CN108593641A - A kind of kit and method quantitatively detecting test substance in whole blood sample - Google Patents
A kind of kit and method quantitatively detecting test substance in whole blood sample Download PDFInfo
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- CN108593641A CN108593641A CN201810311589.5A CN201810311589A CN108593641A CN 108593641 A CN108593641 A CN 108593641A CN 201810311589 A CN201810311589 A CN 201810311589A CN 108593641 A CN108593641 A CN 108593641A
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- test substance
- insoluble carrier
- carrier granular
- whole blood
- reagent composition
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Classifications
-
- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/82—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a precipitate or turbidity
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/82—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a precipitate or turbidity
- G01N2021/825—Agglutination
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4737—C-reactive protein
Abstract
This application involves field of immunological detection.Specifically, this application involves a kind of kits quantitatively detecting test substance in whole blood sample, it includes insoluble carrier granular, the antibody of the test substance and the first reagent composition comprising betaine type amphoteric surfactant are specifically bound.In addition, the application further relates to a kind of method detecting the presence or its amount of test substance in whole blood sample comprising the step of whole blood sample of non-haemolysis being contacted and measured in first reagent composition turbidity variation of the reaction system with the insoluble carrier granular for being coated with the antibody for specifically binding the test substance.
Description
Technical field
This application involves field of immunological detection.Specifically, this application involves a kind of quantitatively detect in whole blood sample to wait for
Survey the kit of substance.In addition, the application further relates to a kind of method detecting the presence or its amount of test substance in whole blood sample.
Background technology
Mankind's c reactive protein (C-reactive protein, CRP) is pentamer albumen, non-by five identical subunits
Covalently connection, relative molecular mass 115-140KD.CRP half lifes about 15h, the concentration of normal person is very low, but is damaged in tissue
6-8h starts to increase after wound, acute infection generation, 24-48h peakings, up to even thousands of times of the hundred times of normal value, increases
Amplitude is directly proportional to gradient of infection, and concentration declines rapidly after inflammation is cured, and can restore normal level within one day.CRP, which persistently increases, to be carried
Showing body, there are chronic inflammation or autoimmune diseases, will not generally be increased when virus infects, and variation is not by of patient
The influence of body difference, fuselage state and medicine.CRP is for antidiastole bacterium or virus infection, the work of monitoring disease
Emotionally the monitoring of condition, curative effect all has good guiding role.
In recent years, with the further investigation to CRP clinical meanings, will be used wider and wider in clinic is general.Mesh
Before, the sample for clinically detecting is generally serum or blood plasma, be both needed to first to whole blood sample carry out pre-separation red blood cell again into
Row test, needs special separation equipment, and complex for operation step, time-consuming and laborious, the sample size needed is big, especially for children and
The blood sampling such as newborn and Patients with Big Area Burn is particularly difficult, cannot be satisfied the demand of clinical quick diagnosis.For this purpose, with whole blood
The quick detection reagent of CRP is carried out for sample seems abnormal urgent and important.
Presently commercially available CRP assay kits are double reagent, by reaction buffer 1 (R1) and the breast of CRP antibody couplings
Glue particle reagents (R2) form.Detection of the kit for sample, the general first step first add R1 to carry out haemolysis to whole blood sample, the
Two steps add R2 to be reacted again, not only influence detection speed in this way, but also increase reagent cost, the two premixes together due to liquid environment
Change the antibody in R2 may be caused to inactivate, to influence the stability of reagent.
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 immunoturbidimetry
It, 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 this
It 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 immunoturbidimetry
Presence or its amount.In certain preferred aspects, the blood sample is selected from whole blood, blood plasma or serum.Certain excellent
In 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 protein
Deng.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, alkylamide
Glycine betaine, azochlorosulfonate propyl lycine, hydroxyl azochlorosulfonate propyl lycine, phosphate ester glycine betaine, and its arbitrary combination.In certain preferred realities
It applies in scheme, first reagent composition includes 3- sulfopropyls-etradecyldimethylamine glycine betaine.In certain preferred embodiment party
In case, the content of the betaine type amphoteric surfactant is 0.01-4% (w/v).In certain preferred aspects, described
The 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 set
It 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 examination
Agent：Buffer solution, preservative, suspending agent, stabilizer and coagulant.
In certain preferred aspects, the buffer solution is selected from glycine buffer, MES buffer solutions, MOPSO bufferings
Liquid, 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 its
Salt, 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 group
It 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 blood
Pure albumen, glycine, gelatin, and its arbitrary combination.In certain preferred aspects, the content of sodium chloride or magnesium chloride
For 0.05-1.5% (w/v), the content of disodium ethylene diamine tetraacetate is 0.01-1% (w/v), bovine serum albumin(BSA) or gelatin
Content is 0.1-3% (w/v).
In certain preferred aspects, the coagulant is selected from Macrogol 6000, PEG 8000, poly- second two
Alcohol 10000, PEG 20000, dextran sulfate, and its arbitrary combination.In certain preferred aspects, described to promote to coagulate
The content of agent is 0.5-3% (w/v).
In certain preferred aspects, first reagent composition consists of the following compositions：Betaine type surface
The 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 16
Water (the example of alkyl betaine, sodium azide, mannitol, polyethylene glycol (for example, Macrogol 6000), glycine and surplus
Such 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 sweet
The 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 granular
It is latex particle.The material of latex particle is not particularly limited, and non-limiting examples include polystyrene, styrene-fourth two
Alkene copolymer, copolymer in cinnamic acrylic ester, styrene-maleic acid copolymer, polyethyleneimine, polyacrylic acid, poly- methyl
Acrylic acid, polymethyl methacrylate etc..In certain preferred aspects, the insoluble carrier granular is polystyrene
Particle.
In certain preferred aspects, second reagent composition also includes for the antibody to be coated in institute
State insoluble carrier particle surface reagent and optional coating buffer solution (for example, carbonate buffer solution, phosphate-buffered
Liquid, 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 art
Face, for example, physical absorption or by functional group's (such as carboxyl, amino, hydroxyl, hydrazides or sulfydryl) change surface realized it is covalent
Coupling.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 Bangs
Laboratories, Inc) webpage on download.Those skilled in the art are known how according to insoluble carrier particle surface institute
Reagent 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, hydroxyl
Base, hydrazide group or sulfydryl modification.In certain exemplary embodiments, the surface of the insoluble carrier granular is repaiied with carboxyl
Decorations.In such embodiment, the antibody can be coated in by the coupling reaction between amino and carboxyl described insoluble
Property carrier particle surface.Therefore, in certain exemplary embodiments, second reagent composition includes EDC or its salt (example
Such as EDCI) and NHS.
In certain preferred aspects, the pan coating of the insoluble carrier granular has and is waited for described in specific binding
Survey the antibody of substance.
In certain preferred aspects, the pan coating of the insoluble carrier granular has and is waited for described in specific binding
Survey 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 preferred
In embodiment, the antibody is polyclonal antibody.
In certain preferred aspects, the kit also includes one or more selected from following reagent or dress
It sets：Standard items (for example, series of samples of the test substance containing different known quantities)；Positive control sample is (for example, containing known
The 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 carries
For.
In certain preferred aspects, the insoluble carrier granular, the specific binding test substance
Antibody, first reagent composition and second reagent composition be provided separately.
In certain preferred aspects, the insoluble carrier granular with first reagent composition as same
Component provides.In such embodiment, the pan coating of the insoluble carrier granular has the specific binding determinand
The antibody of matter；Alternatively, the pan coating of the insoluble carrier granular has the antibody for specifically binding the test substance, and
N6-PEG.In certain preferred aspects, the insoluble carrier granular and first reagent composition are with the shape that is suspended
Formula 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 its
The 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 permitted
Whole 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 turbidity
The 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 described
First 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 protein
Deng.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 excellent
In 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, alkylamide
It is one or more in glycine betaine, azochlorosulfonate propyl lycine, hydroxyl azochlorosulfonate propyl lycine, phosphate ester glycine betaine.Certain preferred
In embodiment, first reagent composition includes 3- sulfopropyls-etradecyldimethylamine glycine betaine.In certain preferred implementations
In 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 examination
Agent：Buffer solution, preservative, suspending agent, stabilizer and coagulant.
In certain preferred aspects, the buffer solution is selected from glycine buffer, MES buffer solutions, MOPSO bufferings
Liquid, 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 its
Salt, 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 group
It 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 blood
Pure albumen, glycine, gelatin, and its arbitrary combination.In certain preferred aspects, the content of sodium chloride or magnesium chloride
For 0.05-1.5% (w/v), the content of disodium ethylene diamine tetraacetate is 0.01-1% (w/v), bovine serum albumin(BSA) or gelatin
Content is 0.1-3% (w/v).
In certain preferred aspects, the coagulant is selected from Macrogol 6000, PEG 8000, poly- second two
Alcohol 10000, PEG 20000, dextran sulfate, and its arbitrary combination.In certain preferred aspects, described to promote to coagulate
The content of agent is 0.5-3% (w/v).
In certain preferred aspects, first reagent composition consists of the following compositions：Betaine type surface
The 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 16
Water (the example of alkyl betaine, sodium azide, mannitol, polyethylene glycol (for example, Macrogol 6000), glycine and surplus
Such 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 sweet
The 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 granular
For 2-5g/L.
In certain preferred aspects, the insoluble carrier granular is latex particle.The material of latex particle does not have
It is particularly limited to, non-limiting examples include polystyrene, styrene-butadiene copolymer, cinnamic acrylic ester copolymerization
Object, 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 system
Collection 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 points
Clock, 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 suitable
It 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 absorbance
It measures (that is, turbidity variation)；Alternatively, (b) absorbance to reaction system at provision wavelengths carries out METHOD FOR CONTINUOUS DETERMINATION, when with unit
Between 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 suction
The 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 difference
For the variable quantity (that is, turbidity variation) of absorbance.
In certain preferred aspects, the provision wavelengths are 800-900nm, such as 840-860nm, such as
850nm.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) starts
The absorbance to reaction system at 850nm carries out 2 measurement respectively, and the variable quantity using its difference as absorbance is (that is, turbid
Degree 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 with
There 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 used
Blood sample.
Detection applications
On the other hand, the purposes the present invention relates to betaine type amphoteric surfactant in reagent preparation box, the examination
Agent box measures the presence of test substance or its amount in the whole blood sample of non-haemolysis by immunoturbidimetry, wherein the kit
Including：
(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 and
Purposes of the antibody of the test substance in reagent preparation box is specifically bound, the kit is surveyed by immunoturbidimetry
The 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 protein
Deng.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, alkylamide
Glycine betaine, azochlorosulfonate propyl lycine, hydroxyl azochlorosulfonate propyl lycine, phosphate ester glycine betaine, and its arbitrary combination.In certain preferred realities
It applies in scheme, the betaine type amphoteric surfactant is 3- sulfopropyls-etradecyldimethylamine glycine betaine.In certain preferred implementations
In scheme, the content of the betaine type amphoteric surfactant is 0.01-4% (w/v).In certain preferred aspects, institute
The 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 set
It 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 carriers
Purposes of the antibody of particle and the specific binding test substance in reagent preparation box, the kit pass through immune ratio
The 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 examination
Agent：Buffer solution, preservative, suspending agent, stabilizer and coagulant.
In certain preferred aspects, the buffer solution is selected from glycine buffer, MES buffer solutions, MOPSO bufferings
Liquid, 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 its
Salt, 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 group
It 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 blood
Pure albumen, glycine, gelatin, and its arbitrary combination.In certain preferred aspects, the content of sodium chloride or magnesium chloride
For 0.05-1.5% (w/v), the content of disodium ethylene diamine tetraacetate is 0.01-1% (w/v), bovine serum albumin(BSA) or gelatin
Content is 0.1-3% (w/v).
In certain preferred aspects, the coagulant is selected from Macrogol 6000, PEG 8000, poly- second two
Alcohol 10000, PEG 20000, dextran sulfate, and its arbitrary combination.In certain preferred aspects, described to promote to coagulate
The content of agent is 0.5-3% (w/v).
In certain preferred aspects, first reagent composition consists of the following compositions：Betaine type surface
The 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 16
Water (the example of alkyl betaine, sodium azide, mannitol, polyethylene glycol (for example, Macrogol 6000), glycine and surplus
Such 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 sweet
The 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 have
It is particularly limited to, non-limiting examples include polystyrene, styrene-butadiene copolymer, cinnamic acrylic ester copolymerization
Object, 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 institute
State insoluble carrier particle surface reagent and optional coating buffer solution (for example, carbonate buffer solution, phosphate-buffered
Liquid, 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, acyl
Diazanyl 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 carboxyl
Particle 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 binding
Survey the antibody of substance.
In certain preferred aspects, the pan coating of the insoluble carrier granular has and is waited for described in specific binding
Survey 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 preferred
In embodiment, the antibody is polyclonal antibody.
In certain preferred aspects, the kit also includes one or more selected from following reagent or dress
It sets：Standard items (for example, series of samples of the test substance containing different known quantities)；Positive control sample is (for example, containing known
The sample of the test substance of amount)；Negative control sample (for example, sample without containing test substance)；Anti-coagulants is (for example, liver
Element)；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 carries
For.
In certain preferred aspects, the insoluble carrier granular, the specific binding test substance
Antibody, first reagent composition and second reagent composition be provided separately.
In certain preferred aspects, the insoluble carrier granular with first reagent composition as same
Component provides.In such embodiment, the pan coating of the insoluble carrier granular has the specific binding determinand
The antibody of matter；Alternatively, the pan coating of the insoluble carrier granular has the antibody for specifically binding the test substance, and
N6-PEG.In certain preferred aspects, the insoluble carrier granular and first reagent composition are with the shape that is suspended
Formula combination provides.
In certain preferred aspects, the kit measures non-haemolysis by the method included the following steps
The 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 not
The 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 turbidity
The 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 protein
Deng.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 system
Collection reaction.
In certain preferred aspects, in step (2), the optically reaction of determination step (1) starts
The 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 afterwards
The 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 when
Between 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 interval
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 suction
The 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 difference
For the variable quantity (that is, turbidity variation) of absorbance.
In certain preferred aspects, the provision wavelengths are 800-900nm, such as 840-860nm, such as
850nm.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) starts
The absorbance to reaction system at 850nm carries out 2 measurement respectively, and the variable quantity using its difference as absorbance is (that is, turbid
Degree 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 with
There 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 used
Blood sample.
Preparation method
On the other hand, the present invention provides a kind of method preparing the coated insoluble carrier granular of antibody, packets
Include 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 insoluble
The surface of carrier.In such embodiment, the surface of the insoluble carrier granular described in step (1) carries functional group's (example
Such 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 as
EDCI) 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 load
Body contacts.In certain preferred aspects, the buffer solution is selected from glycine buffer, MES buffer solutions, MOPSO bufferings
Liquid, 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 technology
The normally understood meaning of personnel institute.Also, virology used herein, biochemistry, Immunology Lab operating procedure are equal
For widely used conventional steps in corresponding field.Meanwhile for a better understanding of the present invention, determining for relational language is provided below
Justice and explanation.
As used herein, term " immunoturbidimetry " refers to, according to the reaction caused by antigen-antibody reaction
The 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 quantify
Former or antibody method, belongs to one kind in immunological detection method.It typically, will in order to improve the sensitivity of detection
The antibody or antigen for enough specifically binding target antigen or target antibody in test sample are fixed on insoluble carrier granular (example
Such as, latex particle) on, to form sensitization particle；Due to the antigen-antibody reaction sensitization particle agglutination, caused by thus
Optical 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 solid
When particle, this method is also referred to as latex enhancing immune turbidimetry (Latex-enhanced turbidimetric
immunoassay,LIA).In the present invention, state " immunoturbidimetry " it is meant that using insoluble carrier granular immunoturbidimetry
Method.
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 can
Use KDValue description.KDValue refers to by the kd (dissociation rates of specific binding molecule-target molecule interaction；Also known as koff) with
Ka (the association rates of particular combination molecule-target molecule interaction；Also known as kon) the ratio between obtained dissociation constant, or refer to
It 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 embodiments
In, 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 to
It 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 obtains
It is handled without any haemolysis.
As used herein, term " betaine type amphoteric surfactant (Betaine type surfactant) " is
Refer to, the amphoteric surfactant being made of quaternary ammonium salt cationic part and carboxylic acid type anion part.The example include but
It is not limited to, alkyl betaine, alkyl amido betaine, azochlorosulfonate propyl lycine, hydroxyl azochlorosulfonate propyl lycine, phosphate ester glycine betaine
Deng.
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 list
End is connected with 6 amino groups, and has chemical formula as follows, wherein m 0,1,2,3 or 4, n are 10~about 200
Integer (be preferably from about 100~about 200 integer, for example, about 100~about 150 integer, for example, about 100~about 120
Integer).Detailed teachings about N6-PEG can be found in, such as Furusho H et al. .Chemistry of
Materials.2009,21,3526-3535.N6-PEG is purchased from Japanese JSR Life Sciences companies, and article No. is
CE510。
As used herein, term " sealer " refers to that can reduce non-specific interaction such as non-specificity
Any 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 blood
Pure 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 particle
The substance of sinking speed.In the present invention, the suspending agent is preferably low molecule suspending agent, such as glycerine, ethylene glycol, sweet dew
Alcohol 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 pH
The 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 for
the 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 tested
As 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 logical
It 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 this
Known to field technology personnel, the example includes but not limited to heparin, EDTA, oxalates (for example, sodium oxalate, potassium oxalate or grass
Sour 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 couplings
Glue 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 again
R2 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 environment
The 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 composition
The 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 sample
Haemolysis processing can be contacted directly with the latex particle of antibody sensitized, to be detected to the variation of its turbidity, therefore be dropped significantly
Low reagent cost, operation is more simple, greatly improves detection speed, as a result accurately and reliably.Therefore, technology of the invention
Scheme 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 art
Member 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 drawings
With the following detailed description of preferred embodiment, various purposes of the invention and advantageous aspect are to those skilled in the art
It will be apparent.
Description of the drawings
Fig. 1 shows the linear regression analysis of the theoretical concentration of CRP calibration objects and practical measurement concentration in embodiment 1.
Fig. 2 shows N6-PEG and CRP calibration curves of the BSA as sealer in embodiment 2.
Fig. 3 shows the detection curve of the CRP calibration objects obtained using different hemolytic agents in embodiment 3.
Fig. 4 shows that the whole blood CRP detection methods in embodiment 4 through the invention are obtained with routine serum CRP detection methods
The regression analysis of the testing result obtained.
Specific implementation mode
It is intended to illustrate the present invention embodiment (rather than limiting the invention) to describe the present invention referring now to following.
Unless specifically stated otherwise, the experimental methods of molecular biology and immunodetection used in the present invention, substantially joins
According to J.Sambrook et al., molecular cloning：Laboratory manual, second edition, CSH Press, 1989, and
F.M.Ausubel et al., fine works molecular biology experiment guide, the 3rd edition, described in John Wiley＆Sons, Inc., 1995
Method carry out；The condition that the use of restriction enzyme is recommended according to goods producer.Those skilled in the art know, implement
Description is of the invention by way of example for example, and is not intended to limit scope of the present invention.
Preparation example 1：The preparation of whole blood c reactive protein detection kit
In this preparation example, caused respectively using with Goat anti-Human CRP antibody (Guilin Immunetech Co, Ltd.)
Quick average diameter is the emulsion reagent (JSR Life Sciences companies) of 68nm and 198nm, then by a certain percentage will be above-mentioned
Reagent mixing is reaction reagent.It is as follows：
3mL is added in the latex particle (with latex particle average diameter 68nm, for solid content 5%) of 1mL carboxylated
Then MES pH of buffer 5.8 is added carbonization 300 μ L of diamine hydrochloride (Aladdin reagent Co., Ltd) 10mg/mL, then adds
Enter n-hydroxysuccinimide (Sinopharm Chemical Reagent Co., Ltd.) 10mg/mL 600uL, 20min is stirred at 25 DEG C, so
400 μ L of 0.2mol/L sodium carbonate liquors are added afterwards, add the 5mL c reactive protein antibody of Goat anti-Human containing 1.6mg/mL
0.02MPBS solution stirs 2h at 25 DEG C, and N6-PEG (the JSR Life Sciences companies of 2mL 2% are added；Article No. is
CE510 it is) sealer, continues 2h, 15300rpm, 4 DEG C of centrifugation 30min of stirring, abandon supernatant, add 12mL pH7.5's
10min, 15300rpm, 4 DEG C of centrifugation 30min are resuspended in 0.02MPBS buffer solution ultrasounds, abandon supernatant, and 12mL is added and preserves liquid ultrasound weight
It is outstanding.Wherein preserving liquid is：Glycine containing 0.01M, 0.05%3- sulfopropyls cetyl betaine, 1g/L sodium azide, 0.5%
Mannitol, 0.5% Macrogol 6000.In addition, in the case where remaining condition is constant, made using bovine serum albumin(BSA) (BSA)
For sealer, control reaction reagent is prepared.
The latex particle that average diameter is 198nm uses preparation method sensitization same as described above.
It is 5 to take little particle (68nm) sensitization latex reagent and bulky grain (198nm) sensitization latex reagent volume ratio later:1
Ratio mixes, to obtain reaction reagent.
Embodiment 1：The assay method of c reactive protein content based on immunoturbidimetry
The 250 μ L of reaction reagent that preparation example 1 obtains are added in reaction cup, it is different to be then respectively adding 8 μ L for 37 DEG C of incubations
The CRP calibration objects of concentration, calibration object and reaction reagent in the 6th second, the 60th second use analyzer (Guilin Uritest after mixing well
Medical Treatment Electron Co., Ltd, BH-5360CRP) absorbance (A1, A2) of reaction system at a wavelength of 850 nm is measured respectively, it calculates
The two difference (A2-A1).Using absorbance difference as ordinate, a concentration of abscissas of corresponding CRP make standard curve.
It is measured in the same method the whole blood sample of patient, c reactive protein content in sample is calculated by standard curve.
Compound concentration is the CRP calibration objects of 175.0mg/L, 120.0mg/L, 60.0mg/L, 25.0mg/L, 5.0mg/L, is led to
The concentration that method as described above detects the CRP calibration objects respectively is crossed, average each concentration is surveyed three times, by theoretical concentration and measurement
The average value of concentration makees linear regression analysis.The results are shown in Figure 1, related coefficient R2=0.9987, in 5-175mg/L ranges
Interior, theoretical value and measured value linear dependence are good.It is good accurate that the above results show that reaction reagent of the invention has
Property.
Embodiment 2：Influence of the different sealers to testing result
The reaction reagent by N6-PEG as sealer that preparation example 1 obtains is used respectively, and with bovine serum albumin(BSA)
(BSA) as the control reaction reagent of sealer, and using the method for embodiment 1 measure respectively series concentration c reactive protein compared with
Quasi- product make CRP standard curves.As a result respectively as shown in table 1 and Fig. 2, when using N6-PEG as sealer the sensitivity of reagent,
Upper limit of detection significantly improves, and the range of linearity is wider, and when using BSA as sealer, and response intensity is only when CRP low value 5mg/mL
0.6 times of N6-PEG, more early there are HOOK effects in high level, and upper limit of detection is low.It can be seen that N6-PEG is significantly excellent as sealer
In BSA.
Table 1：The testing result of CRP calibration objects
Embodiment 3：Influence of the different hemolytic agents to testing result
With respectively containing lauryl sodium sulfate (SDS), 3- sulfopropyls-etradecyldimethylamine glycine betaine
(Sulfobetaine-16), reaction reagent of the cetyl trimethylammonium bromide (CTMAB) as hemolytic agent, with series concentration
CRP calibration objects are 250 by volume:8 ratio mixing, CRP contents are detected by method described in embodiment 1, make CRP calibrations
Curve.The results are shown in Figure 3, and immunoturbidimetry reaction is inhibited to carry out when as a result showing SDS, CTMAB as hemolytic agent, and with
Sulfobetaine-16 has higher degree of reaction, the range of linearity wider as the reaction system of hemolytic agent.
Embodiment 4：The detection of c reactive protein in whole blood sample
CRP contents in the whole blood sample measured using the reaction reagent of preparation example 1.The whole blood sample is new on the day of being obtained from hospital
Blood sample, for the anticoagulated whole blood acquired with vacuum tube.The blood serum sample for obtaining same whole blood sample by centrifuging simultaneously, then makes
With conventional the c reactive protein detection reagent (Mairui Biological Medical Electronic Co., Ltd., Shenzhen, the C reactive protein that have listed
(CRP) assay kit (latex immunoturbidimetry)) content of c reactive protein in the blood serum sample is detected, it has operated
The specification for pressing the kit entirely carries out.The results are shown in Figure 4, and wherein abscissa indicates the CRP measurement results of blood serum sample, indulges
Coordinate is same sample whole blood CRP measurement results.Measurement result consistency is good both known to equation of linear regression, R2=
0.9936.The above results show that kit measurement result of the invention is accurate and reliable.
Although the specific implementation mode of the present invention has obtained detailed description, it will be appreciated by those skilled in the art that：Root
According to all introductions having disclosed, details can be carry out various modifications and be changed, and these change the guarantor in the present invention
Within the scope of shield.The full scope of the present invention is given by the appended claims and any equivalents thereof.
Claims (10)
1. 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；
Preferably, the kit measures the presence of test substance or its amount in blood sample for immunoturbidimetry；Preferably,
The blood sample is selected from whole blood, blood plasma or serum；Preferably, the blood sample is anticoagulated whole blood, and non-haemolysis；
Preferably, the test substance is the albumen in plasma component, such as c reactive protein；Preferably, the test substance is
C reactive protein (CRP)；
Preferably, the betaine type amphoteric surfactant be selected from alkyl betaine, alkyl amido betaine, azochlorosulfonate propyl lycine,
Hydroxyl azochlorosulfonate propyl lycine, phosphate ester glycine betaine, and its arbitrary combination；Preferably, the betaine type amphoteric surfactant is 3-
Sulfopropyl-etradecyldimethylamine glycine betaine；
Preferably, the kit also includes the second reagent composition, and second reagent composition includes sealer；It is preferred that
Ground, the sealer are N6-PEG.
2. kit described in claim 1, wherein first reagent composition further includes one or more selected from following
Reagent：Buffer solution, preservative, suspending agent, stabilizer and coagulant；
Preferably, the buffer solution is selected from glycine buffer, MES buffer solutions, MOPSO buffer solutions, Tris buffer solutions, phosphate
Buffer solution, borate buffer solution, and its arbitrary combination；
Preferably, the preservative is selected from sodium azide, sorbic acid salt, benzoic acid and its salt, sodium nitrite and Prolin-
300；
Preferably, the suspending agent is selected from glycerine, ethylene glycol, mannitol, and its arbitrary combination；
Preferably, the stabilizer sodium chloride, magnesium chloride, disodium ethylene diamine tetraacetate, bovine serum albumin(BSA), glycine, gelatin,
And its arbitrary combination；
Preferably, the coagulant is selected from Macrogol 6000, PEG 8000, polyethylene glycol 10000, polyethylene glycol
20000, dextran sulfate, and its arbitrary combination；
Preferably, first reagent composition consists of the following compositions：Betaine type amphoteric surfactant, preservative, suspending agent,
The water of coagulant, buffer solution and surplus；
Preferably, first reagent composition consists of the following compositions：3- sulfopropyls cetyl betaine, sodium azide,
Mannitol, polyethylene glycol (for example, Macrogol 6000), glycine and surplus water (for example, deionized water, distilled water
Deng)；
Preferably, first reagent composition consists of the following compositions：0.05% (w/v) 3- sulfopropyl cetyl beets
Water (the example of alkali, 1g/L sodium azide, 0.01M mannitol, 0.5% (w/v) Macrogol 6000,0.01M glycine and surplus
Such as, deionized water, distilled water etc.).
3. kit as claimed in claim 1 or 2, wherein the insoluble carrier granular is latex particle, such as polystyrene
Particle；
Preferably, a diameter of 50-400nm of the insoluble carrier granular；
Preferably, the surface of the insoluble carrier granular carries carboxyl, amino, hydroxyl, hydrazide group or sulfydryl modification；It is preferred that
The surface on ground, the insoluble carrier granular carries carboxyl modified；
Preferably, the pan coating of the insoluble carrier granular has the antibody for specifically binding the test substance；Alternatively, institute
The pan coating for stating insoluble carrier granular has the antibody and N6-PEG for specifically binding the test substance.
4. claim 1-3 any one of them kits, wherein second reagent composition also includes for described will resist
Body is coated in the reagent of the insoluble carrier particle surface；
Preferably, second reagent composition includes EDC or its salt (such as EDCI) and NHS；
Preferably, the kit also includes one or more selected from following reagent or device：Standard items are not (for example, containing
With the series of samples of the test substance of known quantity)；Positive control sample (for example, sample of the test substance containing known quantity)；
Negative control sample (for example, sample without containing test substance)；Anti-coagulants (for example, heparin)；With blood-taking device is (for example, nothing
Pyrogen vacuum blood collection tube).
5. claim 1-4 any one of them kits, wherein waited for described in the insoluble carrier granular, specific binding
Antibody, the first reagent composition and the second reagent composition for surveying substance are provided separately；Alternatively, the insoluble carrier granular
It is provided as same component with the first reagent composition, wherein the pan coating of the insoluble carrier granular has specific knot
Close the antibody of the test substance and optional N6-PEG；
Preferably, the insoluble carrier granular combines offer with first reagent composition with suspended form.
6. the presence of test substance or the immunoturbidimetry of its amount in a kind of whole blood sample measuring non-haemolysis comprising following step
Suddenly：
(1) under conditions of allowing antigen-antibody complex formation, by the whole blood of the non-haemolysis in the first reagent composition
Sample is contacted with insoluble carrier granular；
(2) the turbidity variation of the reaction system of determination step (1)；
(3) pass of the turbidity variation obtained step (2) and the known quantity and turbidity variation that indicate the test substance
The standard curve of system compares, and obtains the content of the test substance；
Wherein, the pan coating of the insoluble carrier has the antibody for specifically binding the test substance；Also, described first
Reagent composition includes betaine type amphoteric surfactant；
Preferably, the whole blood sample is anticoagulated whole blood；
Preferably, the test substance is the albumen in plasma component, such as c reactive protein；Preferably, the test substance is
C reactive protein (CRP)；
Preferably, the whole blood sample of the non-haemolysis contains the test substance；Preferably, the whole blood sample of the non-haemolysis contains
There is CRP；
Preferably, the betaine type amphoteric surfactant be selected from alkyl betaine, alkyl amido betaine, azochlorosulfonate propyl lycine,
Hydroxyl azochlorosulfonate propyl lycine, phosphate ester glycine betaine, and its arbitrary combination；It is highly preferred that the betaine type amphoteric surfactant is
3- sulfopropyls-etradecyldimethylamine glycine betaine；
Preferably, in step (1), first reagent composition is as defined in claim any one of 1-5；
Preferably, further include the steps that closing the insoluble carrier granular using N6-PEG before step (1)；Alternatively,
In step (1), the insoluble carrier granular is also coated with N6-PEG；
Preferably, the insoluble carrier granular is latex particle, such as granules of polystyrene.
7. method of claim 6, wherein in step (2), the optically reaction of determination step (1) starts
The 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 afterwards
The variable quantity of light changes using it as the turbidity of the reaction system of the step (1)；
Preferably, in step (2), start 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, the variable quantity using its difference as absorbance is (that is, turbid
Degree variation)；
Preferably, in step (2), reaction system is being provided respectively within the 6th second and the 60th second after the reaction of step (1) starts
Absorbance at wavelength is measured, the variable quantity (that is, turbidity variation) using its difference as absorbance；
Preferably, the provision wavelengths are 800-900nm, such as 840-860nm, such as 850nm；Preferably, the regulation wave
A length of 850nm；
Preferably, in step (2), exist respectively to reaction system within the 6th second and the 60th second after the reaction of step (1) starts
Absorbance at 850nm is measured, the variable quantity (that is, turbidity variation) using its difference as absorbance；
Preferably, further include one or more in the following steps before step (1)：(a) use blood-taking device from subject
Obtain whole blood sample；(b) anti-coagulants (such as heparin) processing blood-taking device or the whole blood sample are used.
8. purposes of the betaine type amphoteric surfactant in reagent preparation box, the kit are measured not by immunoturbidimetry
The presence of test substance or its amount in the whole blood sample of haemolysis, wherein the kit 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；
Preferably, the whole blood sample is anticoagulated whole blood；
Preferably, the test substance is the albumen in plasma component, such as c reactive protein；Preferably, the test substance is
C reactive protein (CRP)；
Preferably, the betaine type amphoteric surfactant be selected from alkyl betaine, alkyl amido betaine, azochlorosulfonate propyl lycine,
Hydroxyl azochlorosulfonate propyl lycine, phosphate ester glycine betaine, and its arbitrary combination；It is highly preferred that the betaine type amphoteric surfactant is
3- sulfopropyls-etradecyldimethylamine glycine betaine；
Preferably, the kit also includes the second reagent composition, and second reagent composition includes sealer；It is preferred that
Ground, the sealer are N6-PEG；
Preferably, first reagent composition is as defined in claim any one of 1-5；
Preferably, the insoluble carrier granular is latex particle, such as granules of polystyrene；
Preferably, a diameter of 50-400nm of the insoluble carrier granular；
Preferably, the surface of the insoluble carrier granular carries carboxyl, amino, hydroxyl, hydrazide group or sulfydryl modification；It is preferred that
The surface on ground, the insoluble carrier granular carries carboxyl modified；
Preferably, the pan coating of the insoluble carrier granular has the antibody for specifically binding the test substance；Alternatively, institute
The pan coating for stating insoluble carrier granular has the antibody and N6-PEG for specifically binding the test substance；
Preferably, second reagent composition also includes for the antibody to be coated in the insoluble carrier particle surface
Reagent and optional coating buffer solution；Preferably, second reagent composition includes EDC or its salt (such as EDCI)
And NHS；
Preferably, the insoluble carrier granular, the antibody of the specific binding test substance, the first reagent composition and
Second reagent composition is provided separately；Alternatively, the insoluble carrier granular is carried with the first reagent composition as same component
There is the antibody for specifically binding the test substance and optional for, wherein the pan coating of the insoluble carrier granular
N6-PEG；
Preferably, the insoluble carrier granular combines offer with first reagent composition with suspended form.
9. purposes according to any one of claims 8, wherein the kit measures non-haemolysis by the method included the following steps
The presence of test substance or its amount in whole blood sample：
(1) under conditions of allowing antigen-antibody complex formation, by the non-haemolysis in first reagent composition
Whole blood sample is contacted with the insoluble carrier granular；
(2) the turbidity variation of the reaction system of determination step (1)；
(3) pass of the turbidity variation obtained step (2) and the known quantity and turbidity variation that indicate the test substance
The standard curve of system compares, and obtains the content of the test substance；
Wherein, the pan coating of the insoluble carrier has the antibody for specifically binding the test substance；
Preferably, the whole blood sample is anticoagulated whole blood；
Preferably, the test substance is the albumen in plasma component, such as c reactive protein；Preferably, the test substance is
C reactive protein (CRP)；
Preferably, the whole blood sample of the non-haemolysis contains the test substance；Preferably, the whole blood sample of the non-haemolysis contains
There is CRP；
Preferably, further include the steps that closing the insoluble carrier granular using N6-PEG before step (1)；Preferably,
In step (1), the insoluble carrier granular is also coated with N6-PEG；
Preferably, in step (2), the optically reaction of determination step (1) starts in rear certain time (for example, 0-5
Minute, 0-2 minute, 5-120 seconds or 5-60 seconds) variable quantity of absorbance at provision wavelengths or scattering light, using it as institute
State the turbidity variation of the reaction system of step (1)；
Preferably, further include one or more in the following steps before step (1)：(a) use blood-taking device from subject
Obtain whole blood sample；(b) anti-coagulants (such as heparin) processing blood-taking device or the whole blood sample are used.
10. a kind of method preparing the coated insoluble carrier granular of antibody comprising 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；
Preferably, in step (1), the antibody is coated in the surface of insoluble carrier by covalent coupling；
Preferably, further include the steps that the product of washing step (2) to remove the substance for having neither part nor lot in reaction after step (2).
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109541241A (en) * | 2019-01-25 | 2019-03-29 | 浙江夸克生物科技有限公司 | A kind of assay kit of lipoprotein (a) |
| CN110007074A (en) * | 2019-04-18 | 2019-07-12 | 桂林优利特医疗电子有限公司 | For detecting kit, the preparation method and the usage of c reactive protein |
| CN110567900A (en) * | 2019-09-29 | 2019-12-13 | 迈克医疗电子有限公司 | method and device for judging antigen excess in sample reaction and optical detection system |
| CN113009161A (en) * | 2021-02-09 | 2021-06-22 | 桂林优利特医疗电子有限公司 | Detection kit for activated partial thromboplastin time and preparation method thereof |
| CN113825984A (en) * | 2019-05-20 | 2021-12-21 | 美国西门子医学诊断股份有限公司 | Method for detecting abnormal results in immunoassays caused by incomplete delivery of polyhapten reagents |
| CN114578040A (en) * | 2022-05-06 | 2022-06-03 | 深圳市帝迈生物技术有限公司 | Sample detection method, sample analyzer and control device thereof |
| CN116466092A (en) * | 2023-03-21 | 2023-07-21 | 浙江夸克生物科技有限公司 | Kit for quantitatively determining uroretinol binding protein |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63298061A (en) * | 1987-05-28 | 1988-12-05 | Hitachi Chem Co Ltd | Determination of human c reactive protein |
| CN102687015A (en) * | 2009-10-30 | 2012-09-19 | 协和梅迪克斯株式会社 | Method for assaying component to be assayed in specimen and assay kit |
| CN103983621A (en) * | 2014-03-31 | 2014-08-13 | 杭州师范大学 | Fluorescence detection method of flavones in hawthorn fruits |
| CN104459127A (en) * | 2013-12-16 | 2015-03-25 | 顾晋元 | Biocarrier and application of biocarrier in detection |
| CN106546455A (en) * | 2015-09-16 | 2017-03-29 | 深圳迈瑞生物医疗电子股份有限公司 | Hemolytic agent, the method for the pretreatment of biological sample, the assay method of object content and kit |
| CN106645749A (en) * | 2016-12-26 | 2017-05-10 | 深圳优迪生物技术有限公司 | Hemolytic agent and application |
| CN107015004A (en) * | 2017-03-21 | 2017-08-04 | 深圳市汇松科技发展有限公司 | A kind of serum amyloid A protein determines kit and preparation method thereof |
| CN107677804A (en) * | 2017-09-30 | 2018-02-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of NBAP detection kit and its application method |
-
2018
- 2018-04-09 CN CN201810311589.5A patent/CN108593641B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63298061A (en) * | 1987-05-28 | 1988-12-05 | Hitachi Chem Co Ltd | Determination of human c reactive protein |
| CN102687015A (en) * | 2009-10-30 | 2012-09-19 | 协和梅迪克斯株式会社 | Method for assaying component to be assayed in specimen and assay kit |
| CN106959367A (en) * | 2009-10-30 | 2017-07-18 | 协和梅迪克斯株式会社 | Method and kit for determining the composition to be determined in sample |
| CN104459127A (en) * | 2013-12-16 | 2015-03-25 | 顾晋元 | Biocarrier and application of biocarrier in detection |
| CN103983621A (en) * | 2014-03-31 | 2014-08-13 | 杭州师范大学 | Fluorescence detection method of flavones in hawthorn fruits |
| CN106546455A (en) * | 2015-09-16 | 2017-03-29 | 深圳迈瑞生物医疗电子股份有限公司 | Hemolytic agent, the method for the pretreatment of biological sample, the assay method of object content and kit |
| CN106645749A (en) * | 2016-12-26 | 2017-05-10 | 深圳优迪生物技术有限公司 | Hemolytic agent and application |
| CN107015004A (en) * | 2017-03-21 | 2017-08-04 | 深圳市汇松科技发展有限公司 | A kind of serum amyloid A protein determines kit and preparation method thereof |
| CN107677804A (en) * | 2017-09-30 | 2018-02-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of NBAP detection kit and its application method |
Non-Patent Citations (2)
| Title |
|---|
| ROBERT M. Q. SHANKS等: "Serratamolide is a Hemolytic Factor Produced by Serratia marcescens", 《PLOS ONE》 * |
| 柳帅 等: "抗体修饰纳米载体表面构建主动靶向制剂常用偶联方法", 《中国医药工业杂志》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109541241A (en) * | 2019-01-25 | 2019-03-29 | 浙江夸克生物科技有限公司 | A kind of assay kit of lipoprotein (a) |
| CN110007074A (en) * | 2019-04-18 | 2019-07-12 | 桂林优利特医疗电子有限公司 | For detecting kit, the preparation method and the usage of c reactive protein |
| CN113825984A (en) * | 2019-05-20 | 2021-12-21 | 美国西门子医学诊断股份有限公司 | Method for detecting abnormal results in immunoassays caused by incomplete delivery of polyhapten reagents |
| CN110567900A (en) * | 2019-09-29 | 2019-12-13 | 迈克医疗电子有限公司 | method and device for judging antigen excess in sample reaction and optical detection system |
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