CN103760358A - Reagent for detecting Von Willebrand factor and preparation method and application thereof - Google Patents
Reagent for detecting Von Willebrand factor and preparation method and application thereof Download PDFInfo
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/22—Haematology
- G01N2800/224—Haemostasis or coagulation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/22—Haematology
- G01N2800/226—Thrombotic disorders, i.e. thrombo-embolism irrespective of location/organ involved, e.g. renal vein thrombosis, venous thrombosis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
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- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/811—Test for named disease, body condition or organ function
Abstract
A reagent for detecting von Willebrand factor. The detection reagent applies latex enhanced immunoturbidimetry method, and comprises a reagent 1, a reagent 2 and a von Willebrand factor standard substance or a standard curve. The reagent 1 is an anti-von Willebrand factor antibody latex reagent, which is formed by combining the anti-von Willebrand factor and latex particles; the reagent 2 is an application liquid; the von Willebrand factor standard substance is a reaction buffer containing von Willebrand factors with different concentrations; and the standard curve is a standard curve of the content of von Willebrand factor in human body. Compared with the prior art, the reagent for detecting von Willebrand factor provided by the invention realizes rapid detection of von Willebrand factor, and has high sensitivity and reliable repeatability, and can be applied to automatic analytical instruments.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of kit that detects vWF (vWF, von Willebrand Factor) in human serum and preparation method thereof.
Background technology
VWF (vWF, von Willebrand Factor), claims again vWF ELISA, is by the vWF gene code on No. 12 chromosome, is a kind of many subunits glycoprotein of macromolecule, the subunit of multiple 220kD, consists of.The degree of polymerization of end-product differs, and molecular weight soprano can be up to 20MD.After having assembled, be stored in the Weibel-Palade corpusculum of hematoblastic α particle, blood vessel endothelium; It is the large-scale adhesion glycoprotein that is present in a kind of multi-functional multivalence in blood.The vWF factor has extremely important physiological action in hemostasis and thrombosis: on the one hand, regulate mediation platelet adhesion reaction, and by with blood platelet on acceptor and the combination of collagen regulate the adhesion of blood platelet on blood vessel endothelium, main process is: thus blood vessel endothelium impaired → expose collagenous fibres → vWF to stick in damaged blood vessels because of sub-connection collagenous fibres → Platelet surface glycoprotein I b receptors bind vWF factor, this function is mainly completed by the polymer of macromolecule; On the other hand; also can with the non-covalent combination of the carrier protein of VIII (FVIII) (the VIII factor); thereby the protection VIII factor avoids suffering the attack of the proteinase including the PROTEIN C of activation; prevent that the VIII factor from being removed too early, normal hemostasis just can not cut because the VIII factor is subject to enzyme extremely.Therefore, in blood, normal vWF is very crucial, and the reduction of its level can cause the disease that blood clotting function is relevant to occur with the disappearance of function, and this type of disease is all because hemostasis and thrombosis cause extremely; Wherein modal is Feng's von Willebrand's disease and hemophilia, and wherein, because the hemophilia due to the vWF factor is modal hemophilia, the morbidity rate of external report is up to 1%～3%.So the content that detects vWF in blood is most important, and the key reference data for above-mentioned medical diagnosis on disease and treatment can be provided.
In addition,, along with the development of scientific research in recent years, other functions of some of vWF are understood by people gradually.It is at Angiogenesis, the propagation of smooth muscle cell, and the diffusion of cancer cell, and in immunologic process, all there is certain regulating and controlling effect.Therefore, it is in some angiocardiopathies, and cancer, can be used as biomarker and studied and use in hepatopathy and immunological diseases.In a word, detect the content of vWF in blood not only in the disease relevant with hemostasis and thrombosis, and have very high value in the diagnosis of other a lot of diseases and treatment.
In prior art, the method for detection vWF has multiple, for example: thus vWF polymer method is that sample agaropectin is separated and judges that whether polymer is normal; Euzymelinked immunosorbent assay (ELISA) (ELISA) thus be that the antigen with sample in is combined the content of detection vWF with the antibody that is coated on plastic sheet surface; Collagen method and euzymelinked immunosorbent assay (ELISA) are similar, and what be only coated on plastic sheet surface is not antibody, but collagen, and this method utilizes the feature that vWF and collagen can efficiency natural combinations to carry out its detection.Wherein, euzymelinked immunosorbent assay (ELISA) is mainly used to detect the content of vWF, and polymer method and collagen method also can detect its activity to a certain extent.
Above detection method differs from one another, and their common maximum limitations are can not be applied to laboratory automatic analysis and detection instrument device as on automatic blood coagulation instrument, cannot carry out a large amount of fast and there is the highly detection of repeatability.Polymer method needs to prepare agaropectin by hand, and the time of whole experiment is also longer.Euzymelinked immunosorbent assay (ELISA) and the collagen method difference of efficiency owing to being coated on plastic sheet surface causes the repeatability of experiment bad.
The advantages such as that latex immunoturbidimetry has is easy and simple to handle, quick, detection sensitivity is high, special, quantitatively accurate are extensively approved in clinical practice.
Early immune analysis forms by observing sediment, and aggegation and haemolysis are analyzed, as immunity diffusion, immunoelectrophoresis, hemagglutination test, complement combination etc.At the beginning of the sixties at the end of the fifties in last century, utilize AG-AB compound to form turbidity is changed, then come than turbid with nephometer, but be not accepted because its susceptibility is poor.To the beginning of the seventies, starting has automatic checkout system, but because of its use be that laser is light source, fixed wave length, sensitivity is lower, and the time generally want 2-3 hour, be difficult to meet clinical needs.The seventies, the appearance of speed nephometer, did not make the reaction of antigen-antibody combination go out result in tens seconds, can be described as the revolutionary development that immunochemistry is measured.
Along with the development of labelling technique, immunochemistry is included clinical chemistry test category in.The ultimate principle of immunity turbidimetric analysis turbidimetry is: antigen-antibody forms fast antigen antibody complex in special damping fluid, makes reactant liquor occur turbidity.When keeping antibody excess in reactant liquor, the compound of formation increases with antigen amount, and the turbidity of reactant liquor also increases thereupon, with a series of standard control, can calculate the content of examined object.
Immunoturbidimetric assay can be divided into two kinds according to the difference of Instrument Design, than turbid instrument, measures (turbidimetermeasure) and scattering turbidimetry instrument mensuration (nephelomitermeasure).Than turbid instrument, measuring is to measure the incident light decay causing due to reflection, absorption or scattering, and its reading represents with absorbance A.A has reflected the ratio of incident light and transmitted light.It is the light quantity of measuring incident light and run into scattering at an angle after particle (compound) that scattering turbidimetry instrument is measured, and this scattered light represents with scattering value after amplifying.
Classical turbidimetric test has 4 shortcomings to overcome, and complex operation, susceptibility low (10～100 μ g/ml), time are long and be difficult to robotization., there is skeptophylaxis precipitation determination method, i.e. Latex-enhanced immunoturbidimetric assay (PETIA) and immune scattering nephelometry the seventies in the principle of quick combination in liquid according to antigen and antibody capable.The all conventional detection for clinical body fluid albumen of these 2 kinds of technology, and createed multiple self-reacting device.
One, traditional immune turbidimetry
When light is when the turbid media solution, owing to there is muddy particle in solution, light is absorbed a part, absorption number be directly proportional to the amount of muddy particle, the method for this mensuration absorbing amount is called turbidimetry.This method is applied to plasma proteins as far back as reports such as nineteen fifty-nine Schultre and Schuick and is combined rear formation compound with its antibody, cause the change of turbidity, carry out again transmission turbidimetric assay, generally adopt the turbidimetry of antibody to antigen quantify, be called immune turbidimetry.Its principle is, utilizes the specific binding of antigen and antibody to form compound, by measure compound formation volume number antigen or antibody are carried out to quantitative method.
Because the time limit that is formed with of immune complex changes, after meeting, antigen-antibody is combined into immediately little compound (<19S), and a few minutes just formed visible compound (>19S) by several hours.As quick turbidimetric assay, this speed is too slow, adds polymerizer (or short poly-agent) can accelerate large immune complex and forms.The short poly-multiplex polyglycol of agent (MW6000～8000) at present, concentration is about 4%.
Turbidimetric analysis turbidimetry also has its weakness.First antigen or antibody amount are prone to soluble complex when greatly superfluous, cause error at measurment, thisly while measuring m protein are more prone to; It two is that should to maintain in reaction tube antibody protein amount superfluous all the time, and this value will be measured in advance, the measurement range that makes instrument lower than physiological range between higher than normal range; It three is the impacts that are subject to blood fat, and especially during low dilutability, the granule of lipoprotein can form turbidity, makes the false rising of measured value.
Two, Latex-enhanced immunoturbidimetric assay
In above-mentioned turbidimetry, the extremely difficult turbidity that forms of a small amount of little antigen antibody complex, unless placed the long period; As formed larger compound, antigen and antibody consumption are also larger, obviously do not meet the requirement of traceization.So developed the Latex-enhanced immunoturbidimetric assay (PETIA) that is widely used in now Biochemical Analyzer: take polyclonal antibody as basic improvement immunoturbidimetry analytic approach, utilize gene engineering method that antibody is combined with latex particle, when combining, antigen-antibody just formed Ag-Ab-latex particle compound, strengthened reaction absorbance, reactant liquor at certain wavelength place than turbid, with the titer comparison of same processing, calculate the content of antigen in sample.Utilize Biochemical Analyzer to carry out turbidimetric assay, whole analytic process only needs a few minutes, and relatively its sensitivity is higher for the method and traditional immunoturbidimetry.
When antigen and antibody specific reacts, generate the amount of bond relevant with the concentration of reactant.No matter in a certain amount of antibody, add the antigen of different amounts or in a certain amount of antigen, add the different antibody of measuring, all can find only when both molecule ratios are suitable, just to occur the strongest reaction.Take precipitation reaction as example, if enter a certain amount of antibody in row's test tube, then successively to the corresponding soluble antigen that adds incremental change in each pipe, according to the proportionate relationship of formed sediment and antigen-antibody, can draw out response curve (as shown in Figure 1).As seen from the figure, the mountain portions of curve is the suitable scope of antigen-antibody molecule ratio, is called the equivalence zone (zone of equivalence) of antigen-antibody reaction.Within the scope of this, the abundant combination of antigen-antibody, sediment forms fast and many.Wherein have a by-reaction the fastest, sediment forms at most, in supernatant, almost without free antigen or antibody, exists, and shows that the ratio of antigen and antibody concentration is the most suitable, is called optimum ratio (optimal ratio).Before and after equivalence zone, be respectively deposit-free formation of antibody excess, this phenomenon is called zoning (zone phenomenon).While appearing at antibody excess, be called front band (prozone), while appearing at antigen excess, be called rear band (postzone) and specifically see Fig. 1.
But in prior art, the kit that application Latex-enhanced immunoturbidimetric assay detects vWF has certain defect.The range of linearity that is mainly detection is less, to testing process, brings a lot of inconvenience.
Summary of the invention
The problems referred to above that exist for prior art, one of object of the present invention is to provide a kind of kit for detection of vWF, to realize fast detecting vWF, and there is very high sensitivity and repeated reliably, and can be applicable on automated analysis instrument.
For achieving the above object, the technical solution used in the present invention is as follows:
For detection of a reagent for vWF, this reagent is the detection reagent of application latex enhancing immune turbidimetry, reagent 1 and reagent 2, consists of; The antibody latex reagent that wherein reagent 1 is anti-vWF, the antibody latex reagent of anti-vWF is the antibody latex reagent of the anti-vWF that is combined into of anti-vWF and latex particle; Reagent 2 is application liquid.
Preferably, this reagent also further comprises vWF standard items or typical curve; VWF standard items are the reaction buffer of the vWF that contains variable concentrations; Typical curve is the typical curve of vWF at people's in-vivo content.
Preferably, the antibody latex reagent of anti-vWF is to obtain by the amino of the antibody of anti-vWF is formed to covalent bond with the carboxyl of latex particle after two steps are reacted; Described two step reactions comprise: the first step is first reacted latex particle with bonding agent, second step is again by latex particle and anti-vWF antibody response.In prior art, be all that employing single step reaction is by latex particle reaction together with bonding agent and anti-vWF antibody while.
More preferably, the antibody latex reagent of anti-vWF is to obtain by the amino of the antibody of anti-vWF is formed to covalent bond with the carboxyl of latex particle after two steps are reacted; First by the carboxyl of latex particle and bonding agent are reacted into latex particle-bonding agent intermediate under faintly acid, then by latex particle-bonding agent intermediate, under alkalescent, make anti-vWF antibody latex reagent with amino reaction of anti-vWF antibody.In prior art, be all adopt single step reaction be by latex particle and bonding agent and anti-vWF antibody simultaneously together with reaction, this reaction difficulty carry out and productive rate low.And two steps reactions provided by the invention have solved latex particle and anti-vWF antibody in this area and are difficult to be reacted into this difficult problem of latex antibody particle, and reaction yield is high, easy operating, and product stable uniform more.
More preferably, latex particle is selected from styrene latex particle, and the best is the styrene latex particle through carboxyl modified.
More preferably, the particle diameter of latex particle is 200～1000nm.
More preferably, the antibody latex reagent of anti-vWF is by after being suspended in latex particle activation and react with bonding agent in damping fluid, then proceeds in binding buffer liquid and add anti-vWF antibody carry out two steps reaction formation covalent bonds and obtain.
More preferably, the antibody latex reagent of anti-vWF is by after being suspended in latex particle activation and reacting with bonding agent in damping fluid, proceed to again in binding buffer liquid and add anti-vWF antibody to carry out two step reactions, then add in the deactivation liquid that contains sealer to add after reaction and preserve buffer memory liquid and remove the latex particle of unreacted and obtain.
More preferably, activation damping fluid is selected from glycine buffer or phosphate buffer, the KH that the best is 0.05M for concentration
2pO
4, its pH is 4.5.Its acid pH is in order to guarantee that carboxyl is in carboxyl coordination steady state (SS), is more conducive to the carrying out of two step reactions.
More preferably, it is 0.1～5% carbodiimide that bonding agent is selected from concentration, and the best is 1% carbodiimide; For forming intermediate with the carboxyl of latex particle, and then react formation covalent bond with the amino of the antibody of anti-vWF.
More preferably, binding buffer liquid is selected from TRIS buffer or borate buffer, and the best is 0.2M borate buffer, and pH is 8.5; Its weakly alkaline pH is amino in amino coordination steady state (SS) in order to guarantee, will more be conducive to the carrying out of two step reactions.
More preferably, anti-vWF antibody is selected from the anti-vWF antibody of goat-anti, mouse-anti or rabbit.
More preferably, sealer is selected from seralbumin or ovalbumin, and concentration is 0.1%～5%, and the best is 1% bovine serum albumin(BSA); Sealer is used for being adsorbed on the not latex particle surface of binding antibody, and reduces the nonspecific reaction in testing process.
More preferably, deactivation liquid is selected from monoethanolamine or glycocoll, and concentration is 5～20mM, and the best is 10mM glycocoll; For the excessive reactive intermediate of neutralization reaction.
More preferably, preserve damping fluid and be selected from glycine buffer, borate buffer or phosphate buffer, the best is 0.1M pH8.0 glycocoll by concentration, concentration is respectively 0.2% Sodium azide, 0.2% bovine serum albumin(BSA) and 0.05% Tween-20 composition.
Preferably, the sodium chloride that application liquid is 0.1%～10% by surfactant 0.05%～0.5%, antiseptic 0.05%～1%, macromolecule accelerator 0.01%～5% and concentration by mass percentage forms, and the mass percent sum of above-mentioned each component is 100%.
More preferably, surfactant is selected from Tween-20 or Triton-X100, and the best is Tween-20; Antiseptic is selected from Proclin300 or Sodium azide, and the best is Sodium azide, and macromolecule accelerator is selected from bovine serum albumin(BSA) or ovalbumin, and buffering agent is selected from trishydroxymethylaminomethane, phosphate or glycocoll, and the best is trishydroxymethylaminomethane.
The reaction buffer of the vWF that preferably, contains variable concentrations is selected from one or more compositions in PBS damping fluid, Hepes damping fluid, glycine buffer, borate buffer solution, acetate buffer or ammonium chloride buffer; Reaction buffer is preferably one or more compositions in PBS damping fluid, Hepes damping fluid, glycine buffer.
More preferably, the concentration of reaction buffer is 20～200mM.
Another object of the present invention is to provide a kind of application of the reagent for detection of vWF, be about to above-mentioned arbitrary reagent for detection of vWF as detecting reagent for the preparation of vWF detection kit, it comprises the reagent bottle of box body and above-mentioned arbitrary detection reagent kit.
Preferably, in reagent bottle placement and box body.
Another object of the present invention is to provide a kind of preparation method of the reagent for detection of vWF, comprises the following steps:
Step a) is prepared anti-vWF antibody latex: will resist vWF antibody through two step reaction post-crosslinkings, to become anti-vWF antibody latex with latex particle;
Step is Application and preparation liquid or dilution b).
Preferably, step concrete operations a) are as follows: after latex particle is cleaned with pure water, be suspended in activation damping fluid; Add bonding agent to be less than stirring reaction at 50 ℃ in temperature, thus activation latex particle; Add again anti-vWF antibody in temperature, to be less than stirring reaction under 50 degree in binding buffer liquid; Be suspended in again in the deactivation liquid that contains sealer and react, after cleaning, be suspended in and preserve in damping fluid, obtain anti-vWF antibody latex suspension, be placed in 4 ℃ of environment and place.
More preferably, step a) middle stirring reaction temperature is preferably room temperature, and the best is 18～25 ℃.
More preferably, it is 10～30 minutes that step adds the stirring reaction time after bonding agent in a), and the best is 15 minutes;
More preferably, step a) in binding buffer liquid the stirring reaction time be 0.5～6 hour, be preferably 1～2 hour, the best is 2 hours.
More preferably, step is suspended in the deactivation liquid that contains sealer in a) reacts 1～3 hour, and the best is 1 hour.
Preferably, step b) Application and preparation liquid is that surfactant, antiseptic, macromolecule accelerator, sodium chloride and buffering agent are dissolved in the water in the lump completely, makes application liquid.
More preferably, step concrete operations b) are as follows: it is 8.0 that buffering agent is adjusted to pH value with hydrochloric acid, then add surfactant, antiseptic, macromolecule accelerator and sodium chloride.
More preferably, step b) middle application liquid pH value is 7.4～8.0, and the best is 8.0; The shared mass percent of surfactant is 0.05%～0.5%, and the best is 0.05%, and the shared mass percent of antiseptic is 0.05%～1%, and the best is 0.2%, and the shared mass percent of macromolecule accelerator is 0.01%～5%, and the best is 0.2%; The shared mass percent of sodium chloride is 0.1%～10%, and the best is 1%, and the mass percent of buffering agent is 10～100mM, and the best is 50mM.
Preferably, under the prerequisite of the typical curve that does not provide vWF in human body, this preparation method also comprises that step c) prepares vWF standard items.
More preferably, step is selected vWF standard items in c), uses reagent 2, apply liquid, carry out serial dilution: S7:40 μ g/ml, S6:20 μ g/ml, S5:10 μ g/ml, S4:5 μ g/ml, S3:2.5 μ g/ml, S2:1 μ g/ml, S1:0.5 μ g/ml, S0: application liquid, packing.
More preferably, step c) in, vWF standard items can be selected commercialization high-purity vWF standard items.
More preferably, step c) in packing can adopt the packing of 0.5ml/ bottle.
The actual conditions that the present invention also provides the particle-enhanced turbidimetric immunoassay kit that adopts this detection vWF to detect on Biochemical Analyzer:
Setup parameter on Biochemical Analyzer:
Temperature of reaction: 37 ℃;
Analytical approach: 2 end-point methods;
Predominant wavelength: 570nm, commplementary wave length: 800nm (can).
Select, after multiple spot calibration, by instrument, automatically to complete calibration, the vWF that then carries out normal plasma sample detects, and result is through the instrument directly report numerical value take μ g/ml as unit of conversion automatically.When using different automatic testers (coaglation analyzer or Biochemical Analyzer), design parameter should adjust accordingly according to instrument difference.
The particle-enhanced turbidimetric immune assay method by being widely used gradually at present that the present invention initiates is applied in the detection of vWF.The detection principle of above-mentioned detection reagent is that a latex particle that has been cross-linked antibody is mixed with detected plasma sample, and the multivalence vWF containing in blood plasma can be because causing the gathering of latex particle with the combination of antibody.The gathering causing can cause the variation of muddiness and the absorbance of sample, and its absorbance is directly proportional to the concentration of vWF, with the calibration solution comparison of same processing, can be calculated and be calculated vWF concentration in sample by absorbance.The present invention adopts particle-enhanced turbidimetric immune assay method to detect vWF; Can avoid the association reaction (plastic plate reacting hole) under solid phase condition of antigen and antibody in traditional euzymelinked immunosorbent assay (ELISA) to carry out, the two one of be fixed and cannot move freely; And the reaction that realizes antibody and latex is to carry out under liquid-phase condition, be swift in response thoroughly, more approach state of nature, the mode strengthening by particle, amplified antigen-antibody reaction, made up the not high deficiency of common turbidimetry sensitivity, turbidimetry good stability, advantage have easily and fast been inherited again simultaneously, overcome ELISA and RIA method complicated operation, bad etc. the shortcoming of repeatability, realize human blood vWF fast detecting blood vWF level in enormous quantities in biochemical instruments.
Experimental result shows provided by the invention and has the remarkable advantage of the following aspects: (1) is simple to operate; (2) sensitivity is higher; (3) be not subject to manual operation and extraneous factor and disturb, detect stability and reproducible, can reflect more truly the content of measured matter; (4) can utilize Biochemical Analyzer to detect, easily realize robotization, be suitable for clinical application; Compare polymer method, euzymelinked immunosorbent assay (ELISA) and collagen method, the present invention can be applied on automatic blood coagulation instrument, and has feature easy and simple to handle, highly sensitive and reproducible.Therefore, the present invention can be used to carry out a large amount of and clinical vitro detection fast, has good application prospect.
Accompanying drawing explanation
Fig. 1 is the response curve figure that in prior art, latex particle immunity strengthens turbidimetry.
Embodiment
Below in conjunction with embodiment, the present invention is done the explanation of further detailed complete.If no special instructions, in following examples, agents useful for same or instrument are commercially available prod, and use according to its instructions operation.
Embodiment 1
A) preparation of reagent 1
Reagent 1 reacts the antibody latex reagent that must resist vWF for the latex particle that carboxylic group is contained in surface under bonding agent effect with the amino group of antibody; Concrete preparation method is as follows:
Preparation 0.05M KH
2pO
4the activation damping fluid of pH4.5, is suspended in 10ml activation damping fluid after the latex particle pure water of getting particle diameter and be the carboxyl modified of 500nm cleans three times, and latex particle final concentration quality percent by volume counts 1% with g/ml.The acid pH of activation damping fluid is in order to guarantee that carboxyl is in carboxyl coordination steady state (SS), and this is conducive to the carrying out of reaction.
Fresh preparation 2% carbodiimide again, gets 10ml and adds in equal-volume latex particle, at room temperature (18-25 ℃) stirring reaction 15 minutes; Thereby activation latex particle.
The binding buffer liquid of preparation 0.2M borate buffer pH8.5; The latex particle that cleans three times activation with pure water, is suspended in 10ml binding buffer liquid; Then add isopyknic mouse-anti vWF monoclonal antibody, in binding buffer liquid, in room temperature (18-25 ℃), descend stirring reaction 2 hours, complete two steps reactions.The alkalescent pH of binding buffer liquid is amino in amino coordination steady state (SS) in order to guarantee, this is conducive to the carrying out of reaction.
With being suspended in the deactivation liquid that contains sealer and reacting 1 hour after three times latex particles of pure water cleaning; Sealer is wherein 1% bovine serum albumin(BSA), is in order to be adsorbed on the not latex particle surface of binding antibody, and reduces the nonspecific reaction in testing process; Deactivation liquid is 10mM glycocoll, is in order to neutralize reactive intermediate excessive in reaction.Preserving damping fluid is 0.1M glycocoll pH8.0,0.2% Sodium azide, 0.2% bovine serum albumin(BSA) and 0.05% Tween-20.After cleaning, be suspended in and preserve in damping fluid.
B) preparation of reagent 2
Reagent 2 is application liquid, and its preparation method is: trishydroxymethylaminomethane (Tris) concentration is 50mM, and after regulating with hydrochloric acid, pH value is 8.0.Add following composition, its final concentration is respectively again: sodium chloride 1%, Sodium azide 0.2%, bovine serum albumin(BSA) 0.2% and Tween-20 0.05%.
C) configuration vWF standard items, are used application liquid to carry out serial dilution: S7:40 μ g/ml, S6:20 μ g/ml, S5:10 μ g/ml, S4:5 μ g/ml, S3:2.5 μ g/ml, S2:1 μ g/ml, S1:0.5 μ g/ml, S0: application liquid.
Detect specificity, sensitivity and the range of linearity of gained kit and see following experiment.
Embodiment 2
The difference of the present embodiment and embodiment 1 is only that the particle diameter of described latex particle is 1000nm, and other operations and proportioning are consistent with embodiment 1.
Detect specificity, sensitivity and the range of linearity of gained kit and see following experiment.
The detection of specificity, sensitivity and the range of linearity of kit
1. the detection running program on automatic tester, specific as follows:
(1) venous blood of fresh extraction mixes rear centrifugally in the ratio of 9:1 and citrate anticoagulation liquid, separates and obtains blood plasma;
(2) setup parameter on automatic tester: 37 ℃ of temperature of reaction; Analytical approach: 2 end-point methods; Predominant wavelength: 570nm, commplementary wave length: 800nm; Sample size/reagent 1/ reagent 2:20 μ l/100 μ l/100 μ l; The Direction of Reaction: rise;
(3) select vWF standard items, use application liquid to carry out serial dilution: S7:40 μ g/ml, S6:20 μ g/ml, S5:10 μ g/ml, S4:5 μ g/ml, S3:2.5 μ g/ml, S2:1 μ g/ml, S1:0.5 μ g/ml, S0: application liquid.
(4) in automatic tester, add the standard items of variable concentrations, (standard items/reagent 1/ reagent 2:20 μ l/100 μ l/100 μ l) and open the reaction of beginning for reagent 1 and reagent 2.Automatic tester will automatically be measured absorbance and make " concentration-absorbance " typical curve.
(5) get plasma sample to be measured, measure as stated above absorbance.Automatic tester will directly be reported the vWF numerical value of sample by result through automatically converting according to " concentration-absorbance " typical curve take μ g/ml as unit.
(6) when using different automatic testers (coaglation analyzer or Biochemical Analyzer), design parameter should adjust accordingly according to instrument difference.
2. the specific detection of kit
Table 1 detects vWF content method specificity
Result shows: the specificity of this detection kit is high, and with haemoglobin, cholerythrin, the multiple factor such as triglyceride is not intersected.
3. the detection of kit sensitivity and linear measurement range
(1) sensitivity
The sensitivity of table 2 vWF detection method
Result shows: vWF content when 40-0.5 μ g/ml, OD value >=negative control * 2.1, the detection sensitivity of the method is 0.5 μ g/ml.
(2) sensing range and linearity:
The table 3 vWF detection method range of linearity
Result shows: vWF content when 0.5-40 μ g/ml, linear >=0.98, the vWF scope 0.5-40 μ g/ml of detection.
Embodiment 3
A) preparation of reagent 1
Reagent 1 reacts the antibody latex reagent that must resist vWF for the latex particle that carboxylic group is contained in surface under bonding agent effect with the amino group of antibody; Concrete preparation method is as follows:
Preparation 0.0SM KH
2pO
4the activation damping fluid of pH4.5, is suspended in 10ml activation damping fluid after the latex particle pure water of getting particle diameter and be the carboxyl modified of 500nm cleans three times, and latex particle final concentration quality percent by volume counts 1% with g/ml.The acid pH of activation damping fluid is in order to guarantee that carboxyl is in carboxyl coordination steady state (SS), and this is conducive to the carrying out of reaction.
Fresh preparation 2% carbodiimide again, gets 10ml and adds in equal-volume latex particle, at room temperature (18-25 ℃) stirring reaction 10 minutes; Thereby activation latex particle.
The binding buffer liquid of preparation 0.2M borate buffer pH8.5; The latex particle that cleans three times activation with pure water, is suspended in 10ml binding buffer liquid; Then add isopyknic mouse-anti vWF monoclonal antibody, in binding buffer liquid, in room temperature (18-25 ℃), descend stirring reaction 0.5 hour, complete two steps reactions.The alkalescent pH of binding buffer liquid is amino in amino coordination steady state (SS) in order to guarantee, this is conducive to the carrying out of reaction.
With being suspended in the deactivation liquid that contains sealer and reacting 2 hours after three times latex particles of pure water cleaning; Sealer is wherein 1% bovine serum albumin(BSA), is in order to be adsorbed on the not latex particle surface of binding antibody, and reduces the nonspecific reaction in testing process; Deactivation liquid is 10mM glycocoll, is in order to neutralize reactive intermediate excessive in reaction.Preserving damping fluid is 0.1M glycocoll pH8.0,0.2% Sodium azide, 0.2% bovine serum albumin(BSA) and 0.05% Tween-20.After cleaning, be suspended in and preserve in damping fluid.
B) preparation of reagent 2
Reagent 2 is application liquid, and its preparation method is: trishydroxymethylaminomethane (Tris) concentration is 10mM, and after regulating with hydrochloric acid, pH value is 7.7.Add following composition, its final concentration is respectively again: sodium chloride 0.1%, Sodium azide 0.05%, bovine serum albumin(BSA) 0.01% and Tween-20 0.2%.
C) configuration vWF standard items, are used application liquid to carry out serial dilution: S7:40 μ g/ml, S6:20 μ g/ml, S5:10 μ g/ml, S4:5 μ g/ml, S3:2.5 μ g/ml, S2:1 μ g/ml, S1:0.5 μ g/ml, S0: application liquid.
There is the consistance within the scope of theoretical error in the kit data that specificity, sensitivity and the range of linearity the data obtained of detection gained kit and embodiment 1 make.
Embodiment 4
A) preparation of reagent 1
Reagent 1 reacts the antibody latex reagent that must resist vWF for the latex particle that carboxylic group is contained in surface under bonding agent effect with the amino group of antibody; Concrete preparation method is as follows:
Preparation 0.05M KH
2pO
4the activation damping fluid of pH4.5, is suspended in 10ml activation damping fluid after the latex particle pure water of getting particle diameter and be the carboxyl modified of 500nm cleans three times, and latex particle final concentration quality percent by volume counts 1% with g/ml.The acid pH of activation damping fluid is in order to guarantee that carboxyl is in carboxyl coordination steady state (SS), and this is conducive to the carrying out of reaction.
Fresh preparation 2% carbodiimide again, gets 10ml and adds in equal-volume latex particle, at room temperature (18-25 ℃) stirring reaction 30 minutes; Thereby activation latex particle.
The binding buffer liquid of preparation 0.2M borate buffer pH8.5; The latex particle that cleans three times activation with pure water, is suspended in 10ml binding buffer liquid; Then add isopyknic mouse-anti vWF monoclonal antibody, in binding buffer liquid, in room temperature (18-25 ℃), descend stirring reaction 6 hours, complete two steps reactions.The alkalescent pH of binding buffer liquid is amino in amino coordination steady state (SS) in order to guarantee, this is conducive to the carrying out of reaction.
With being suspended in the deactivation liquid that contains sealer and reacting 3 hours after three times latex particles of pure water cleaning; Sealer is wherein 1% bovine serum albumin(BSA), is in order to be adsorbed on the not latex particle surface of binding antibody, and reduces the nonspecific reaction in testing process; Deactivation liquid is 10mM glycocoll, is in order to neutralize reactive intermediate excessive in reaction.Preserving damping fluid is 0.1M glycocoll pH8.0,0.2% Sodium azide, 0.2% bovine serum albumin(BSA) and 0.05% Tween-20.After cleaning, be suspended in and preserve in damping fluid.
B) preparation of reagent 2
Reagent 2 is application liquid, and its preparation method is: trishydroxymethylaminomethane (Tris) concentration is 100mM, and after regulating with hydrochloric acid, pH value is 7.4.Add following composition, its final concentration is respectively again: sodium chloride 10%, Sodium azide 0.1%, bovine serum albumin(BSA) 5% and Tween-20 0.5%.
C) configuration vWF standard items, are used application liquid to carry out serial dilution: S7:40 μ g/ml, S6:20 μ g/ml, S5:10 μ g/ml, S4:5 μ g/ml, S3:2.5 μ g/ml, S2:1 μ g/ml, S1:0.5 μ g/ml, S0: application liquid.
There is the consistance within the scope of theoretical error in the kit data that specificity, sensitivity and the range of linearity the data obtained of detection gained kit and embodiment 1 make.
Above-mentioned experimental data shows: employing particle-enhanced turbidimetric immune assay method provided by the invention detects reagent and the method for vWF, compare polymer method, euzymelinked immunosorbent assay (ELISA) and collagen method, detection reagent provided by the invention or kit can be applied on automatic blood coagulation instrument, and there is feature easy and simple to handle, highly sensitive and reproducible.Therefore, testing product provided by the invention can be used to carry out a large amount of and clinical vitro detection fast, has significantly reduced testing cost, has significant economic benefit.
Finally be necessary described herein: above embodiment is only for being described in more detail technical scheme of the present invention; can not be interpreted as limiting the scope of the invention, some nonessential improvement that those skilled in the art's foregoing according to the present invention is made and adjustment all belong to protection scope of the present invention.
Claims (10)
1. the reagent for detection of vWF, it is characterized in that: the described reagent for detection of vWF is the detection reagent of application latex enhancing immune turbidimetry, by reagent 1 and reagent 2, formed, the antibody latex reagent that wherein reagent 1 is anti-vWF, the antibody latex reagent of anti-vWF is the antibody latex reagent of the anti-vWF that is combined into of anti-vWF and latex particle; Reagent 2 is application liquid.
2. the reagent for detection of vWF according to claim 1, is characterized in that: the described reagent for detection of vWF also comprises vWF standard items or typical curve; Described vWF standard items are the reaction buffer of the vWF that contains variable concentrations; Typical curve is the typical curve of vWF at people's in-vivo content.
3. the reagent for detection of vWF according to claim 1, is characterized in that: the antibody latex reagent of described anti-vWF is to obtain by the amino of the antibody of anti-vWF is formed to covalent bond with the carboxyl of latex particle after two steps are reacted; Described two step reactions comprise: the first step is first reacted latex particle with bonding agent, second step is again by latex particle and anti-vWF antibody response.
4. the reagent for detection of vWF according to claim 3, is characterized in that: the antibody latex reagent of described anti-vWF is to obtain by the amino of the antibody of anti-vWF is formed to covalent bond with the carboxyl of latex particle after two steps are reacted; First by the carboxyl of latex particle and bonding agent are reacted into latex particle-bonding agent intermediate under faintly acid, then by latex particle-bonding agent intermediate, under alkalescent, make anti-vWF antibody latex reagent with amino reaction of anti-vWF antibody.
5. the reagent for detection of vWF according to claim 1, is characterized in that: described latex particle is styrene latex particle.
6. the reagent for detection of vWF according to claim 1, is characterized in that: the particle diameter of described latex particle is 200～1000nm.
7. the reagent for detection of vWF according to claim 8, is characterized in that: it is 0.1～5% carbodiimide that described bonding agent is selected from concentration.
8. a method of preparing the arbitrary described reagent for detection of vWF of claim 1～7, is characterized in that, comprises the following steps:
Step a) is prepared anti-vWF antibody latex: will resist vWF antibody and glue
Breast particle becomes anti-vWF antibody latex through two step reaction post-crosslinkings;
Step is Application and preparation liquid or dilution b).
9. the method for the reagent for the preparation of detection vWF according to claim 8, is characterized in that, step concrete operations a) are as follows: after latex particle is cleaned with pure water, be suspended in activation damping fluid; Add bonding agent to be less than stirring reaction at 50 ℃ in temperature, thus activation latex particle; Add again anti-vWF antibody in temperature, to be less than stirring reaction under 50 degree in binding buffer liquid; Be suspended in again in the deactivation liquid that contains sealer and react, after cleaning, be suspended in and preserve in damping fluid, obtain anti-vWF antibody latex suspension, be placed in 4 ℃ of environment and place.
10. prepare an application for the arbitrary described reagent for detection of vWF of claim 1～7, it is characterized in that: using arbitrary claim 1～7 described reagent for detection of vWF as detecting reagent for the preparation of vWF testing product.
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| WO2009026551A1 (en) * | 2007-08-23 | 2009-02-26 | Bloodcenter Of Wisconsin, Inc. | Methods and kits for measuring von willebrand factor |
| CN101932606A (en) * | 2008-01-23 | 2010-12-29 | 格兰马克药品股份有限公司 | Humanized antibodies specific for von willebrand factor |
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| CN102608325A (en) * | 2012-02-24 | 2012-07-25 | 南京诺尔曼生物技术有限公司 | Fatty acid-binding protein (H-FABP) determination kit (latex enhanced immunoturbidimetric assay) |
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| WO2009026551A1 (en) * | 2007-08-23 | 2009-02-26 | Bloodcenter Of Wisconsin, Inc. | Methods and kits for measuring von willebrand factor |
| CN101932606A (en) * | 2008-01-23 | 2010-12-29 | 格兰马克药品股份有限公司 | Humanized antibodies specific for von willebrand factor |
| CN102532316A (en) * | 2010-12-24 | 2012-07-04 | 神州细胞工程有限公司 | Anti-vWF (von Willebrand factor) monoclonal antibody and application thereof |
| CN102608325A (en) * | 2012-02-24 | 2012-07-25 | 南京诺尔曼生物技术有限公司 | Fatty acid-binding protein (H-FABP) determination kit (latex enhanced immunoturbidimetric assay) |
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