S., particle size 130-170nm, carboxyl-content 160-170 μ eq/g) in this centrifuge tube, constantly piping and druming, is distributed in MES activation damping fluid latex particle uniformly.
Weigh solid EDCA (purchased from Shanghai covalent chemical Science and Technology Ltd.) and be dissolved in MES activation damping fluid, concentration is 0.001mg/ul.In above-mentioned latex particle damping fluid, add the above-mentioned EDCA solution of 10ul, with the piping and druming of rifle head, in a moment slightly, observe latex system immediately.
Found that, adding after the activator of MES buffering, in latex system, occur immediately a small amount of flocky precipitate, after approximately 30 seconds, occur a large amount of flocculent deposits, placed 5-6 as a child, this system layering.
To this, detect described in use MES buffering activating solution and embodiment 2 and activated damping fluid, latex system is adding the variation of activator EDC front and back absorbance.Result is as shown in the table:
Table 2: the impact of different activation damping fluids on latex system stability
| Activating solution composition | Absorbance 1 | Absorbance 2 | ΔA |
| The activation damping fluid of embodiment 2 | 2.56 | 2.57 | 0.01 |
| MES | 2.53 | 3.85 | 1.32 |
Note: absorbance 1 is for latex is with after the dilution of each activation damping fluid, the absorbance recording under 540nm; ;
Absorbance 2 is reacted after 10s for each latex dilution adds activator EDC, the absorbance recording in 540nm;
Δ A is absorbance 2-absorbance 1.
As shown in data in table 1, use MES damping fluid latex system absorbance before and after adding EDCA to change greatly, approximately 1.32, this illustrates this system generation coagulation sedimentation, MES damping fluid is also not suitable for the latex microsphere of activation zone carboxyl.
And use the present invention to activate damping fluid, and latex system absorbance before and after adding EDCA is almost unchanged, and this system is more stable, does not produce agglutination phenomenon.
Embodiment 6: for detection of 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%);
The latex microsphere solution of the coated melamine antibody of R2:1%, prepares 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.Detect wavelength: 540nm.
Detecting step: two-point method.
The R1 reagent 250ul that adds in embodiment 1 preparation in cuvette, gets 2.5ul standard items or liquid milk sample with pipettor, join in cuvette, with described R1 reagent mix and under 37 degree incubation 5min; The R2 reagent 50ul that adds in embodiment 3 preparation, mixes immediately, starts timing after 10S, and to survey absorbance be A1, and after 5min reaction, surveys absorbance is A2, calculating absorbance difference A2-A1, i.e. absorbance difference Δ A.
Draw a typical curve according to the absorbance difference of variable concentrations standard items.Can calculate according to typical curve the concentration of melamine in described liquid milk sample.
Embodiment 8: detection kit recovery detected value
Detect the sample that has added concentration known melamine according to the method described in embodiment 7.According to the melamine recovery of detectable concentration and interpolation concentration calculating kit of the present invention.
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: sample melamine concentration and the kit recovery calculated according to typical curve
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:
Prepare after R2 reagent according to embodiment 3, it is carried out to stability test, method of testing is as follows:
Choose 40ppb melamine standard items, test absorbance with two-point method, and according to the figure that runs a curve that changes of absorbance difference under different time, the results are shown in Figure in 3 detection curve first.
Kit described in embodiment 4 is placed under 37 ° of C and is deposited after 7 days (simulation accelerated experiment is equivalent to deposit 1 year under 4 ° 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, two curves of result almost overlap, this confirmation, and this latex conjugate does not come off, and antibody activity is better, and kit set forth in the present invention has good stability.
Claims (10)
1. a preparation method for the latex microsphere solution of coated melamine antibody, it comprises:
1) surface is diluted in activation damping fluid with the latex microsphere of carboxyl, wherein, described activation damping fluid is the 10～40mM phosphate buffer that comprises 0.5～1g/L polysorbas20, pH7.4-7.8;
2) add activator, hybrid reaction, wherein said activator is carbodiimides;
3) add melamine monoclonal antibody, be coated with reaction;
4) add cancellation liquid cessation reaction; With
5) add confining liquid to seal free carboxyl, thereby obtain the latex microsphere solution of coated melamine antibody.
2. the method for claim 1, wherein use a step EDC activation method to activate the carboxyl on described latex microsphere surface.
3. method as claimed in claim 1 or 2, wherein, described activation damping fluid is the 10mM phosphate buffer that comprises 0.7g/L polysorbas20, pH7.4.
4. the method as described in any one in claim 1-3, wherein said latex microsphere is that particle diameter is 100-200nm, carboxyl-content is the polystyrene latex microballoon of 100-200 μ eq/g.
5. the method as described in any one in claim 1-4, wherein said method is further comprising the steps of:
6) latex microsphere solution centrifugal step 5 being obtained is removed supernatant, use redissolution liquid to clean, wherein said redissolution liquid comprises 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/L EDTANa2, pH7.8-8.2;
7) repeating step 6) three times, latex solution low temperature under 2-8 ° of C that will be dissolved in subsequently described redissolution liquid is preserved.
6. method as claimed in claim 5, wherein, 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/L EDTANa2, pH8.0.
7. the latex microsphere solution of the coated melamine antibody of preparing by method described in any one in claim 1-6.
8. for detection of the kit of melamine in sample, it comprises:
1) R1 reagent: the phosphate buffer that comprises PEG6000 and polysorbas20, pH7.0-7.6;
2) R2 reagent: the latex microsphere solution of coated melamine antibody claimed in claim 7; With
3) melamine standard items.
9. for detection of a method for melamine concentration in sample, it comprises the following steps:
1) by described sample and the phosphate buffer (pH7.0-7.6) that comprises PEG6000 and polysorbas20, mix incubation;
2) add the latex microsphere solution of coated melamine antibody claimed in claim 7;
3) measure gained potpourri and adding after described latex microsphere solution the absorbance difference Δ A540 of any two time points between 10s to 5 minute; With
4) determine the melamine concentration in sample according to the typical curve that uses melamine standard items to draw.
10. method as claimed in claim 9, wherein said sample is selected from fresh milk, milk powder, cheese, cream and Yoghourt.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310074726.5A CN104034893B (en) | 2013-03-08 | 2013-03-08 | A kind of melamine rapid detection method based on latex and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310074726.5A CN104034893B (en) | 2013-03-08 | 2013-03-08 | A kind of melamine rapid detection method based on latex and kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104034893A true CN104034893A (en) | 2014-09-10 |
| CN104034893B CN104034893B (en) | 2016-04-20 |
Family
ID=51465740
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310074726.5A Active CN104034893B (en) | 2013-03-08 | 2013-03-08 | A kind of melamine rapid detection method based on latex and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104034893B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105911293A (en) * | 2016-05-26 | 2016-08-31 | 安徽伊普诺康生物技术股份有限公司 | Kit for determining immunoglobulin A and preparation method thereof |
| CN106405110A (en) * | 2016-09-06 | 2017-02-15 | 北京华科泰生物技术有限公司 | Activated fluorescent latex microspheres for fluorescence immunochromatography test strip for myoglobin and application |
| CN106405109A (en) * | 2016-09-06 | 2017-02-15 | 北京华科泰生物技术有限公司 | Activated fluorescent latex microsphere used for ferritin fluorescence immunoassay chromatography detection card |
| CN107741492A (en) * | 2017-09-30 | 2018-02-27 | 安徽伊普诺康生物技术股份有限公司 | A kind of preparation method for the latex microsphere for being coated with N akrencephalon natriuretic peptide precursor antibodies |
| CN111077304A (en) * | 2019-12-31 | 2020-04-28 | 深圳市瀚德标检生物工程有限公司 | Coupling method of carboxyl latex microspheres and antibody |
| CN111721937A (en) * | 2019-03-29 | 2020-09-29 | 北京九强生物技术股份有限公司 | Human epididymis protein 4 immunoturbidimetry kit |
| CN112485438A (en) * | 2020-11-10 | 2021-03-12 | 深圳市科曼医疗设备有限公司 | Specific protein reaction detection method and device |
| CN114137223A (en) * | 2021-11-25 | 2022-03-04 | 安徽大千生物工程有限公司 | Latex enhanced immunoturbidimetry assay kit for rapidly assaying IGF-1 and preparation and use methods thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0600936A1 (en) * | 1991-07-29 | 1994-06-15 | Serex, Inc. | Differential binding affinities and dissociation assays based thereon |
| US6096563A (en) * | 1996-03-29 | 2000-08-01 | Strategic Diagnostics Inc. | Dual particle immunoassay method and kit |
| CN1742096A (en) * | 2002-11-26 | 2006-03-01 | 国防科研与发展组织 | A process for preparation of an agglutination reagent for rapid detection of typhoid |
| CN1761880A (en) * | 2003-04-03 | 2006-04-19 | 金伯利-克拉克环球有限公司 | Reduction of the hook effect in assay devices |
| CN101680894A (en) * | 2007-03-14 | 2010-03-24 | 国防研究与发展组织总干事 | reagent for detection of analyte and processes thereof |
| CN101819202A (en) * | 2010-05-06 | 2010-09-01 | 上海太阳生物技术有限公司 | D-Dimer measuring kit (latex immunonephelometry method) |
| CN102854314A (en) * | 2012-10-10 | 2013-01-02 | 深圳康美生物科技股份有限公司 | Kit for detecting helicobacter pylori antibody by using latex immunoturbidimetry assay |
| CN102879559A (en) * | 2011-07-12 | 2013-01-16 | 上海执诚生物科技股份有限公司 | Real-time quantitative detection reagent and method of time-resolved fluorescence immune chromatography |
-
2013
- 2013-03-08 CN CN201310074726.5A patent/CN104034893B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0600936A1 (en) * | 1991-07-29 | 1994-06-15 | Serex, Inc. | Differential binding affinities and dissociation assays based thereon |
| US6096563A (en) * | 1996-03-29 | 2000-08-01 | Strategic Diagnostics Inc. | Dual particle immunoassay method and kit |
| CN1742096A (en) * | 2002-11-26 | 2006-03-01 | 国防科研与发展组织 | A process for preparation of an agglutination reagent for rapid detection of typhoid |
| CN1761880A (en) * | 2003-04-03 | 2006-04-19 | 金伯利-克拉克环球有限公司 | Reduction of the hook effect in assay devices |
| CN101680894A (en) * | 2007-03-14 | 2010-03-24 | 国防研究与发展组织总干事 | reagent for detection of analyte and processes thereof |
| CN101819202A (en) * | 2010-05-06 | 2010-09-01 | 上海太阳生物技术有限公司 | D-Dimer measuring kit (latex immunonephelometry method) |
| CN102879559A (en) * | 2011-07-12 | 2013-01-16 | 上海执诚生物科技股份有限公司 | Real-time quantitative detection reagent and method of time-resolved fluorescence immune chromatography |
| CN102854314A (en) * | 2012-10-10 | 2013-01-02 | 深圳康美生物科技股份有限公司 | Kit for detecting helicobacter pylori antibody by using latex immunoturbidimetry assay |
Non-Patent Citations (2)
| Title |
|---|
| YUJI TERAMURA ET AL: "Surface plasmon resonance-based highly sensitive immunosensing for brain natriuretic peptide using nanobeads for signal amplification", 《ANALYTICAL BIOCHEMISTRY》 * |
| 龙腾镶等: "颗粒增强免疫比浊法测定血清中同型半胱氨酸的研究", 《中国优秀硕士学位论文全文数据库（电子期刊）医药卫生科技辑》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105911293A (en) * | 2016-05-26 | 2016-08-31 | 安徽伊普诺康生物技术股份有限公司 | Kit for determining immunoglobulin A and preparation method thereof |
| CN106405110A (en) * | 2016-09-06 | 2017-02-15 | 北京华科泰生物技术有限公司 | Activated fluorescent latex microspheres for fluorescence immunochromatography test strip for myoglobin and application |
| CN106405109A (en) * | 2016-09-06 | 2017-02-15 | 北京华科泰生物技术有限公司 | Activated fluorescent latex microsphere used for ferritin fluorescence immunoassay chromatography detection card |
| CN106405109B (en) * | 2016-09-06 | 2018-01-05 | 北京华科泰生物技术有限公司 | Ferritin fluorescence immune chromatography detection card activation fluorescent latex microballoon |
| CN106405110B (en) * | 2016-09-06 | 2018-01-16 | 北京华科泰生物技术有限公司 | Myoglobins fluorescence immune chromatography test paper bar activation fluorescent latex microballoon and application |
| CN107741492A (en) * | 2017-09-30 | 2018-02-27 | 安徽伊普诺康生物技术股份有限公司 | A kind of preparation method for the latex microsphere for being coated with N akrencephalon natriuretic peptide precursor antibodies |
| CN111721937A (en) * | 2019-03-29 | 2020-09-29 | 北京九强生物技术股份有限公司 | Human epididymis protein 4 immunoturbidimetry kit |
| CN111077304A (en) * | 2019-12-31 | 2020-04-28 | 深圳市瀚德标检生物工程有限公司 | Coupling method of carboxyl latex microspheres and antibody |
| CN112485438A (en) * | 2020-11-10 | 2021-03-12 | 深圳市科曼医疗设备有限公司 | Specific protein reaction detection method and device |
| CN114137223A (en) * | 2021-11-25 | 2022-03-04 | 安徽大千生物工程有限公司 | Latex enhanced immunoturbidimetry assay kit for rapidly assaying IGF-1 and preparation and use methods thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104034893B (en) | 2016-04-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104034893B (en) | A kind of melamine rapid detection method based on latex and kit | |
| CN104198723A (en) | Rapid NGAL (Neutrophil Gelatinase Associated Lipocalin) detection kit based on amino acid spacer arm | |
| CN110862881A (en) | Special cleaning solution or diluent for full-automatic chemiluminescence determinator and preparation method thereof | |
| CN110736837B (en) | Latex immunoturbidimetric assay kit for neuron-specific enolase | |
| CN104897904A (en) | Method for marking protein by carboxyl microsphere | |
| CN102901810A (en) | Preparation method of latex particles coated with prostate specific antigen-antibody and PSA enhanced turbidimetric immunophelometry kit | |
| CN108593641A (en) | A kind of kit and method quantitatively detecting test substance in whole blood sample | |
| CN111122874A (en) | Kit for determining LN based on latex enhanced immunoturbidimetry, and preparation and use methods thereof | |
| CN108089007A (en) | A kind of kit and preparation method for quantitatively detecting c reactive protein | |
| CN105277717A (en) | Magnetic particle separation chemiluminescence immunoassay method for thyroglobulin | |
| CN110907639A (en) | Serum amyloid protein A detection kit and preparation method thereof | |
| CN104820102A (en) | Kit of detecting the content of Lp-PLA2 and CRP on the basis of chemiluminescence, and method and application thereof | |
| CN108196043A (en) | Kit of microdose urine protein content and preparation method thereof in a kind of detection serum | |
| CN106324254A (en) | Anti-insulin antibody detection kit and detection method thereof | |
| CN103048476A (en) | Thyroxine nanometer magnetic particle chemiluminiscence determinstion kit as well as preparation method and detection method thereof | |
| CN106970226A (en) | The kit of the ECD concentration levels of HER 2 in a kind of measure human serum | |
| CN111912990B (en) | Neutrophil gelatinase-associated lipocalin assay kit | |
| CN104833810A (en) | Complement C3 detection method | |
| CN103048477A (en) | Nanometer magnetic particle chemiluminescence detection kit for triiodothyronine as well as preparation method and detecting method of same | |
| CN101446586A (en) | Immunological assay reagents and assay method | |
| CN102369441B (en) | Immunoassay method and reagent therefor | |
| CN102341707A (en) | Test reagent, and method for measuring analyte in test sample using same | |
| CN106990234A (en) | A kind of lipoprotein（a）Detection reagent and method | |
| CN103383354B (en) | Detection method of magnetic particle chemiluminescence kit for detecting enterotoxin SEA | |
| CN106093419A (en) | A kind of mensuration dimeric test kit of D and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |