CN108458999A - The method and its kit of a variety of cardiac biomarkers of joint-detection - Google Patents
The method and its kit of a variety of cardiac biomarkers of joint-detection Download PDFInfo
- Publication number
- CN108458999A CN108458999A CN201810123322.3A CN201810123322A CN108458999A CN 108458999 A CN108458999 A CN 108458999A CN 201810123322 A CN201810123322 A CN 201810123322A CN 108458999 A CN108458999 A CN 108458999A
- Authority
- CN
- China
- Prior art keywords
- reagent
- cardiac biomarkers
- blood
- detection
- joint
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The present invention provides a kind of kits of a variety of cardiac biomarkers of joint-detection, including melt blood reagent, microballoon reagent and fluorescent reagent, and described melt in blood reagent contains surfactant；The microballoon reagent includes the different N kind microballoons of diameter, the first monoclonal antibody of corresponding coupling N kind cardiac biomarkers respectively on the N kinds microballoon, and mark and have in first monoclonal antibody, wherein the value range of N for 2~10 positive integer；The fluorescent reagent includes the second monoclonal antibody of the N kinds cardiac biomarkers, and being marked on the second monoclonal antibody has, and first monoclonal antibody, the second monoclonal antibody are different from the binding site of antigen.
Description
Technical field
The invention belongs to the sides of field of molecular biotechnology more particularly to a kind of a variety of cardiac biomarkers of joint-detection
Method and its kit.
Background technology
Immunofluorescence technique is using corresponding in fluorescein-labeled antibody (or antigen) detection tissue, cell or serum
The method of antigen (or antibody).Due to fluorescence antibody have the characteristics that safety, it is sensitive, be widely used in Immunofluorescence test
With flow cytometry field.Currently used immunofluorescence technique includes：Radio-immunity, enzyme linked immunological, latex is than turbid, dry type
Fluorescence immunoassay, time-resolved fluoroimmunoassay, chemiluminescence immunoassay etc..Chemiluminescence immunoassay is the immune detection of current most mainstream
Method, testing principle are usually：Using magnetic microsphere as solid phase material conjugated monoclonal antibodies (primary antibody), after antigen-reactive,
The monoclonal antibody (secondary antibody) of enzyme label is added, fully removes unreacted reagent through washing, separation and purifying after reaction.Then
Luminous substrate (luminous color developing agent) is added, under incubation conditions appropriate, substrate is developed the color by enzymatic.It is finally whole according to reaction solution
Body luminous intensity judges antigen concentration, is quantitative determined to single project.This method detection speed is fast, high sensitivity, extensively
It applies in central laboratory of clinical laboratory.But on the one hand, since detection-phase must measure the Integral luminous value of reaction solution, anti-
The antigen-antibody reaction stage needs that excessive secondary antibody reagent is added, and to obtain abundant reaction, and then to carry out multistep after incubation
Rapid washing and Magneto separate, to remove excessive unreacted secondary antibody reagent.This so that entire apparatus structure is complicated, and cost is got higher,
Body machine becomes larger, and detection speed is slack-off.On the other hand, chemiluminescence immunoassay can only use serum as sample, clinic is measured and receive
Blood layering and serum extracting are also carried out after collection whole blood, increase the complexity and pollution risk of Clinical practice, Wu Fa
Emergency call and basic medical unit are carried out extensively.In addition, an item can only be measured by being detected every time using Chemiluminescence Immunoassay
Mesh detects while cannot achieve multiple projects.
Dry type fluorescence immunoassay is due to its easy to operate, extensive use.The method of dry type immunofluorence technic is usually：Whole blood
Or serum sample enters sample pad after filter screen, if any determined antigen in sample, determined antigen first under bonding pad with it is glimmering
The monoclonal antibody (primary antibody) of light element label combines, and forms Ag-Ab * luciferin complexes, and lean on capillarity to test
Line moves.Another monoclonal antibody (secondary antibody) is fixed on p-wire, it, can further shape when reaction solution is chromatographed to p-wire
At the double antibodies sandwich compound for being marked with fluorescein, generated with the reactant built up in the laser irradiation detection line of respective wavelength glimmering
Light, to be quantitative determined.But using dry type fluorescence immunoassay detection when, due to solid phase liquid phase reactor itself there are biggers not
Certainty, and reactant has thickness in the accumulation of test line position, and laser is only capable of the fluorescein p-wire surface
Excitation, therefore the withinrun precision CV of dry type fluorescence strip is usually more than the 10% (standard of 20 measurement of the same samples of CV=
Difference/average value * 100%), i.e., precision is insufficient, limits the application of dry type immunofluorence technic.In addition, dry type strip is each
Test will have solid phase carrier, NC films, dry type reaction to need a large amount of antibody, therefore reagent cost is far above the reagent of liquid phase
(such as chemiluminescence immunoassay, suspension array are immune).
Invention content
The purpose of the present invention is to provide the method and its kit of a kind of a variety of cardiac biomarkers of joint-detection, purports
In the immunologic detection method for solving existing cardiac marker, can only be detected for serum sample, and detection time length,
It is complicated for operation, price is high, and the problem of each test can only measure a project.
For achieving the above object, the technical solution adopted by the present invention is as follows：
One aspect of the present invention provides a kind of kit of a variety of cardiac biomarkers of joint-detection, including melt blood reagent,
Microballoon reagent and fluorescent reagent,
Described melt in blood reagent contains surfactant；
The microballoon reagent includes the different N kind microballoons of diameter, corresponds to coupling N kind hearts life on the N kinds microballoon respectively
First monoclonal antibody of object marker, and mark and have in first monoclonal antibody, wherein the value range of N
For 2~10 positive integer；
The fluorescent reagent includes the second monoclonal antibody of the N kinds cardiac biomarkers, second monoclonal
There is label, and the binding site of first monoclonal antibody, the second monoclonal antibody and antigen is not on antibody
Together.
And a kind of method of a variety of cardiac biomarkers of joint-detection, it at least includes the following steps：
Whole blood sample, serum sample and the kit such as a variety of cardiac biomarkers of above-mentioned joint-detection are provided；
N kinds business antigen corresponding with the N kinds cardiac biomarkers is added in the serum sample, is configured to ladder
The Working Standard Solution of concentration is spent, concentration-Standardization curve for fluorescence intensity of N kind cardiac biomarkers is obtained；
It is added in the whole blood sample and melts blood reagent, carried out melting blood incubation processing, obtain melting blood sample；Melt blood described
Microballoon reagent and fluorescent reagent are added in sample, sample to be tested is obtained through immune response；Identical with the working standard
Under testing conditions, the sample to be tested is detected using suspension array fluorescence immunity analyzer, by testing result with it is described
Concentration-fluorescence standard curve is compared, and obtains the concentration of N kind cardiac biomarkers.
The kit of a variety of cardiac biomarkers of joint-detection provided by the invention, using microsphere volume as coding,
Coupling is marked with the monoclonal antibody (one of the N kind cardiac biomarkers of biotin respectively on the microballoon of multiple and different diameters
It is anti-), when there is these cardiac biomarkers to be measured in sample, the microballoon of different-diameter captures different determinands respectively,
After addition is marked with the second monoclonal antibody of N kind cardiac biomarkers of fluorescein, it is respectively formed on microballoon for difference
The double antibodies sandwich reactant of cardiac biomarkers.It is quantitative determined using suspension array fluorescence immunity analyzer, by each
The microballoon of volume is analyzed one by one and accumulation calculating is as a result, quantified results that are final while obtaining N kind cardiac biomarkers.
Specifically, the kit of a variety of cardiac biomarkers of joint-detection provided by the invention has the following advantages：
First, the joint-detection of N kind cardiac biomarkers can be realized by suspension array fluorescence immunity analyzer, and is had
Have the advantages that at low cost, efficient, precision is high.
Secondly, the kit of a variety of cardiac biomarkers of joint-detection provided by the invention can be used whole blood as sample
This, reaction process is carried out in liquid phase environment, and the washing without multistep isolates and purifies, can be under the premise of being detached without serum
Quickly, the joint-detection for conveniently, accurately carrying out cardiac marker, has filled up the market vacancy.
Again, the kit of a variety of cardiac biomarkers of joint-detection provided by the invention, reagent prepare core former material
Expect that dosage is few, testing cost can be greatly reduced.
The method of a variety of cardiac biomarkers of joint-detection provided by the invention is based on a variety of heart biologies of joint-detection
The operation principle of the kit of marker, has the following advantages：
First, the present invention can be easy to operate, detection is quick using whole blood as pattern detection, and can one-stop completion it is more
The detection of a cardiac marker project makes the detection level of cardiac biomarkers improve a step.
Second, it is quantitative determined, result is added up later, therefore need not be to micro- for each microballoon in detection-phase
Ball carries out washing and separating step, has the advantages that equipment cost is lower, body machine smaller.To realize two heart biology marks simultaneously
For the detection of will object, microballoon and antibody levels that suspension array immunological technique uses probably are the one of existing chemiluminescence
Half, 1/10th of dry type immunofluorence technic, moreover it is possible to the packing cost of outer packaging material is greatly reduced, therefore, present invention tool
There is apparent cost advantage.
Third, the present invention is based on the liquid phase reactor that suspension array immunological technique uses double antibodies sandwich, precision reaches existing
The highest level of immunoassay, CV<5%.
Specific implementation mode
In order to make technical problems, technical solutions and advantageous effects to be solved by the present invention be more clearly understood, below in conjunction with
Embodiment, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used to explain
The present invention is not intended to limit the present invention.
In the description of the present invention, it is to be understood that, term " first ", " second " are used for description purposes only, and cannot
It is interpreted as indicating or implies relative importance or implicitly indicate the quantity of indicated technical characteristic.Define as a result, " the
One ", the feature of " second " can explicitly or implicitly include one or more this feature.In the description of the present invention,
The meaning of " plurality " is two or more, unless otherwise specifically defined.
An embodiment of the present invention provides a kind of kits of a variety of cardiac biomarkers of joint-detection, including melt blood examination
Agent, microballoon reagent and fluorescent reagent,
Described melt in blood reagent contains surfactant；
The microballoon reagent includes the different N kind microballoons of diameter, corresponds to coupling N kind hearts life on the N kinds microballoon respectively
First monoclonal antibody of object marker, and mark and have in first monoclonal antibody, wherein the value range of N
For 2~10 positive integer；
The fluorescent reagent includes the second monoclonal antibody of the N kinds cardiac biomarkers, second monoclonal
There is label, and the binding site of first monoclonal antibody, the second monoclonal antibody and antigen is not on antibody
Together.
The kit of a variety of cardiac biomarkers of joint-detection provided in an embodiment of the present invention, using microsphere volume as volume
Code, the monoclonal for being coupled the N kind cardiac biomarkers for being marked with biotin respectively on the microballoon of multiple and different diameters are anti-
Body (primary antibody), when there is these cardiac biomarkers to be measured in sample, the microballoon of different-diameter captures different to be measured respectively
Object is respectively formed on microballoon and is directed to after addition is marked with the second monoclonal antibody of N kind cardiac biomarkers of fluorescein
The double antibodies sandwich reactant of different cardiac biomarkers.It is quantitative determined using suspension array fluorescence immunity analyzer, by right
The microballoon of each volume is analyzed one by one and accumulation calculating is as a result, quantitative determination that is final while obtaining N kind cardiac biomarkers
As a result.Specifically, the kit of a variety of cardiac biomarkers of joint-detection provided in an embodiment of the present invention has the following advantages：
First, the joint-detection of N kind cardiac biomarkers can be realized by suspension array fluorescence immunity analyzer, and is had
Have the advantages that at low cost, efficient, precision is high.
Secondly, whole blood can be used in the kit of a variety of cardiac biomarkers of joint-detection provided in an embodiment of the present invention
As sample, reaction process is carried out in liquid phase environment, the washing without multistep isolates and purifies, and can detached without serum
Under the premise of quickly, conveniently, accurately carry out cardiac marker joint-detection, filled up the market vacancy.
Again, the kit of a variety of cardiac biomarkers of joint-detection provided in an embodiment of the present invention, reagent prepare core
Heart raw material usage is few, and testing cost can be greatly reduced.
In the embodiment of the present invention, described melt in blood reagent contains surfactant, thin for destroying the blood in whole blood sample
The cell membrane of born of the same parents realizes the detection of whole blood sample to remove interference of the haemocyte for detecting system.Specifically, described melt blood
Reagent includes melting blood Reagent Concentrate and melting blood reagent dilutions, wherein the blood Reagent Concentrate that melts is as functional liquid, mainly
Cell membrane for destroying the haemocyte in whole blood sample；The blood reagent dilutions that melt are described for diluting whole blood sample
The work for melting blood Reagent Concentrate is prepared.Further, described to melt blood Reagent Concentrate and the blood reagent dilutions of melting
Volume ratio is (1:20)~(1:5), i.e., described to melt blood Reagent Concentrate with the blood reagent dilutions that melt according to volume ratio (1:
20)~(1:5) it is configured to finished product and melts blood reagent.The suitable volume ratio can substantially effectively disperse the blood in whole blood sample
Cell, and then be conducive to fully, efficiently destroy the cell membrane of the haemocyte in whole blood sample, removal haemocyte is for detection system
The interference of system.
Preferably, it is in terms of 100% by the total weight for melting blood Reagent Concentrate, the blood Reagent Concentrate that melts includes matter
Measure the following following component of percentage composition：
The suitable component and content for melting blood Reagent Concentrate, is conducive to the destruction of haemocyte in whole blood sample, meanwhile, no
Increase post-processing difficulty (not introducing new influence factor), to ensure the accuracy of test result.
It is further preferred that the surfactant is lauryl sodium sulfate, the preservative is Proclin300.It is excellent
Blood Reagent Concentrate is melted in choosing, has and preferably melts blood effect, to which haemocyte to be preferably minimized the interference of testing result, carries
The accuracy of high detection.
In the embodiment of the present invention, the PBS buffer solution melted blood reagent dilutions and use 200mM, and contain quality percentage
Content is 0.9% sodium chloride, and the blood reagent dilutions pH that melts is conducive to the abundant of haemocyte in whole blood sample between 7-9
Dispersion, and then the surfactant is contributed to be destroyed.
In the embodiment of the present invention, the microballoon reagent includes the different N kind microballoons of diameter, i.e. the embodiment of the present invention passes through right
The volume of microballoon is encoded, and different cardiac biomarkers are corresponding with the volume of microballoon, distinguishes different heart biology marks
The testing result of will object.The microballoon is the microballoon for the antibody that can be coupled cardiac biomarkers, i.e., contains on the described microballoon
It can be coupled the modification structure of the antibody of cardiac biomarkers, specifically, on the modification structure and antibody on the microballoon
Biotin is coupled.Preferably, the microballoon is selected from polystyrene microsphere, hydroxyl or the amido modified macromolecule of streptomysin modification
One kind in microballoon, hydroxyl or amido modified metallic microspheres.The preferred microballoon, can not only securely be coupled heart biology
The antibody of marker, and the factor of other influences detection accuracy and timeliness will not be introduced in detection process, it improves
The accuracy of detection and efficiency.
In the embodiment of the present invention, the value range of N is 2~10 positive integer, i.e., the described a variety of heart biologies of joint-detection
The kit of marker can be realized simultaneously the detection of 2~10 cardiac biomarkers.It is further preferred that the value of the N
Ranging from 2~6 positive integer, and the cardiac biomarkers are selected from cTnI (Troponin I), (the amino ends NT-proBNP
End-brain natriuretic peptide is former), cTnT (troponin T), CK-MB (creatine kinase isoenzymes), BNP (brain natriuretic peptide), Myo (flesh red eggs
In vain).As a specific embodiment, kit joint-detection cTnI, NT-proBNP, respectively in two kinds of different volumes
Two kind monoclonal antibodies of the microsphere surface coupling for people cTnI antigens and people's NT-proBNP antigens.
As an implementation, the microballoon reagent can be prepared by following methods.Specifically, the microballoon reagent
Preparation method include the following steps：
Pretreatment：Microsphere suspensions are provided, centrifugal treating after being diluted with PBS buffer solution removes supernatant, collects microballoon；
Label：After being diluted with PBS buffer solution, life is added in the first monoclonal antibody solution for providing cardiac biomarkers
Object element solution obtains the first monoclonal antibody solution of biotin labeling；
Coupling：First monoclonal antibody solution of the biotin labeling is mixed with the microballoon, mixing processing after from
The heart removes supernatant, obtains the first monoclonal antibody coupling microballoon；
Closing：Seal treatment is carried out in first monoclonal antibody coupling microballoon；
It is quenched：First monoclonal antibody coupling microballoon after closure is quenched, as microballoon Reagent Concentrate
It stores for future use.
The microballoon reagent of different volumes microballoons (corresponding different cardiac biomarkers) is completed using above-mentioned steps, then into
Row mixed processing.
As a specific embodiment, using cTnI and people NT-proBNP as detection object, joint-detection cTnI and people
The preparation method of the microballoon reagent of the kit of NT-proBNP includes the following steps：
Pretreatment：The raw material microsphere suspensions for taking certain volume on demand, (contain 0.9wt% with the PBS buffer solution of 200mM
Sodium chloride, pH is between 7-9) 10 times of dilution, it is centrifuged 10 minutes in 10000G room temperatures, removes supernatant, after addition dilution in equal volume
Above-mentioned PBS buffer solution is vortexed 1 minute, is centrifuged 10 minutes in 10000G room temperatures, goes supernatant spare.
Label：The first monoclonal antibody solution of certain volume is taken, (PBS buffer solution of 200mM, contains with PBS buffer solution
0.9wt% sodium chloride, pH is between 7-9) 5-10 times of dilution, the appropriate biotin solution (mass ratio of biotin and antibody is added
Not less than 1:10) it, is stored at room temperature 6 hours.
Coupling：The first monoclonal antibody solution after label is added in the spare microballoon of supernatant, room temperature mixing 4-8
Hour, it is centrifuged 10 minutes in 10000G room temperatures, goes supernatant spare.
Closing：Isometric PBS buffer solution is added, and (PBS buffer solution of 200mM contains 0.9wt% sodium chloride, 0.5wt%
BSA, pH are between 7-9), it mixes, is vortexed 1 minute with the microballoon after coupling, 36 DEG C of constant temperature blendings 18-24 hours, in 10000G
Room temperature centrifuges 10 minutes, goes supernatant spare.
It is quenched：Isometric PBS buffer solution is added, and (PBS buffer solution of 200mM contains 0.1M containing 0.9wt% sodium chloride
Biotin, pH is between 7-9), it mixes, is vortexed 1 minute with the microballoon after coupling, 36 DEG C of constant temperature blendings 4-8 hours, in 10000G
Room temperature centrifuges 10 minutes, removes supernatant, is added containing 0.5wt%BSA, 0.02-0.05wt%Tween80,0.05-0.1wt%
The PBS buffer solution (PBS buffer solution of 200mM, containing 0.9wt% sodium chloride, pH is between 7-9) of PC300, as microballoon reagent
Concentrate stores for future use.
The microballoon Reagent Concentrate for being respectively completed cTnI and NT-proBNP is mixed according to a certain percentage (such as according to cTnI
Ball number ratio NT-proBNP microballoon numbers are 1:6) microballoon Reagent Concentrate semi-finished product, are obtained after allotment, 100-400 times of dilution obtains
Finished product microballoon reagent.
In the embodiment of the present invention, the fluorescent reagent includes the second monoclonal antibody of the N kinds cardiac biomarkers,
There is label, but first monoclonal antibody, the second monoclonal antibody are different on the second monoclonal antibody,
That is, the site of second monoclonal antibody connection antigen connect the position of antigen with the first monoclonal antibody used in microballoon reagent
Point is different, to ensure to form the reaction structure of double antibodies sandwich.The embodiment of the present invention forms the reaction structure of double antibodies sandwich, Cai Nengbao
After card antigen is coupled the microballoon capture of first monoclonal antibody, the second monoclonal antibody with fluorescein is made
It is connect with antigen for marker.The fluorescein is glimmering using the fluorescein that can be detected system detectio as signal tracer
The selection of light element does not limit strictly specifically.
The preparation method of the fluorescent reagent is as follows：First the monoclonal antibody of detection disparity items is individually marked respectively
Note, then two kinds of semi-finished product are mixed to get finished product in proportion, pretreatment is specifically included, marks, be quenched, purifying, mixing five ranks
Section.
As a specific embodiment, using cTnI and people NT-proBNP as detection object, joint-detection cTnI and people
The preparation method of the fluorescent reagent of the kit of NT-proBNP includes the following steps：
Pretreatment：The solvent that raw material antibody-solutions are replaced with the PBS buffer solution (pH=7-9) of 100mM, can be used super
The methods of filter, dialysis are completed, finally that the concentration constant volume of antibody-solutions is spare to 5mg/ml.
Label：The antibody-solutions of certain volume are taken on demand, and with PBS buffer solution, (PBS buffer solution of 200mM, contains
0.9wt% sodium chloride, pH is between 7-9) 5-10 times of dilution, appropriate luciferin solution (fluorescein molecule and antibody molecule is added
Molar ratio be not less than 100:1), it is protected from light room temperature mixing 18-24 hours.
It is quenched：According to isometric PBS buffer solution of the addition containing 1M glycine, (PBS buffer solution of 200mM, contains
0.9wt% sodium chloride, pH is between 7-9), room temperature is protected from light standing 4 hours.
Purifying：Solution after being quenched, to PBS buffer solution, (PBS buffer solution of 200mM, containing 0.9wt% sodium chloride, pH exists
Between 7-9), according to 1:1000 volumes are dialysed 48 hours, replace dialyzate for every eight hours, the fluorescent reagent concentrate after dialysis is kept away
Light stores for future use.
Mixing：The fluorescent reagent concentrate for being respectively completed cTnI and NT-proBNP mix according to a certain percentage (such as according to
CTnI antibody mole ratio NT-proBNP antibody molal quantitys are 1:1) fluorescent reagent concentrate semi-finished product, dilution, are obtained after allotment
50-200 times obtains finished product fluorescent reagent.
Correspondingly, an embodiment of the present invention provides a kind of method of a variety of cardiac biomarkers of joint-detection, at least wrap
Include following steps：
S01., whole blood sample, serum sample and the kit such as a variety of cardiac biomarkers of above-mentioned joint-detection are provided；
S02. N kinds business antigen corresponding with the N kinds cardiac biomarkers, configuration are added in the serum sample
At the Working Standard Solution of gradient concentration, concentration-Standardization curve for fluorescence intensity of N kind cardiac biomarkers is obtained；
S03. it is added in the whole blood sample and melts blood reagent, carried out melting blood incubation processing, obtain melting blood sample；Described
Melt and microballoon reagent and fluorescent reagent are added in blood sample, sample to be tested is obtained through immune response；With the working stamndard condition
With testing conditions under, the sample to be tested is detected using suspension array fluorescence immunity analyzer, by testing result with
Concentration-the fluorescence standard curve is compared, and obtains the concentration of N kind cardiac biomarkers.
The method of a variety of cardiac biomarkers of joint-detection provided in an embodiment of the present invention is based on a variety of hearts of joint-detection
The operation principle of the kit of dirty biomarker, has the following advantages：
First, the embodiment of the present invention can be easy to operate, detection is quick using whole blood as pattern detection, and can be one-stop
The detection for completing multiple cardiac marker projects makes the detection level of cardiac biomarkers improve a step.
Second, it is quantitative determined, result is added up later, therefore need not be to micro- for each microballoon in detection-phase
Ball carries out washing and separating step, has the advantages that equipment cost is lower, body machine smaller.To realize two heart biology marks simultaneously
For the detection of will object, microballoon and antibody levels that suspension array immunological technique uses probably are the one of existing chemiluminescence
Half, 1/10th of dry type immunofluorence technic, moreover it is possible to which the packing cost of outer packaging material is greatly reduced, therefore, the present invention is real
Applying example has apparent cost advantage.
Third, the embodiment of the present invention use the liquid phase reactor of double antibodies sandwich, precision to reach based on suspension array immunological technique
To the highest level of existing immunoassay, CV<5%.
Specifically, in above-mentioned steps S01, the whole blood sample, the serum sample are in vitro blood sample.Joint inspection
The kit for surveying a variety of cardiac biomarkers is as described above, and in order to save length, details are not described herein again.
In above-mentioned steps S02, it is bent to obtain the corresponding concentration of serum sample N kind cardiac biomarkers-fluorescence intensity standard
Line.Specifically, N kinds business antigen corresponding with the N kinds cardiac biomarkers is added in the serum sample, it is configured to
The Working Standard Solution of gradient concentration is detected the Working Standard Solution using suspension array fluorescence immunity analyzer,
Obtain relative concentration-fluorescence standard curve of N kind cardiac biomarkers；It is the standard items of matrix to the phase to use human serum
Concentration-fluorescence standard curve is calibrated, obtains concentration-fluorescence standard curve of N kind cardiac biomarkers respectively.Into one
Step, use human serum to be calibrated the relative concentration-fluorescence standard curve for the standard items of matrix, including use people's blood
It is clear that the corresponding relative concentration-fluorescence standard curve of N kind cardiac biomarkers is determined respectively for the N kinds standard items of matrix
Mark.
In above-mentioned steps S03, it is added in the whole blood sample and melts blood reagent, destroyed haemocyte film, avoid haemocyte pair
Detect the interference brought.Preferably, the whole blood sample is diluted 10-30 times using the blood reagent that melts, under the conditions of 37 DEG C
Constant-temperature incubation 1-5min is conducive to substantially effectively destroy haemocyte film.Further, microballoon is added in blood sample in described melt
Reagent and fluorescent reagent obtain the sample to be tested of the reaction structure with double antibodies sandwich through immune response.Preferably, the microballoon
The volume ratio of reagent and the fluorescent reagent is 1:1, the condition of the immune response is constant-temperature incubation 2- under the conditions of 37 DEG C
15min.Under testing conditions identical with the working standard, using suspension array fluorescence immunity analyzer to described to be measured
Sample is detected, and testing result is compared with the concentration-fluorescence standard curve, obtains N kind cardiac biomarkers
Concentration.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement etc., should all be included in the protection scope of the present invention made by within refreshing and principle.
Claims (10)
1. a kind of kit of a variety of cardiac biomarkers of joint-detection, which is characterized in that including melting blood reagent, microballoon reagent
And fluorescent reagent,
Described melt in blood reagent contains surfactant；
The microballoon reagent includes the different N kind microballoons of diameter, and coupling N kind heart biology marks are corresponded to respectively on the N kinds microballoon
First monoclonal antibody of will object, and mark and have in first monoclonal antibody, wherein the value range of N is 2~
10 positive integer；
The fluorescent reagent includes the second monoclonal antibody of the N kinds cardiac biomarkers, the second monoclonal antibody
Upper label has, and first monoclonal antibody, the second monoclonal antibody are different from the binding site of antigen.
2. the kit of a variety of cardiac biomarkers of joint-detection as described in claim 1, which is characterized in that the microballoon
One in streptomysin, hydroxyl or amido modified polymer microsphere, streptomysin, hydroxyl or amido modified metallic microspheres
Kind.
3. the kit of a variety of cardiac biomarkers of joint-detection as described in claim 1, which is characterized in that the N's
Value range be 2~6 positive integer, and the cardiac biomarkers be selected from cTnI, NT-proBNP, cTnT, CK-MB, BNP,
Myo。
4. the kit of a variety of cardiac biomarkers of joint-detection as described in any one of claims 1-3, which is characterized in that
The blood reagent that melts includes melting blood Reagent Concentrate and to melt blood reagent dilutions, and described melt blood Reagent Concentrate and described melt blood
The volume ratio of reagent dilutions is (1:20)~(1:5).
5. the kit of a variety of cardiac biomarkers of joint-detection as claimed in claim 4, which is characterized in that melted with described
The total weight of blood Reagent Concentrate is 100% meter, and the blood Reagent Concentrate that melts includes following following group of mass percentage
Point：
6. the kit of a variety of cardiac biomarkers of joint-detection as claimed in claim 5, which is characterized in that the surface
Activating agent is lauryl sodium sulfate, and the preservative is Proclin300.
7. a kind of method of a variety of cardiac biomarkers of joint-detection, which is characterized in that at least include the following steps：
Whole blood sample, serum sample and a variety of cardiac biomarkers of joint-detection as described in claim any one of 1-6 are provided
Kit；
N kinds business antigen corresponding with the N kinds cardiac biomarkers is added in the serum sample, it is dense to be configured to gradient
The Working Standard Solution of degree obtains concentration-Standardization curve for fluorescence intensity of N kind cardiac biomarkers；
It is added in the whole blood sample and melts blood reagent, carried out melting blood incubation processing, obtain melting blood sample；Melt blood sample described
Middle addition microballoon reagent and fluorescent reagent obtain sample to be tested through immune response；In detection identical with the working standard
Under the conditions of, the sample to be tested is detected using suspension array fluorescence immunity analyzer, by testing result and the concentration-
Fluorescence standard curve is compared, and obtains the concentration of N kind cardiac biomarkers.
8. the method for a variety of cardiac biomarkers of joint-detection as claimed in claim 7, which is characterized in that melt blood described
In the step of microballoon reagent and fluorescent reagent are added in sample, sample to be tested is obtained through immune response, the microballoon reagent and institute
The volume ratio for stating fluorescent reagent is 1:1, the condition of the immune response is constant-temperature incubation 2-15min under the conditions of 37 DEG C.
9. the method for a variety of cardiac biomarkers of joint-detection as claimed in claim 7, which is characterized in that in the whole blood
It is added in sample and melts blood reagent, carry out melting the step of blood is incubated processing, including：Using the blood reagent that melts by the whole blood sample
10-30 times of dilution, constant-temperature incubation 1-5min under the conditions of 37 DEG C.
10. such as the method for a variety of cardiac biomarkers of claim 7-9 any one of them joint-detections, which is characterized in that
N kinds business antigen corresponding with the N kinds cardiac biomarkers is added in the serum sample, is configured to gradient concentration
After Working Standard Solution, further include；The Working Standard Solution is detected using suspension array fluorescence immunity analyzer, is obtained
Take relative concentration-fluorescence standard curve of N kind cardiac biomarkers；It is the standard items standard items of matrix to institute to use human serum
It states relative concentration-fluorescence standard curve to be calibrated, obtains concentration-fluorescence standard curve of N kind cardiac biomarkers respectively.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810123322.3A CN108458999A (en) | 2018-02-07 | 2018-02-07 | The method and its kit of a variety of cardiac biomarkers of joint-detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810123322.3A CN108458999A (en) | 2018-02-07 | 2018-02-07 | The method and its kit of a variety of cardiac biomarkers of joint-detection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108458999A true CN108458999A (en) | 2018-08-28 |
Family
ID=63238764
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810123322.3A Pending CN108458999A (en) | 2018-02-07 | 2018-02-07 | The method and its kit of a variety of cardiac biomarkers of joint-detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108458999A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112782136A (en) * | 2020-12-24 | 2021-05-11 | 湖南博奥瑞康生物科技有限公司 | Biomarker combined detection method and kit based on peroxyoxalate chemiluminescence system |
| CN113189071A (en) * | 2021-04-29 | 2021-07-30 | 上海交通大学 | Kit and imaging method for accurate imaging of three-dimensional network of blood vessel of complete organ |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1181551A1 (en) * | 1999-05-04 | 2002-02-27 | Dan A. Pankowsky | Products and methods for single parameter and multiparameter phenotyping of cells |
| EP2065707A1 (en) * | 2007-11-28 | 2009-06-03 | Laboratorios S.A.L.V.A.T., S.A. | Detection of endometrial secretion markers for assesment of endometriosis |
| CN101825627A (en) * | 2009-03-02 | 2010-09-08 | 江苏迈迪基因生物科技有限公司 | Combined parallel detection method for cardiac failure biomarkers and diagnostic reagent kit |
| CN102062735A (en) * | 2009-11-18 | 2011-05-18 | 江苏迈迪基因生物科技有限公司 | Biomarker detection method and diagnostic kit for acute coronary syndrome |
| CN103713120A (en) * | 2013-12-20 | 2014-04-09 | 浙江大学 | Method for judging cell state in body liquid environment based on liquid-phase chip technology |
| CN103837675A (en) * | 2014-03-07 | 2014-06-04 | 常艳敏 | Homogeneous luminescence immunoassay method for quantitatively analyzing multiple components simultaneously and kit used for method |
| CN106950381A (en) * | 2017-03-21 | 2017-07-14 | 中国人民解放军第四五八医院 | A kind of kit of joint-detection acute myocardial infarction AMI biomarker |
| CN107340395A (en) * | 2017-07-05 | 2017-11-10 | 深圳开立生物医疗科技股份有限公司 | A kind of Immunoturbidimetric kit for detecting Procalcitonin |
-
2018
- 2018-02-07 CN CN201810123322.3A patent/CN108458999A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1181551A1 (en) * | 1999-05-04 | 2002-02-27 | Dan A. Pankowsky | Products and methods for single parameter and multiparameter phenotyping of cells |
| EP2065707A1 (en) * | 2007-11-28 | 2009-06-03 | Laboratorios S.A.L.V.A.T., S.A. | Detection of endometrial secretion markers for assesment of endometriosis |
| CN101825627A (en) * | 2009-03-02 | 2010-09-08 | 江苏迈迪基因生物科技有限公司 | Combined parallel detection method for cardiac failure biomarkers and diagnostic reagent kit |
| CN102062735A (en) * | 2009-11-18 | 2011-05-18 | 江苏迈迪基因生物科技有限公司 | Biomarker detection method and diagnostic kit for acute coronary syndrome |
| CN103713120A (en) * | 2013-12-20 | 2014-04-09 | 浙江大学 | Method for judging cell state in body liquid environment based on liquid-phase chip technology |
| CN103837675A (en) * | 2014-03-07 | 2014-06-04 | 常艳敏 | Homogeneous luminescence immunoassay method for quantitatively analyzing multiple components simultaneously and kit used for method |
| CN106950381A (en) * | 2017-03-21 | 2017-07-14 | 中国人民解放军第四五八医院 | A kind of kit of joint-detection acute myocardial infarction AMI biomarker |
| CN107340395A (en) * | 2017-07-05 | 2017-11-10 | 深圳开立生物医疗科技股份有限公司 | A kind of Immunoturbidimetric kit for detecting Procalcitonin |
Non-Patent Citations (1)
| Title |
|---|
| 钱学贤等: "《现代心血管病学》", 31 May 1999 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112782136A (en) * | 2020-12-24 | 2021-05-11 | 湖南博奥瑞康生物科技有限公司 | Biomarker combined detection method and kit based on peroxyoxalate chemiluminescence system |
| CN113189071A (en) * | 2021-04-29 | 2021-07-30 | 上海交通大学 | Kit and imaging method for accurate imaging of three-dimensional network of blood vessel of complete organ |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108459162A (en) | Detect the method and its kit of inflammation biomarker | |
| Chon et al. | Simultaneous immunoassay for the detection of two lung cancer markers using functionalized SERS nanoprobes | |
| US7618792B2 (en) | Multiplexed detection of anti-red cell alloantibodies | |
| CN105403693B (en) | A kind of preparation method of magnetic microparticle chemiluminescence reagent | |
| EP1255111A1 (en) | Immunochromato device and method for measuring samples using the same | |
| CN107817232A (en) | For the automation immunoassay system for the diagnostic assay for carrying out allergy and autoimmune disease | |
| JPH01163662A (en) | Detection of antigen and/or antibody and test kit for detection | |
| TWI475229B (en) | A reagent group for transfusion test and its test method | |
| CN106645709A (en) | Aptamer test strip adopting dual competitive method and application of aptamer detection strip | |
| FI95752B (en) | Determination kit and method for immunological measurement of whole cells | |
| CN104535770A (en) | Myoglobin determination kit of compound antibody | |
| CN108603880A (en) | The detection method of tested substance and the detection kit of tested substance | |
| CN108802360A (en) | Commutative copper and one step of ceruloplasmin are detected with kit, preparation method and application simultaneously in a kind of serum | |
| CN108458999A (en) | The method and its kit of a variety of cardiac biomarkers of joint-detection | |
| EP1673635B1 (en) | Hemoglobin assay for neonatal screening | |
| CN109298178A (en) | Cardiac myosin binding protein C(cMyBP-C based on immunomagnetic beads) time-resolved fluoroimmunoassay kit | |
| Lou et al. | A signal amplifying fluorescent nanoprobe and lateral flow assay for ultrasensitive detection of cardiac biomarker troponin I | |
| JP2010281595A (en) | Method for detecting ligand molecule | |
| Huang et al. | Receptor antibody time‐resolved A2 phospholipase bead immunochromatography and its application in idiopathic membranous nephropathy | |
| CN107271656B (en) | A kind of protein immunoblotting membrane regeneration liquor | |
| CN104569430B (en) | A kind of homogeneous fluorescent immunoreagent group of Quantitative detection H-FABP and preparation method thereof | |
| CN111505303A (en) | Kit for detecting heart-type fatty acid binding protein by chemiluminescence method and use method thereof | |
| CN103323586A (en) | Blood platelet magnetizing and immunolabeling analysis method | |
| CN106124771A (en) | A kind of step homogeneous cTnT detection kit and application thereof | |
| EP2557423B1 (en) | Immunoassay of carboxymethylarginine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Han Di Inventor after: Ye Lei Inventor before: Ye Lei |
|
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180828 |