Invention content
The present inventor passes through a large amount of experimental study, it was thus unexpectedly found that：In the whole blood sample based on immunoturbidimetryIt, can will not by using specific reagent composition in the quantitative detection process of middle test substance (for example, c reactive protein)The whole blood sample of haemolysis is directly contacted with the latex particle of antibody sensitized, to be detected to the variation of its turbidity.Based on thisIt was found that inventor developed the new immue quantitative detection reagent boxes and detection method for test substance in whole blood sample.
Kit
Therefore, in one aspect, the present invention provides a kind of kit, it includes：
(a) insoluble carrier granular；
(b) antibody of the test substance is specifically bound；With
(c) the first reagent composition, it includes betaine type amphoteric surfactants.
In certain preferred aspects, the kit measures test substance in blood sample for immunoturbidimetryPresence or its amount.In certain preferred aspects, the blood sample is selected from whole blood, blood plasma or serum.Certain excellentIn the embodiment of choosing, the blood sample is anticoagulated whole blood, and non-haemolysis.
In certain preferred aspects, the test substance is the albumen in plasma component, such as c reactive proteinDeng.In certain exemplary embodiments, the test substance is c reactive protein (CRP).
In certain preferred aspects, the betaine type amphoteric surfactant is selected from alkyl betaine, alkylamideGlycine betaine, azochlorosulfonate propyl lycine, hydroxyl azochlorosulfonate propyl lycine, phosphate ester glycine betaine, and its arbitrary combination.In certain preferred realitiesIt applies in scheme, first reagent composition includes 3- sulfopropyls-etradecyldimethylamine glycine betaine.In certain preferred embodiment partyIn case, the content of the betaine type amphoteric surfactant is 0.01-4% (w/v).In certain preferred aspects, describedThe content of betaine type amphoteric surfactant is 0.05-0.5% (w/v).
In certain preferred aspects, the kit also includes the second reagent composition, second reagent setIt includes sealer to close object.In certain preferred aspects, the sealer is N6-PEG.
In certain preferred aspects, first reagent composition further includes one or more selected from following examinationAgent：Buffer solution, preservative, suspending agent, stabilizer and coagulant.
In certain preferred aspects, the buffer solution is selected from glycine buffer, MES buffer solutions, MOPSO bufferingsLiquid, Tris buffer solutions, phosphate buffer, borate buffer solution, and its arbitrary combination.In certain preferred aspects,The content of the buffer solution is 0.02-0.1mol/L.
In certain preferred aspects, the preservative be selected from sodium azide, sorbic acid salt, benzoic acid and itsSalt, sodium nitrite and Prolin-300.In certain preferred aspects, the content of the preservative is 0.5-1.5g/L.
In certain preferred aspects, the suspending agent is selected from glycerine, ethylene glycol, mannitol and its arbitrary groupIt closes.In certain preferred aspects, the content of the suspending agent is 1-30% (v/v).
In certain preferred aspects, the stabilizer sodium chloride, magnesium chloride, disodium ethylene diamine tetraacetate, ox bloodPure albumen, glycine, gelatin, and its arbitrary combination.In certain preferred aspects, the content of sodium chloride or magnesium chlorideFor 0.05-1.5% (w/v), the content of disodium ethylene diamine tetraacetate is 0.01-1% (w/v), bovine serum albumin(BSA) or gelatinContent is 0.1-3% (w/v).
In certain preferred aspects, the coagulant is selected from Macrogol 6000, PEG 8000, poly- second twoAlcohol 10000, PEG 20000, dextran sulfate, and its arbitrary combination.In certain preferred aspects, described to promote to coagulateThe content of agent is 0.5-3% (w/v).
In certain preferred aspects, first reagent composition consists of the following compositions：Betaine type surfaceThe water of activating agent, preservative, suspending agent, coagulant, buffer solution and surplus.
In certain exemplary implementation schemes, first reagent composition consists of the following compositions：3- sulfopropyls 16Water (the example of alkyl betaine, sodium azide, mannitol, polyethylene glycol (for example, Macrogol 6000), glycine and surplusSuch as, deionized water, distilled water etc.).
In certain exemplary implementation schemes, first reagent composition consists of the following compositions：0.05% (w/v) 3-Sulfopropyl cetyl betaine, 1g/L sodium azide, 0.01M mannitol, 0.5% (w/v) Macrogol 6000,0.01M are sweetThe water (for example, deionized water, distilled water etc.) of propylhomoserin and surplus.
In the present invention, the insoluble carrier granular includes being known to be used in appointing for immunoturbidimetry (enhancing turbidimetry)What carrier granular, such as latex particle or particles of polylactic acid.In certain preferred aspects, the insoluble carrier granularIt is latex particle.The material of latex particle is not particularly limited, and non-limiting examples include polystyrene, styrene-fourth twoAlkene copolymer, copolymer in cinnamic acrylic ester, styrene-maleic acid copolymer, polyethyleneimine, polyacrylic acid, poly- methylAcrylic acid, polymethyl methacrylate etc..In certain preferred aspects, the insoluble carrier granular is polystyreneParticle.
In certain preferred aspects, second reagent composition also includes for the antibody to be coated in instituteState insoluble carrier particle surface reagent and optional coating buffer solution (for example, carbonate buffer solution, phosphate-bufferedLiquid, Tris-HCL buffer solutions or borate buffer solution etc.).
In the present invention, antibody can be coated in insoluble carrier granular table with various technologies well known by persons skilled in the artFace, for example, physical absorption or by functional group's (such as carboxyl, amino, hydroxyl, hydrazides or sulfydryl) change surface realized it is covalentCoupling.The detailed teachings of covalent coupling can be found in such as TechNote 205, Rev.003, for example March, and 2002," Covalent Coupling " (being incorporated herein by reference) can test company (Bangs from BangsLaboratories, Inc) webpage on download.Those skilled in the art are known how according to insoluble carrier particle surface instituteReagent of the functional group's selection of modification suitably for antibody to be coated in the insoluble carrier particle surface.
Therefore, in certain preferred aspects, the surface of the insoluble carrier granular is with carboxyl, amino, hydroxylBase, hydrazide group or sulfydryl modification.In certain exemplary embodiments, the surface of the insoluble carrier granular is repaiied with carboxylDecorations.In such embodiment, the antibody can be coated in by the coupling reaction between amino and carboxyl described insolubleProperty carrier particle surface.Therefore, in certain exemplary embodiments, second reagent composition includes EDC or its salt (exampleSuch as EDCI) and NHS.
In certain preferred aspects, the pan coating of the insoluble carrier granular has and is waited for described in specific bindingSurvey the antibody of substance.
In certain preferred aspects, the pan coating of the insoluble carrier granular has and is waited for described in specific bindingSurvey the antibody and N6-PEG of substance.
In certain exemplary embodiments, the test substance is c reactive protein (CRP).In such embodiment,The kit includes the antibody of specific binding CRP.
In certain preferred aspects, the antibody is monoclonal antibody or polyclonal antibody.Certain preferredIn embodiment, the antibody is polyclonal antibody.
In certain preferred aspects, the kit also includes one or more selected from following reagent or dressIt sets：Standard items (for example, series of samples of the test substance containing different known quantities)；Positive control sample is (for example, containing knownThe sample of the test substance of amount)；Negative control sample (for example, sample without containing test substance)；Anti-coagulants (such as heparin)；With blood-taking device (for example, apyrogeneity vacuum blood collection tube).
In certain preferred aspects, each component as described above that kit of the invention is included separately carriesFor.
In certain preferred aspects, the insoluble carrier granular, the specific binding test substanceAntibody, first reagent composition and second reagent composition be provided separately.
In certain preferred aspects, the insoluble carrier granular with first reagent composition as sameComponent provides.In such embodiment, the pan coating of the insoluble carrier granular has the specific binding determinandThe antibody of matter；Alternatively, the pan coating of the insoluble carrier granular has the antibody for specifically binding the test substance, andN6-PEG.In certain preferred aspects, the insoluble carrier granular and first reagent composition are with the shape that is suspendedFormula combination provides.
Detection method
On the other hand, the present invention provides a kind of presence of test substance in whole blood sample measuring non-haemolysis or itsThe immunoturbidimetry of amount comprising following steps：
It (1), will the non-haemolysis in the first reagent composition under the conditions of antigen-antibody complex forms permittedWhole blood sample contacted with insoluble carrier granular；
(2) the turbidity variation of the reaction system of determination step (1)；
(3) the turbidity variation obtained step (2) changes with the known quantity of the test substance is indicated with the turbidityThe standard curve of relationship compare, and obtain the content of the test substance；
Wherein, the pan coating of the insoluble carrier has the antibody for specifically binding the test substance；Also, it is describedFirst reagent composition includes betaine type amphoteric surfactant.
In certain preferred aspects, the whole blood sample is anticoagulated whole blood.
In certain preferred aspects, the test substance is the albumen in plasma component, such as c reactive proteinDeng.In certain exemplary embodiments, the test substance is c reactive protein (CRP).
In certain preferred aspects, the whole blood sample of the non-haemolysis contains the test substance.Certain excellentIn the embodiment of choosing, the whole blood sample of the non-haemolysis contains CRP.
In certain preferred aspects, method of the invention is used for non-diagnostic purpose.
In certain preferred aspects, the betaine type amphoteric surfactant is selected from alkyl betaine, alkylamideIt is one or more in glycine betaine, azochlorosulfonate propyl lycine, hydroxyl azochlorosulfonate propyl lycine, phosphate ester glycine betaine.Certain preferredIn embodiment, first reagent composition includes 3- sulfopropyls-etradecyldimethylamine glycine betaine.In certain preferred implementationsIn scheme, the content of the betaine type amphoteric surfactant is 0.01-4% (w/v).
In certain preferred aspects, in step (1), the pan coating of the insoluble carrier granular has N6-PEG。
In certain preferred aspects, first reagent composition further includes one or more selected from following examinationAgent：Buffer solution, preservative, suspending agent, stabilizer and coagulant.
In certain preferred aspects, the buffer solution is selected from glycine buffer, MES buffer solutions, MOPSO bufferingsLiquid, Tris buffer solutions, phosphate buffer, borate buffer solution, and its arbitrary combination.In certain preferred aspects,The content of the buffer solution is 0.02-0.1mol/L.
In certain preferred aspects, the preservative be selected from sodium azide, sorbic acid salt, benzoic acid and itsSalt, sodium nitrite and Prolin-300.In certain preferred aspects, the content of the preservative is 0.5-1.5g/L.
In certain preferred aspects, the suspending agent is selected from glycerine, ethylene glycol, mannitol and its arbitrary groupIt closes.In certain preferred aspects, the content of the suspending agent is 1-30% (v/v).
In certain preferred aspects, the stabilizer sodium chloride, magnesium chloride, disodium ethylene diamine tetraacetate, ox bloodPure albumen, glycine, gelatin, and its arbitrary combination.In certain preferred aspects, the content of sodium chloride or magnesium chlorideFor 0.05-1.5% (w/v), the content of disodium ethylene diamine tetraacetate is 0.01-1% (w/v), bovine serum albumin(BSA) or gelatinContent is 0.1-3% (w/v).
In certain preferred aspects, the coagulant is selected from Macrogol 6000, PEG 8000, poly- second twoAlcohol 10000, PEG 20000, dextran sulfate, and its arbitrary combination.In certain preferred aspects, described to promote to coagulateThe content of agent is 0.5-3% (w/v).
In certain preferred aspects, first reagent composition consists of the following compositions：Betaine type surfaceThe water of activating agent, preservative, suspending agent, coagulant, buffer solution and surplus.
In certain exemplary implementation schemes, first reagent composition consists of the following compositions：3- sulfopropyls 16Water (the example of alkyl betaine, sodium azide, mannitol, polyethylene glycol (for example, Macrogol 6000), glycine and surplusSuch as, deionized water, distilled water etc.).
In certain exemplary implementation schemes, first reagent composition consists of the following compositions：0.05% (w/v) 3-Sulfopropyl cetyl betaine, 1g/L sodium azide, 0.01M mannitol, 0.5% (w/v) Macrogol 6000,0.01M are sweetThe water (for example, deionized water, distilled water etc.) of propylhomoserin and surplus.
In certain preferred aspects, in the reaction system of step (1), the content of the insoluble carrier granularFor 2-5g/L.
In certain preferred aspects, the insoluble carrier granular is latex particle.The material of latex particle does not haveIt is particularly limited to, non-limiting examples include polystyrene, styrene-butadiene copolymer, cinnamic acrylic ester copolymerizationObject, styrene-maleic acid copolymer, polyethyleneimine, polyacrylic acid, polymethylacrylic acid, polymethyl methacrylate etc..In certain preferred embodiments, the insoluble carrier granular is granules of polystyrene.
In certain preferred aspects, in step (1), the surface institute of the insoluble carrier granular is coated anti-With test substance immune response can occur for body, and then it is solidifying to occur so that the insoluble carrier granular is aggregated in the reaction systemCollection reaction.
In the present invention, the method for the turbidity variation of the reaction system of determination step (1) is that those skilled in the art are known's.For example, can the reaction of optically determination step (1) start in rear certain time (for example, 0-5 minutes, 0-2 pointsClock, 5-120 seconds or 5-60 seconds) absorbance at provision wavelengths or the variable quantity of light is scattered, the variation using it as turbidity.
Therefore, in certain preferred aspects, in step (2), after the reaction of step (1) starts：(a) with suitableIt is measured when time interval carries out 2 times to absorbance of the reaction system at provision wavelengths, using its difference as the variation of absorbanceIt measures (that is, turbidity variation)；Alternatively, (b) absorbance to reaction system at provision wavelengths carries out METHOD FOR CONTINUOUS DETERMINATION, when with unitBetween variable quantity (that is, turbidity variation) of the absorbance change rate as absorbance.
In certain preferred aspects, in step (2), start (example in 0-120 seconds latter in the reaction of step (1)Such as 5-120 seconds, such as 5-60 seconds) 2 measurement are carried out to absorbance of the reaction system at provision wavelengths, using its difference as suctionThe variable quantity (that is, turbidity variation) of luminosity.In certain preferred aspects, in step (2), in the anti-of step (1)To reaction system, absorbance at provision wavelengths carries out 2 measurement respectively within the 6th second and the 60th second after should starting, and is made with its differenceFor the variable quantity (that is, turbidity variation) of absorbance.
In certain preferred aspects, the provision wavelengths are 800-900nm, such as 840-860nm, such as850nm.In certain exemplary embodiments, the provision wavelengths are 850nm.
In certain exemplary embodiments, in step (2), the 6th second and the 60th second after the reaction of step (1) startsThe absorbance to reaction system at 850nm carries out 2 measurement respectively, and the variable quantity using its difference as absorbance is (that is, turbidDegree variation).
In certain preferred aspects, further include closing the insoluble load using N6-PEG before step (1)The step of body particle.Therefore, in certain preferred aspects, in step (1), the insoluble carrier granular is also coated withThere is N6-PEG.
In certain preferred aspects, further include one or more in the following steps before step (1)：(a)Using blood-taking device whole blood sample is obtained from subject；(b) anti-coagulants (such as heparin) processing blood-taking device or described complete is usedBlood sample.
Detection applications
On the other hand, the purposes the present invention relates to betaine type amphoteric surfactant in reagent preparation box, the examinationAgent box measures the presence of test substance or its amount in the whole blood sample of non-haemolysis by immunoturbidimetry, wherein the kitIncluding：
(a) insoluble carrier granular；
(b) antibody of the test substance is specifically bound；With
(c) the first reagent composition, it includes betaine type amphoteric surfactants.
In certain preferred aspects, the present invention relates to betaine type amphoteric surfactant, insoluble carrier granular andPurposes of the antibody of the test substance in reagent preparation box is specifically bound, the kit is surveyed by immunoturbidimetryThe presence of test substance or its amount in the whole blood sample of fixed non-haemolysis.
In certain preferred aspects, the whole blood sample is anticoagulated whole blood.
In certain preferred aspects, the test substance is the albumen in plasma component, such as c reactive proteinDeng.In certain exemplary embodiments, the test substance is c reactive protein (CRP).
In certain preferred aspects, the betaine type amphoteric surfactant is selected from alkyl betaine, alkylamideGlycine betaine, azochlorosulfonate propyl lycine, hydroxyl azochlorosulfonate propyl lycine, phosphate ester glycine betaine, and its arbitrary combination.In certain preferred realitiesIt applies in scheme, the betaine type amphoteric surfactant is 3- sulfopropyls-etradecyldimethylamine glycine betaine.In certain preferred implementationsIn scheme, the content of the betaine type amphoteric surfactant is 0.01-4% (w/v).In certain preferred aspects, instituteThe content for stating betaine type amphoteric surfactant is 0.05-0.5% (w/v).
In certain preferred aspects, the kit also includes the second reagent composition, second reagent setIt includes sealer to close object.In certain preferred aspects, the sealer is N6-PEG.
In certain preferred aspects, the present invention relates to betaine type amphoteric surfactant, N6-PEG, insoluble carriersPurposes of the antibody of particle and the specific binding test substance in reagent preparation box, the kit pass through immune ratioThe presence of test substance or its amount in whole blood sample of the turbid method to measure non-haemolysis.
In certain preferred aspects, first reagent composition further includes one or more selected from following examinationAgent：Buffer solution, preservative, suspending agent, stabilizer and coagulant.
In certain preferred aspects, the buffer solution is selected from glycine buffer, MES buffer solutions, MOPSO bufferingsLiquid, Tris buffer solutions, phosphate buffer, borate buffer solution, and its arbitrary combination.In certain preferred aspects,The content of the buffer solution is 0.02-0.1mol/L.
In certain preferred aspects, the preservative be selected from sodium azide, sorbic acid salt, benzoic acid and itsSalt, sodium nitrite and Prolin-300.In certain preferred aspects, the content of the preservative is 0.5-1.5g/L.
In certain preferred aspects, the suspending agent is selected from glycerine, ethylene glycol, mannitol and its arbitrary groupIt closes.In certain preferred aspects, the content of the suspending agent is 1-30% (v/v).
In certain preferred aspects, the stabilizer sodium chloride, magnesium chloride, disodium ethylene diamine tetraacetate, ox bloodPure albumen, glycine, gelatin, and its arbitrary combination.In certain preferred aspects, the content of sodium chloride or magnesium chlorideFor 0.05-1.5% (w/v), the content of disodium ethylene diamine tetraacetate is 0.01-1% (w/v), bovine serum albumin(BSA) or gelatinContent is 0.1-3% (w/v).
In certain preferred aspects, the coagulant is selected from Macrogol 6000, PEG 8000, poly- second twoAlcohol 10000, PEG 20000, dextran sulfate, and its arbitrary combination.In certain preferred aspects, described to promote to coagulateThe content of agent is 0.5-3% (w/v).
In certain preferred aspects, first reagent composition consists of the following compositions：Betaine type surfaceThe water of activating agent, preservative, suspending agent, coagulant, buffer solution and surplus.
In certain exemplary implementation schemes, first reagent composition consists of the following compositions：3- sulfopropyls 16Water (the example of alkyl betaine, sodium azide, mannitol, polyethylene glycol (for example, Macrogol 6000), glycine and surplusSuch as, deionized water, distilled water etc.).
In certain exemplary implementation schemes, first reagent composition consists of the following compositions：0.05% (w/v) 3-Sulfopropyl cetyl betaine, 1g/L sodium azide, 0.01M mannitol, 0.5% (w/v) Macrogol 6000,0.01M are sweetThe water (for example, deionized water, distilled water etc.) of propylhomoserin and surplus.
In certain preferred aspects, the insoluble carrier granular is latex particle.The material of latex particle does not haveIt is particularly limited to, non-limiting examples include polystyrene, styrene-butadiene copolymer, cinnamic acrylic ester copolymerizationObject, styrene-maleic acid copolymer, polyethyleneimine, polyacrylic acid, polymethylacrylic acid, polymethyl methacrylate etc..In certain preferred embodiments, the insoluble carrier granular is granules of polystyrene.
In certain preferred aspects, second reagent composition also includes for the antibody to be coated in instituteState insoluble carrier particle surface reagent and optional coating buffer solution (for example, carbonate buffer solution, phosphate-bufferedLiquid, Tris-HCL buffer solutions or borate buffer solution etc.).
In certain preferred aspects, the surface of the insoluble carrier granular is with carboxyl, amino, hydroxyl, acylDiazanyl or sulfydryl modification.In certain exemplary embodiments, the surface of the insoluble carrier granular carries carboxyl modified.In such embodiment, the antibody can be coated in the insoluble carrier by the coupling reaction between amino and carboxylParticle surface.Therefore, in certain exemplary embodiments, second reagent composition include EDC or its salt (such as) and NHS EDCI.
In certain preferred aspects, the pan coating of the insoluble carrier granular has and is waited for described in specific bindingSurvey the antibody of substance.
In certain preferred aspects, the pan coating of the insoluble carrier granular has and is waited for described in specific bindingSurvey the antibody and N6-PEG of substance.
In certain exemplary embodiments, the test substance is c reactive protein (CRP).In such embodiment,The kit includes the antibody of specific binding CRP.
In certain preferred aspects, the antibody is monoclonal antibody or polyclonal antibody.Certain preferredIn embodiment, the antibody is polyclonal antibody.
In certain preferred aspects, the kit also includes one or more selected from following reagent or dressIt sets：Standard items (for example, series of samples of the test substance containing different known quantities)；Positive control sample is (for example, containing knownThe sample of the test substance of amount)；Negative control sample (for example, sample without containing test substance)；Anti-coagulants is (for example, liverElement)；With blood-taking device (for example, apyrogeneity vacuum blood collection tube).
In certain preferred aspects, each component as described above that kit of the invention is included separately carriesFor.
In certain preferred aspects, the insoluble carrier granular, the specific binding test substanceAntibody, first reagent composition and second reagent composition be provided separately.
In certain preferred aspects, the insoluble carrier granular with first reagent composition as sameComponent provides.In such embodiment, the pan coating of the insoluble carrier granular has the specific binding determinandThe antibody of matter；Alternatively, the pan coating of the insoluble carrier granular has the antibody for specifically binding the test substance, andN6-PEG.In certain preferred aspects, the insoluble carrier granular and first reagent composition are with the shape that is suspendedFormula combination provides.
In certain preferred aspects, the kit measures non-haemolysis by the method included the following stepsThe presence of test substance or its amount in whole blood sample：
(1) under the conditions of antigen-antibody complex forms permitted, in first reagent composition by described in notThe whole blood sample of haemolysis is contacted with the insoluble carrier granular；
(2) the turbidity variation of the reaction system of determination step (1)；
(3) the turbidity variation obtained step (2) changes with the known quantity of the test substance is indicated with the turbidityThe standard curve of relationship compare, and obtain the content of the test substance；
Wherein, the pan coating of the insoluble carrier has the antibody for specifically binding the test substance.
In certain preferred aspects, the whole blood sample is anticoagulated whole blood.
In certain preferred aspects, the test substance is the albumen in plasma component, such as c reactive proteinDeng.In certain exemplary embodiments, the test substance is c reactive protein (CRP).
In certain preferred aspects, the whole blood sample of the non-haemolysis contains CRP.
In certain preferred aspects, in step (1), the surface institute of the insoluble carrier granular is coated anti-With test substance immune response can occur for body, and then it is solidifying to occur so that the insoluble carrier granular is aggregated in the reaction systemCollection reaction.
In certain preferred aspects, in step (2), the optically reaction of determination step (1) startsThe absorbance or scattering of (for example, 0-5 minutes, 0-2 minutes, 5-120 seconds or 5-60 seconds) at provision wavelengths in certain time afterwardsThe variable quantity of light is changed using it as turbidity.
In certain preferred aspects, in step (2), after the reaction of step (1) starts：(a) with it is appropriate whenBetween interval 2 measurement are carried out to absorbance of the reaction system at provision wavelengths, variable quantity using its difference as absorbance (That is, turbidity changes)；Alternatively, (b) absorbance to reaction system at provision wavelengths carries out METHOD FOR CONTINUOUS DETERMINATION, with the unit intervalVariable quantity (that is, turbidity variation) of the absorbance change rate as absorbance.
In certain preferred aspects, in step (2), start (example in 0-120 seconds latter in the reaction of step (1)Such as 5-120 seconds, such as 5-60 seconds) 2 measurement are carried out to absorbance of the reaction system at provision wavelengths, using its difference as suctionThe variable quantity (that is, turbidity variation) of luminosity.In certain preferred aspects, in step (2), in the anti-of step (1)To reaction system, absorbance at provision wavelengths carries out 2 measurement respectively within the 6th second and the 60th second after should starting, and is made with its differenceFor the variable quantity (that is, turbidity variation) of absorbance.
In certain preferred aspects, the provision wavelengths are 800-900nm, such as 840-860nm, such as850nm.In certain exemplary embodiments, the provision wavelengths are 850nm.
In certain exemplary embodiments, in step (2), the 6th second and the 60th second after the reaction of step (1) startsThe absorbance to reaction system at 850nm carries out 2 measurement respectively, and the variable quantity using its difference as absorbance is (that is, turbidDegree variation).
In certain preferred aspects, further include closing the insoluble load using N6-PEG before step (1)The step of body particle.Therefore, in certain preferred aspects, in step (1), the insoluble carrier granular is also coated withThere is N6-PEG.
In certain preferred aspects, further include one or more in the following steps before step (1)：(a)Using blood-taking device whole blood sample is obtained from subject；(b) anti-coagulants (such as heparin) processing blood-taking device or described complete is usedBlood sample.
Preparation method
On the other hand, the present invention provides a kind of method preparing the coated insoluble carrier granular of antibody, packetsInclude following steps：
(1) antibody is coated in the surface of insoluble carrier granular；
(2) sealer and the product of step (1) are incubated with, the sealer is N6-PEG.
In certain preferred aspects, in step (1), the antibody is coated in by covalent coupling insolubleThe surface of carrier.In such embodiment, the surface of the insoluble carrier granular described in step (1) carries functional group's (exampleSuch as carboxyl, amino, hydroxyl, hydrazides or sulfydryl) modification.
It in certain preferred aspects, will be described by the coupling reaction between amino and carboxyl in step (1)Antibody is coated in insoluble carrier particle surface.In such embodiment, insoluble carrier granular described in step (1)Surface carries carboxyl modified.Therefore, in certain exemplary embodiments, in step (1), EDC or its salt (such asEDCI) and NHS it is existing under the conditions of, the antibody is contacted with the insoluble carrier.In certain exemplary embodiments,In step (1), in containing EDC or its salt (such as EDCI) and the buffer solution of NHS, by the antibody and the insoluble loadBody contacts.In certain preferred aspects, the buffer solution is selected from glycine buffer, MES buffer solutions, MOPSO bufferingsLiquid, Tris buffer solutions, phosphate buffer and borate buffer solution, and its arbitrary combination.
In certain preferred aspects, in step (2), the content of the sealer is 0.4%-2% (w/v).In certain preferred aspects, the content of the sealer is 1% (w/v).
In certain preferred aspects, further include the product of washing step (2) to remove not after step (2)The step of participating in the substance of reaction.In certain preferred aspects, the product of the step (2) is washed using buffer solution.In certain exemplary embodiments, the buffer solution is selected from phosphate buffer, MES buffer solutions, Tris buffer solutions etc..
Abbreviation and term definition
Following abbreviation used herein：
EDC 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides (EDC)
EDCI 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDCI)
NHS n-hydroxysuccinimides
MES 2- (N- morpholines) ethanesulfonic acid
MOPSO 3- (N- morpholinyls) 2- hydroxy-propanesulfonic acids
Tris trishydroxymethylaminomethanes
In the present invention, unless otherwise stated, Science and Technology noun used herein has art technologyThe normally understood meaning of personnel institute.Also, virology used herein, biochemistry, Immunology Lab operating procedure are equalFor widely used conventional steps in corresponding field.Meanwhile for a better understanding of the present invention, determining for relational language is provided belowJustice and explanation.
As used herein, term " immunoturbidimetry " refers to, according to the reaction caused by antigen-antibody reactionThe variation of the turbidity (for example, the optical properties such as absorbance or scattering light) of system, it is anti-in test sample to detect or quantifyFormer or antibody method, belongs to one kind in immunological detection method.It typically, will in order to improve the sensitivity of detectionThe antibody or antigen for enough specifically binding target antigen or target antibody in test sample are fixed on insoluble carrier granular (exampleSuch as, latex particle) on, to form sensitization particle；Due to the antigen-antibody reaction sensitization particle agglutination, caused by thusOptical property variation is detected analyte or quantifies, and this method is also referred to as particle enhancing immunoturbidimetry (Particle-enhanced turbidimetric immunoassay,PETIA)；Particularly, use latex particle as insoluble solidWhen particle, this method is also referred to as latex enhancing immune turbidimetry (Latex-enhanced turbidimetricimmunoassay,LIA).In the present invention, state " immunoturbidimetry " it is meant that using insoluble carrier granular immunoturbidimetryMethod.
As used herein, term " specific binding " refers to, between two molecules (i.e. binding molecule and target molecule)Nonrandom association reaction, such as the reaction between antibody and its targeted antigen.Binding affinity between two molecules canUse KDValue description.KDValue refers to by the kd (dissociation rates of specific binding molecule-target molecule interaction；Also known as koff) withKa (the association rates of particular combination molecule-target molecule interaction；Also known as kon) the ratio between obtained dissociation constant, or refer toIt is expressed as the kd/ka of molar concentration (M).KDIt is worth smaller, two molecule combinations are closer, and affinity is higher.In certain embodimentsIn, the antibody (or having the antibody of specificity to certain antigen) for specifically binding certain antigen refers to that antibody is to be less than about 10-5M,It is, for example, less than about 10-6M、10-7M、10-8M、10-9M or 10-10M or smaller affinity (KD) combine the antigen.KDValue can lead toIt crosses method well known in the art to determine, such as is measured in BIACORE instrument using surface plasma body resonant vibration art (SPR).
As used herein, statement " the non-haemolysis of whole blood sample " refers to that the whole blood sample is after subject obtainsIt is handled without any haemolysis.
As used herein, term " betaine type amphoteric surfactant (Betaine type surfactant) " isRefer to, the amphoteric surfactant being made of quaternary ammonium salt cationic part and carboxylic acid type anion part.The example include butIt is not limited to, alkyl betaine, alkyl amido betaine, azochlorosulfonate propyl lycine, hydroxyl azochlorosulfonate propyl lycine, phosphate ester glycine betaineDeng.
As used herein, term " N6-PEG " refers to a kind of " methoxypolyethylene glycol-pentaethylene hexamine (α-Methoxy-poly (ethyleneglycol)-pentaethylenehexamin e) " polymer, polyethylene glycol segment listEnd is connected with 6 amino groups, and has chemical formula as follows, wherein m 0,1,2,3 or 4, n are 10~about 200Integer (be preferably from about 100~about 200 integer, for example, about 100~about 150 integer, for example, about 100~about 120Integer).Detailed teachings about N6-PEG can be found in, such as Furusho H et al. .Chemistry ofMaterials.2009,21,3526-3535.N6-PEG is purchased from Japanese JSR Life Sciences companies, and article No. isCE510。
As used herein, term " sealer " refers to that can reduce non-specific interaction such as non-specificityAny substance that antibody combines.This substance is well known to those skilled in the art, and the example includes but not limited to gelatin, ox bloodPure albumen, ovalbumin, casein and skim milk etc..
As used herein, term " coagulant " refers to the substance that can speed up antigen-antibody complex formation.This substance is well known to those skilled in the art, and the example includes but not limited to water soluble polymer, such as polyethylene glycol, poly-Vinyl alcohol, dextran, sodium chondroitin sulfate etc..
As used herein, term " suspending agent " refers to that can increase the viscosity of decentralized medium, to reduce particleThe substance of sinking speed.In the present invention, the suspending agent is preferably low molecule suspending agent, such as glycerine, ethylene glycol, sweet dewAlcohol etc..
As used herein, term " stabilizer " refer to can control or inhibit insoluble carrier granular (for example,Latex particle) self-solidifying substance, this substance is well known to those skilled in the art, the example include but not limited to sodium chloride,Magnesium chloride, disodium ethylene diamine tetraacetate, bovine serum albumin(BSA), glycine, gelatin etc..
As used herein, term " buffer solution " refers to that can match the effect of component by its soda acid to prevent pHThe solution of significant changes.This substance is well known to those skilled in the art, reference can be made to such as Buffers.A Guide forthe Preparation and Use of Buffers in Biological Systems,Gueffroy,D.,ed.Calbiochem Corporation(1975).The non-limiting examples of buffer solution include MES, MOPS, MOPSO, Tris,HEPES, phosphate, acetate, citrate, succinate, ammonium salt etc..
As used herein, term " positive control sample " refers to the sample of the c reactive protein containing known quantity.In some embodiments, the amount of test substance (such as c reactive protein) can be by by the detection of sample to be tested in sample to be testedAs a result it is compared to calculate with the testing result of the positive control sample, such methods are well known in the art, such as logicalIt crosses the positive control sample of detection various concentration and builds standard curve to carry out.
As used herein, term " anti-coagulants " refers to that can prevent the substance of blood clotting.This substance is thisKnown to field technology personnel, the example includes but not limited to heparin, EDTA, oxalates (for example, sodium oxalate, potassium oxalate or grassSour ammonium), trifoliate orange acid sodium etc..
Advantageous effect of the invention
Presently commercially available CRP assay kits are double reagent, by reaction buffer 1 (R1) and the breast of CRP antibody couplingsGlue particle reagents (R2) form.In practical application, needing the first step that R1 is first added to carry out haemolysis to whole blood sample, second step adds againR2 is reacted.Detection speed is not only influenced in this way, but also increases reagent cost；And the two premix is together due to the change of liquid environmentThe antibody in R2 may be caused to inactivate, to influence the stability of reagent.
The present invention provides a kind of quantitative detecting reagents of test substance in whole blood sample comprising particular agent compositionThe quantitative detecting method of test substance in box and the whole blood sample established based on the reagent composition.Compared with prior art,Technical scheme of the present invention can directly be detected the whole blood sample of non-haemolysis, namely without being carried out formerly to whole blood sampleHaemolysis processing can be contacted directly with the latex particle of antibody sensitized, to be detected to the variation of its turbidity, therefore be dropped significantlyLow reagent cost, operation is more simple, greatly improves detection speed, as a result accurately and reliably.Therefore, technology of the inventionScheme is especially suitable for quick diagnosis patient gradient of infection and prognostic evaluation.
Embodiment of the present invention is described in detail below in conjunction with drawings and examples, but people in the artMember will be understood that following drawings and embodiment are merely to illustrate the present invention, rather than to the restriction of the scope of the present invention.With reference to the accompanying drawingsWith the following detailed description of preferred embodiment, various purposes of the invention and advantageous aspect are to those skilled in the artIt will be apparent.