CN105911284A - Cup-type time-resolved fluorescent analysis kit for creatine jubase-MB based on microspheres, and preparation method and application thereof - Google Patents
Cup-type time-resolved fluorescent analysis kit for creatine jubase-MB based on microspheres, and preparation method and application thereof Download PDFInfo
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Abstract
The invention especially relates to a cup-type time-resolved fluorescent analysis kit for creatine jubase-MB based on microspheres, and a preparation method and application thereof, belonging to the field of clinical medical diagnosis. The kit is composed of a detection reaction cup, a fluorescence labeled antibody and a cleaning solution. In concrete detection, a standard curve is drafted at first, then a to-be-detected sample is added into the detection reaction cup, then the fluorescence labeled antibody is added, incubation at 30 to 40 DEG C is carried out, and unbonded antigen and fluorescence labeled antibody are removed through cleaning via the cleaning solution; and the fluorescence signals are compared with the standard curve so as to obtain the concentration of creatine jubase-MB in the to-be-detected sample. The detection method for the creatine jubase-MB is easy to realize automatic operation due to its superhigh sensitivity, uniform in reaction temperature, free of influence by environmental factors, better in accuracy and excellent in precision.
Description
Technical field
The invention belongs to clinical diagnose field, particularly to a kind of cup type time-resolved fluorescence based on microsphere
Creatine kinase isozyme assay kit and its preparation method and application.
Background technology
Cardiovascular disease is harm human health and the serious disease of life, it has also become 21 century human health
Number one killer.Bayer medicine reported in ESC's annual meeting and pointed out the whole world in 2005 in August, 2009
There are about 17,500,000 people and die from cardiovascular disease, it is contemplated that these digital 20,000,000 people of riseing in 2015, greatly
There are about half case to occur in the Asian-Pacific area.The improvement of living condition and the transformation of life style, make Chinese Hair
The risk of raw cardiovascular disease catches up with and surpasses rapidly the developed countries such as the U.S..
China's nineteen ninety urbanite cardiovascular disease mortality rate is 92/,100,000 people, within 2000, is 106/10
Ten thousand people, dead rate of increase is 1.3% every year on average.2008 annual death rate have reached 1,21/,100,000 people, compare
When 2000, Mean Death rate of increase is 1.4%, and wherein the mortality rate of acute myocardial infarction accounts for therein three
/ mono-, illustrate that myocardial infarction mortality rate is still in continuous growth trend.Along with Chinese population aging trend, press
Mean Death rate of increase is 1.5% calculating, and by 2015, angiopathy mortality rate was up to 1,41/,100,000 people,
The mortality rate of acute myocardial infarction is up to 47/,100,000 people.
The mortality rate of the acute myocardial infarction that ministry of Health of China is announced for 2008 is 39.72/10 ten thousand people, anxious every year
The number of the infected of myocardial inyaretion is more than 5,000,000 people, and at least the crowd more than 2,000,000 goes to hospital to seek medical advice to carry out
The coherent detection of myocardial infarction disease, this does not comprise every year because chest pain suspects the patient's number for myocardial infarction,
Actual hospital detects number every year and has reached ten million person-time.
The typical clinical symptom of myocardial infarction is chest pain, arrhythmia, heart failure etc..But breast in actual clinical
The Symptoms complexity of pain is various, and dangerous difference is the biggest, and also heart infarction patient is without classical symptom.Medically
Using within after acute myocardial infarction 6 hours as " prime time " rescued, exceed this time period,
Therapeutic effect will be had a greatly reduced quality, and even produces serious consequence threat to life.Chinese medical checks branch 2006
" cardiac marker Clinical detection application proposal when coronary artery disease and heart failure " that year formulates is pointed out, suitable
Should have preferably diagnosis, risk stratification and prognosis estimated value for clinical cardiac marker, analyze inspection
Survey method should be special, sensitive, quick, and the detection turnaround time ＜ 60min of cardiac marker.
Creatine kinase isozyme (CK-MB) is a kind of middle and advanced stage Applications of Cardiac Markers, has the heaviest in clinical diagnosis
The meaning wanted, when various pathological changes include amyotrophy and myocardial infarction occurs, creatine kinase water in the serum of people
Flat improve rapidly, it is believed that in the diagnosis of myocardial infarction, measure the specific activity of creatine kinase have an electro-cardiogram and more may be used
Lean on.During myocardial infarction, creatine kinase raises in onset 6 hours, within 24 hours, reaches peak, recovers in 3-4 day
Normally.Creatine kinase has important physiological function because of it and clinical value has caused people to pay attention to widely
And in-depth study.
The multiplex chemoluminescence method of CK-MB and colloidal gold immunity chromatography or fluorescence immune chromatography method etc. traditionally
Method measures.Chemoluminescence method requires height to technology, and operating procedure is the most loaded down with trivial details, needs multistep reagent to add
Add process.Although it is few that colloidal gold immunity chromatography has specimen consumption, easy to be quick, cheap advantage, but
When running into that in some sample, antigen or antibody content are extremely low, the color of gold colloidal will be the most shallow even without developing the color, very
, easily there is erroneous judgement in difficulty with the naked eye judged result, and sensitivity is relatively low.Although fluorescence immune chromatography method sensitivity
Higher than colloidal gold immunity chromatography, but its detection precision is unsatisfactory, and sensitivity is luminous with giant chemical
Or electrochemiluminescence instrument still has gap.
Time-resolved fluorescence (Time-resolved Fluorescence, TRF) is a kind of heterotope fluorescence mark
The features such as note thing, compared with common fluorescent, has stock displacement big, fluorescence lifetime length, can effectively keep away
Exempt from the background fluorescence in sample, and the impact of the veiling glare such as exciting light, therefore compare common fluorescent and have higher
Sensitivity and capacity of resisting disturbance.
The reagent using time-resolved fluorescence detection the most on the market all uses the enhancing lanthanide series fluorescence that dissociates to exempt from
Epidemic disease analyzes (DELFIA), and it uses the chelate with bifunctional group structure so that it is one section and europium (Eu)
Connecting, the other end is connected with the free amino group on antibody/antigen molecule, forms the antibody/antigen of EU labelling,
After immunoreation, form immune complex, owing to this complex fluorescence intensity in water is the most weak, need
One to be added is dissociated reinforcing agent so that europium ion disintegrates down from complex, and with another in reinforcing agent
Planting chelating agen and form micell, this micel can send the strongest fluorescence under the exciting of ultraviolet light.
Summary of the invention
It is an object of the invention to provide a kind of cup type time-resolved fluorescence creatine kinase isozyme based on microsphere to divide
Analysis test kit, this test kit can complete the detection of creatine kinase isozyme within the shorter detection time, during detection
Between short, detection sensitivity is high.
Present invention also offers cup type time-resolved fluorescence creatine kinase isozyme assay kit based on microsphere
Preparation method.
Present invention also offers the detection method of cup type time-resolved fluorescence creatine kinase isozyme based on microsphere.
In order to realize above technique effect, the present invention is to be achieved by the steps of:
A kind of cup type time-resolved fluorescence creatine kinase isozyme assay kit based on microsphere, its feature exists
In: this test kit is formed by detecting reaction cup, fluorescent-labeled antibody and cleanout fluid；The detection basis of this test kit
It it is the immunoreation of double-antibody sandwich.
The solid phase surface of described detection reaction cup is coated with creatine kinase isozyme monoclonal antibody or Anti-TNF-α
Body；
Described fluorescent-labeled antibody is time-resolved fluorescence microsphere and creatine kinase isozyme monoclonal antibody or many
Clonal antibody is by covalently cross-linked.
Being filled with lanthanide chelate in described time-resolved fluorescence microsphere, its particle diameter is 100-1000nm.
Preferably, described lanthanide chelate is Europium chelate.It is further preferred that this Europium chelate is
Eu (TTA) 3/TOPO or Eu (TTA) 3/Phen.
The preparation method of above-mentioned cup type time-resolved fluorescence creatine kinase isozyme assay kit based on microsphere,
Its step includes,
(1) preparation of reaction cup, is detected: creatine kinase isozyme monoclonal antibody or polyclonal antibody are diluted
Latter 37 DEG C are coated, and close with Block buffer, pat dry；It is placed in the drying baker of 37 DEG C and dries, seal guarantor
Deposit；
Preferably, the phosphoric acid of creatine kinase isozyme monoclonal antibody or polyclonal antibody 0.2mol/L is delayed
Rushing liquid and be diluted to 40 μ g/mL, 100 μ L/ holes, 37 DEG C are coated 4 hours, and with Block buffer by 300 μ L/
Hole adds reaction cup, closes 4 hours, discards the confining liquid being coated in reaction cup, pat dry, be placed in 37 DEG C for 37 DEG C
Drying baker in dry, seal preservation；
(2), the preparation of fluorescent-labeled antibody: time-resolved fluorescence microsphere is activated；It is subsequently adding creatine kinase
Isozyme monoclonal antibody or polyclonal antibody mix, close, clean, and dilute with reaction buffer, it is thus achieved that
Final concentration is to the fluorescent-labeled antibody of 20-50 μ g/mL；
(3) preparation of cleanout fluid: containing PB, NaCl, Tween 20 in this cleanout fluid, this cleanout fluid
PH value is 7-9.Preferably, cleanout fluid contains PB, 0.9%NaCl, the 0.05%Tween 20 of 5mmol/L,
The pH value of this cleanout fluid is 7.2.
In described step (2), the step of time-resolved fluorescence microsphere activation is, at carboxyl time-resolved fluorescence
Microsphere adds MES and EDC and carries out primary activation process, add aminocaproic acid room temperature mixing 15-60min,
Add MES, NHS and EDC and carry out activation processing again；Every 1mg carboxyl time-resolved fluorescence microsphere is corresponding
10-100 μ L 50mmol/L aminocaproic acid.
Preferably, described primary activation processes and includes: every 1mg carboxyl time-resolved fluorescence microsphere, adds 60 μ L
The MES of 500mmol/L pH5.0-7.0,0.02-0.2mg 1-(3-dimethylamino-propyl)-3-ethyl carbon two is sub-
Amine hydrochlorate, adds purified water to final volume 600 μ L, room temperature mixing 15～60min；
Described activation processing again includes: after adding the mixing of described aminocaproic acid room temperature, add 1mL 100mmol/L
MES pH6.0, eccentric cleaning 15000rpm 20min, supernatant discarded；Add the 100mmol/L of 400 μ L
MES pH6.0, ultrasonic Separation, add 0.02-0.2mg N-hydroxy-succinamide, add 0.01-0.1mgEDC,
Room temperature mixing 15min；Add the MES pH5.0-7.0 buffer of 1mL 100mmol/L, eccentric cleaning 15000rpm
20min, supernatant discarded；100mmol/L MES pH5.0-7.0 buffer returns to 1mL.Fluorescent particle is lived
Aminocaproic acid is added as arm so that the distance between antibody and microsphere increases, and effectively reduces during change
Space steric effect, improves detecting system sensitivity.
The detection method of above-mentioned cup type time-resolved fluorescence creatine kinase isozyme based on microsphere, its step bag
Include,
(A), the drafting of standard curve: use cup type time-resolved fluorescence creatine kinase isozyme based on microsphere
Assay kit measures the calibration object of variable concentrations, and according to the fluorescence signal value on Fluorescent reader, with calibration
Product concentration is abscissa, with fluorescence signal value as vertical coordinate, is depicted as standard curve；
(B), by testing sample add in detection reaction cup, add fluorescent-labeled antibody, 30 DEG C of-40 DEG C of temperature
Educate, clean with cleanout fluid and remove unconjugated antigen and fluorescent-labeled antibody；
(C) under 340-380nm excitation, fluorescence signal in test detection reaction cup, detects wavelength
For 600-630nm；
(D) by described fluorescence signal and the standard curve comparison in step (A), it is thus achieved that described testing sample
Creatine kinase isozyme concentration.
In described step (A), concentration is is 0,0.5,2,5,10,25,50, the calibration of 100ng/mL
Product add fluorescent-labeled antibody, 37 DEG C of incubation 5-25min, then cleans with cleanout fluid and remove unconjugated resisting
Former and fluorescent-labeled antibody, described calibration object and fluorescent-labeled antibody volume ratio is 1:4-6.
In described step (B), the volume ratio that testing sample resists with fluorescent labeling is 1:4-6, and heated culture temperature is
37℃。
The invention has the beneficial effects as follows:
1) fluorescent marker prepared by the present invention is time-resolved fluorescence latex beads, is filled with in each microsphere
Several ten thousand arrive hundreds of thousands rare earth element ion chelate, be not required to enhancing process of dissociating, just may be used during detection
Sending the strongest fluorescence, simplify time-resolved fluorescence detecting step, the Enhanced time resolved fluorometric that dissociates detection is past
Just can obtain testing result toward about 1 hour time of needs, and use time-resolved fluorescence microsphere as labelling
Thing, owing to being filled with substantial amounts of lanthanide chelate in each microsphere, fluorescence signal amplifies more than thousands of times,
Therefore can complete detection within the detection time of 5-20 minute, shorten the detection time, and effectively improve
Detection sensitivity.
2) fluorescent particle activation process is added aminocaproic acid as arm so that the distance between antibody and microsphere
Increase, effectively reduce space steric effect, improve detecting system sensitivity.
3) detection method of based on microsphere the cup type time-resolved fluorescence creatine kinase isozyme that the present invention provides,
Sensitivity due to its superelevation, it is easy to accomplish automation mechanized operation, and reaction temperature is homogeneous, not by environmental factors
Impact, accuracy is more preferable, and precision is the most excellent.
Accompanying drawing explanation
Fig. 1 is the principle schematic of the present invention.
Fig. 2 be the present invention according to the fluorescence signal value on Fluorescent reader, with calibration object concentration as abscissa, with
Fluorescence signal value is vertical coordinate, the standard curve being depicted as.
Fig. 3 is to use the reagent set synthesis creatine kinase isozyme time-resolved fluorescence prepared in embodiment 1 quantitative
Test kit, measures clinical sample, with the comparing result figure of chemiluminescence clinical sample test result.
In Fig. 1: 1 is fluorescent-labeled antibody, 2 is creatine kinase isozyme antigen, and 3 is coated antibody, 4
For reaction cup solid phase surface, 5 is light immune complex.
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described:
Equipment used in experiment: the multi-functional microplate reader of Victor X4 of PerkinElmer company.
The principle schematic of the present invention as it is shown in figure 1, coated antibody 3 is combined on reaction cup solid phase surface 4,
Adding a certain amount of sample in reaction cup makes the creatine kinase isozyme antigen 2 in sample solid with reaction cup
The antibody 3 that phase surface combines combines, and adds fluorescent-labeled antibody 1, and incubation forms electrochemiluminescent immunoassay complex 5,
Use cleanout fluid to clean after reaction cup and remove unconjugated antigen and fluorescent-labeled antibody, glimmering in detection reaction cup
Optical signal, contrasts with standard curve, obtains the concentration of creatine kinase isozyme in sample to be tested.
Embodiment 1
(1), the preparation of detection reaction cup:
A, detection reaction cup are coated: creatine kinase isozyme antibody 7501 (Medix company) uses 0.2mol/L
Phosphate buffer (pH7.8) be diluted to 40 μ g/ml, 100 μ L/ holes, 37 DEG C are coated 4 hours, wash plate.
B, detection reaction cup are closed: adding reaction cup with Block buffer by 200 μ L/ holes, 37 DEG C of closings 4 are little
Time, discard the confining liquid being coated in reaction cup, pat dry.
C, detection reaction cup are dried: the reaction cup that above-mentioned closing is good is positioned over 37 DEG C less than 30% of humidity and does
Drying 8 hours in dry case, hermetically drying preserves.
(2), the preparation of fluorescent-labeled antibody:
A, fluorescent particle activate:
Take 1mg carboxyl time-resolved fluorescence microsphere (200nm, 0.1ml, 10mg/mL, Bangslab company),
Add MES (2-(N-morpholine) ethyl sulfonic acid) buffer of 60 μ L 500mmol/L, pH6.0, add 0.2mg 1-(3-
Dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC), add purified water to final volume 600 μ L, room temperature
Mixing 15min.Add 100 μ L 50mmol/L aminocaproic acids, room temperature mixing 30min.Add 1mL 100mmol/L
MES pH6.0, eccentric cleaning 15000rpm 20min, supernatant discarded.Add 400 μ L 100mmol/L MES pH6.0,
Ultrasonic Separation, adds 0.2mg N-hydroxy-succinamide (NHS), adds 0.1mgEDC, room temperature mixing 15min.
Add 1mL 100mmol/L MES pH6.0 buffer, eccentric cleaning 15000rpm 20min, supernatant discarded.
100mmol/L MES pH6.0 buffer returns to 1ml.
It is B, antibody linked: to add the creatine kinase isozyme monoclonal antibody 7502 (Medix company) of 0.2mg,
Room temperature 25 DEG C mixing 60min.
C, closing: add BSA confining liquid, to final concentration 10mg/mL BSA, 25 DEG C of mixing overnight of room temperature.
D, cleaning: add 3mL cross-linking buffer, 25000rpm eccentric cleaning 30min, supernatant discarded.Add 500 μ L
Buffer, ultrasonic Separation, final concentration 2mg/mL.
E, work fluorescent-labeled antibody are prepared:
Preparation reaction buffer: containing 50mmol/L tris, 1%BSA, 0.9%NaCl, 1% Portugal in buffer
Polysaccharide, 0.5%tween 20, pH7.2.
Above-mentioned cleaned fluorescent-labeled antibody is diluted to final concentration to 30 μ g/mL with reaction buffer, as
Detection fluorescent-labeled antibody.
(3), the preparation of cleanout fluid
Preparation cleaning buffer solution, containing 5mmol/L PB, 0.9%NaCl, 0.05%Tween 20, pH7.2.
Embodiment 2
(1) drafting of standard curve:
Use the reagent set synthesis creatine kinase isozyme time-resolved fluorescence quantitative reagent of preparation in embodiment 1
Box, measures calibration object, and each concentration is repeated 10 times.
Each detection adds calibration object 10 μ L, fluorescent-labeled antibody 50 μ L, 37 DEG C of incubation 20min, then
Clean detection cup, the Victor X4 Fluorescent reader of PerkinElmer company carries out reading (excitation wave
Long 340-380nm, detects wavelength 600-630nm) concrete data are as shown in table 1.
Table 1
According to the data in table 1, with calibration object concentration as abscissa, with fluorescence signal average as vertical coordinate, paint
Make standard curve.Standard curve is as shown in Figure 2.This standard curve is linearly good, can be bent by this standard
Line carries out quantitative analysis to creatine kinase isozyme concentration contained in sample.
By table 1 result, the withinrun precision of detection kit is good, calibration object each concentration point precision
It is respectively less than 10%, and reagent sensitivity is good (except 0 value calibration product), in 0.5ng/mL level and 0 value school
Quasi-product have notable differentiation.
(2), pattern detection
Use the reagent set synthesis creatine kinase isozyme time-resolved fluorescence quantitative reagent of preparation in embodiment 1
Box, measures clinical sample, carries out correlation analysis with Beckman access2 system test result.
Each detection adds sample 10 μ L, fluorescent-labeled antibody 50 μ L, 37 DEG C of incubation 20min, then cleans inspection
Survey cup, the Victor X4 Fluorescent reader of PerkinElmer company carries out reading (excitation wavelength
340-380nm, detects wavelength 600-630nm), test result is brought into Fig. 2 standard curve, calculates sample and survey
Examination concentration, test result is as shown in table 2, carries out dependency with Beckman access2 system test result
Analyzing, correlation curve is as shown in Figure 3.
Table 2 clinical sample test result
As can be known from Fig. 3, the kit measurement result of the present invention measures with Beckman instrument and matched reagent box
Result coefficient R2Being 0.960, dependency is fine.Beckman access2 system and supporting creatine swash
Enzyme isoenzyme test kit is well-known chemiluminescence detection kit, but it is expensive, general the most large-scale by three
Just there is configuration in the central laboratory of first hospital, and its precision and result are generally acknowledged reliable.But reagent prepared by the present invention
No matter box considers from price or from accuracy in detection, can be used for clinical diagnosis and uses.
Embodiment 3
(1), the preparation of detection reaction cup:
A, detection reaction cup are coated: creatine kinase isozyme antibody 7501 (Medix company) uses 0.2mol/L
Phosphate buffer (pH7.8) be diluted to 40 μ g/ml, 100 μ L/ holes, 37 DEG C are coated 4 hours, wash plate.
B, detection reaction cup are closed: adding reaction cup with Block buffer by 200 μ L/ holes, 37 DEG C of closings 4 are little
Time, discard the confining liquid being coated in reaction cup, pat dry.
C, detection reaction cup are dried: the reaction cup that above-mentioned closing is good is positioned over 37 DEG C less than 30% of humidity and does
Drying 8 hours in dry case, hermetically drying preserves.
(2), the preparation of fluorescent-labeled antibody:
A, fluorescent particle activate:
Take 1mg carboxyl time-resolved fluorescence microsphere (200nm, 0.1ml, 10mg/mL, Bangslab company),
Add 60ul 500mM MES (2-(N-morpholine) ethyl sulfonic acid) buffer, pH6.0, add 0.2mg 1-(3-bis-
Methylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC), add 0.4mg N-hydroxy-succinamide (NHS,
40 μ L, 10mg/mL), add purified water and mix 15min to final volume 600 μ L, room temperature.Add 1mL 100mmol/L
MES pH6.0, eccentric cleaning 15000rpm 20min, supernatant discarded.
It is B, antibody linked: to add the creatine kinase isozyme monoclonal antibody 7502 (Medix company) of 0.2mg,
Room temperature 25 DEG C mixing 60min.
C, closing: add BSA confining liquid, to final concentration 10mg/mL BSA, 25 DEG C of mixing overnight of room temperature.
D, cleaning: add 3mL cross-linking buffer, 25000rpm eccentric cleaning 30min, supernatant discarded.Add 500 μ L
Buffer, ultrasonic Separation, final concentration 2mg/mL.
E, work fluorescent-labeled antibody are prepared:
Preparation reaction buffer: containing 50mmol/L tris, 1%BSA, 0.9%NaCl, 1% Portugal in buffer
Polysaccharide, 0.5%tween 20, pH7.2.
Above-mentioned cleaned fluorescent-labeled antibody is diluted to final concentration to 30 μ g/mL with reaction buffer, as
Detection fluorescent-labeled antibody.
(3), the preparation of cleanout fluid
Preparation cleaning buffer solution, containing 5mmol/L PB, 0.9%NaCl, 0.05%Tween 20, pH7.2.
(4), calibration object test
Use reagent set synthesis creatine kinase isozyme time-resolved fluorescence quantification kit prepared by said method,
Measuring calibration object, each concentration is repeated 10 times.
Each detection adds calibration object 10 μ L, fluorescent-labeled antibody 50 μ L, 37 DEG C of incubation 20min, then
Clean detection cup, the Victor X4 Fluorescent reader of PerkinElmer company carries out reading.
By table 3 result, the withinrun precision of detection kit is good, and calibration object each concentration point fluorescence is believed
Number precision is respectively less than 10% (in addition to 0 and 0.5ng/ml level), for reagent sensitivity compares embodiment 1
Decrease, only have with 0 value calibration product in 2ng/mL level and significantly distinguish.Locate before fluorescent particle is described
The effective sensitivity promoting detection can be had by the way of adding arm during reason.
Table 3 creatine kinase isozyme quantitative measurement standard curve data
Claims (10)
1. a cup type time-resolved fluorescence creatine kinase isozyme assay kit based on microsphere, its feature
It is: this test kit is formed by detecting reaction cup, fluorescent-labeled antibody and cleanout fluid；
The solid phase surface of described detection reaction cup is coated with creatine kinase isozyme monoclonal antibody or Anti-TNF-α
Body；
Described fluorescent-labeled antibody is time-resolved fluorescence microsphere and creatine kinase isozyme monoclonal antibody or many
Clonal antibody is by covalently cross-linked.
Cup type time-resolved fluorescence creatine kinase isozyme based on microsphere the most according to claim 1 divides
Analysis test kit, it is characterised in that: described time-resolved fluorescence microsphere is filled with lanthanide chelate, its grain
Footpath is 100-1000nm.
Cup type time-resolved fluorescence creatine kinase isozyme based on microsphere the most according to claim 2 divides
Analysis test kit, it is characterised in that: described lanthanide chelate is Europium chelate.
Cup type time-resolved fluorescence creatine kinase isozyme based on microsphere the most according to claim 1 divides
The preparation method of analysis test kit, its step includes,
(1) preparation of reaction cup, is detected: creatine kinase isozyme monoclonal antibody or polyclonal antibody are diluted
Latter 37 DEG C are coated, and close with Block buffer, pat dry；It is placed in the drying baker of 37 DEG C and dries, seal guarantor
Deposit；
(2), the preparation of fluorescent-labeled antibody: time-resolved fluorescence microsphere is activated；It is subsequently adding creatine kinase
Isozyme monoclonal antibody or polyclonal antibody mix, close, clean, and dilute with reaction buffer, it is thus achieved that
Final concentration is to the fluorescent-labeled antibody of 20-50 μ g/mL；
(3) preparation of cleanout fluid: containing PB, NaCl, Tween 20 in this cleanout fluid, this cleanout fluid
PH value is 7-9.
Cup type time-resolved fluorescence creatine kinase isozyme based on microsphere the most according to claim 4 divides
The preparation method of analysis test kit, it is characterised in that: in described step (2), time-resolved fluorescence microsphere activates
Step be, in carboxyl time-resolved fluorescence microsphere add MES and EDC carry out primary activation process, add
Aminocaproic acid room temperature mixing 15-60min, adds MES, NHS and EDC and carries out activation processing again；Every 1mg
Carboxyl time-resolved fluorescence microsphere correspondence 10-100 μ L 50mmol/L aminocaproic acid.
Cup type time-resolved fluorescence creatine kinase isozyme based on microsphere the most according to claim 5 divides
The preparation method of analysis test kit, it is characterised in that: described primary activation processes and includes: every 1mg carboxyl time divides
Distinguish fluorescent microsphere, add MES, 0.02-0.2mg 1-(the 3-diformazan ammonia of 60 μ L 500mmol/L pH5.0-7.0
Base propyl group)-3-ethyl-carbodiimide hydrochloride, add purified water and mix 15-60min to final volume 600 μ L, room temperature；
Described activation processing again includes: after adding the mixing of described aminocaproic acid room temperature, add 1mL 100mmol/L
MES pH6.0, eccentric cleaning 15000rpm 20min, supernatant discarded；Add the 100mmol/L of 400 μ L
MES pH6.0, ultrasonic Separation, add 0.02-0.2mg N-hydroxy-succinamide, add 0.01-0.1mgEDC,
Room temperature mixing 15min；Add the MES pH5.0-7.0 buffer of 1mL 100mmol/L, eccentric cleaning 15000rpm
20min, supernatant discarded；100mmol/L MES pH5.0-7.0 buffer returns to 1mL.
Cup type time-resolved fluorescence creatine kinase isozyme based on microsphere the most according to claim 4 divides
The preparation method of analysis test kit, it is characterised in that: containing 5mmol/L in the cleanout fluid in described step (3)
PB, 0.9%NaCl, 0.05%Tween 20, the pH value of this cleanout fluid is 7.2.
8. the detection of based on microsphere the cup type time-resolved fluorescence creatine kinase isozyme described in claim 1
Method, its step includes,
(A), the drafting of standard curve: use cup type time-resolved fluorescence creatine kinase isozyme based on microsphere
Assay kit measures the calibration object of variable concentrations, and according to the fluorescence signal value on Fluorescent reader, with calibration
Product concentration is abscissa, with fluorescence signal value as vertical coordinate, is depicted as standard curve；
(B), by testing sample add in detection reaction cup, add fluorescent-labeled antibody, 30 DEG C of-40 DEG C of temperature
Educate, clean with cleanout fluid and remove unconjugated antigen and fluorescent-labeled antibody；
(C) under 340-380nm excitation, fluorescence signal in test detection reaction cup, detects wavelength
For 600-630nm；
(D) by described fluorescence signal and the standard curve comparison in step (A), it is thus achieved that described testing sample
The concentration of creatine kinase isozyme.
9. the detection of based on microsphere the cup type time-resolved fluorescence creatine kinase isozyme described in claim 8
Method, it is characterised in that: in described step (A), concentration is 0,0.5,2,5,10,25,50,100ng/mL
Calibration object in add fluorescent-labeled antibody, 37 DEG C of incubation 5-25min, then with cleanout fluid clean remove do not tie
The antigen closed and fluorescent-labeled antibody, described calibration object is 1:4-6 with the volume ratio of fluorescent-labeled antibody.
10. the detection of based on microsphere the cup type time-resolved fluorescence creatine kinase isozyme described in claim 8
Method, it is characterised in that: in described step (B), the volume ratio that testing sample resists with fluorescent labeling is 1:4-6,
Heated culture temperature is 37 DEG C.
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