CN110275023A - The method of joint-detection lung cancer tumor marker based on flow cytometry - Google Patents
The method of joint-detection lung cancer tumor marker based on flow cytometry Download PDFInfo
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Abstract
The method of joint-detection lung cancer tumor marker based on flow cytometry, comprising the following steps: the preparation of probe 1: prepare different types of fluorescence-encoded micro-beads；Marked by streptavidin fluorescence-encoded micro-beads are used respectively；Antibody is captured with biotin labeling；By the capture antibody coupling of the coding microball of marked by streptavidin and biotin labeling；The preparation of probe 2: antibody is detected with fluorochrome label；The reaction system of fluorescence resonance energy transfer system progress multicomponent biological marker detection are as follows:It in above-mentioned reaction system, is added in sample there are when tumor markers, probe 1 is combined with probe 2, forms fluorescence resonance energy transfer system；Working curve is brought into the fluorescence intensity of 2 maximum emission wavelength of probe by the coding microball that will test, the concentration of you can get it variety classes tumor markers.This method is sensitive, quick, high-throughput, price is low.
Description
Technical field
The invention belongs to immunodiagnosis detection fields, are related to the detection method of tumor markers, and in particular to one kind is based on
The method of the joint-detection lung cancer tumor marker of flow cytometry.
Background technique
Tumor markers are chemicals existing for reflection tumour, their presence or quantitative change can prompt the property of tumour
Matter, so as to understanding the tissue generation of tumour, cell differentiation, cell function, with help the diagnosis, classification, Index for diagnosis of tumour with
And treatment guidance.
Since same tumour can contain one or more markers, the higher tumor markers joint of some specificity is selected
Detection is conducive to improve the sensibility and specificity of lesion detection.
Chemiluminescence immune assay (CLIA) and Electrogenerated chemiluminescent immunoassay (ECLIA) are hospital's detection tumor markers
Common method.CLIA has the instrument and reagent of production domesticization, but monitoring type is less, and popularity rate is low, and current vast hospital still adopts
It is detected with the instrument of the imports such as Roche, Beckman producer and reagent.The instrument and reagent of ECLIA is all import, detection essence
Degree is high, easy to operate, therefore large hospital carries out the detection of immune or biochemical indicator using this method, but detects expensive.
Flow cytometry is the new technology just to grow up in recent years, is able to use a sample and carries out height to many indexes
Flux detection, detection speed is fast, and it is the new tool in life science that precision is high.
Summary of the invention
The method of the purpose of the present invention is to provide a kind of joint-detection lung cancer tumor marker based on flow cytometry,
Lung cancer tumor marker CEA, SCC, CYFRA21-1, NSE, the party are quickly homogeneously detected using fluorescence resonance energy transfer system
Method is sensitive, quick, high-throughput, price is low, effectively solution above-mentioned technical problem.
To achieve the above object, the present invention is achieved by the following technical scheme:
The method of joint-detection lung cancer tumor marker based on flow cytometry, specifically includes the following steps:
One, the preparation of probe 1
A, different types of fluorescence-encoded micro-beads, the type of the fluorescence-encoded micro-beads and tumor markers to be detected are prepared
Quantity it is corresponding；
B, respectively with the fluorescence-encoded micro-beads of marked by streptavidin step A preparation；
C, antibody is captured with biotin labeling, the capture antibody is the capture antibody of tumor markers to be detected；
D, different by the capture antibody coupling of the coding microball of step B marked by streptavidin and step C biotin labeling
Tumor markers are combined with different types of fluorescence-encoded micro-beads, identify tumour mark by the fluorescence intensity of fluorescence-encoded micro-beads
Will object；
Two, the preparation of probe 2
Antibody is detected with fluorochrome label, the detection antibody and capture antibody are pairing antibody；
Three, fluorescence resonance energy transfer system carries out the reaction system of multicomponent biological marker detection are as follows:
It, will be by corresponding when being added in sample or standard items there are when tumor markers in above-mentioned reaction system
Fluorescence-encoded micro-beads capture, probe 1 are combined with probe 2, form fluorescence resonance energy transfer system；At this point, passing through volume
The fluorescence intensity of code wavelength determines coding microball, determines the type of tested tumor markers；Existed by the coding microball that will test
The fluorescence intensity of 2 maximum emission wavelength of probe brings working curve into, the concentration of you can get it variety classes tumor markers；
Four, working curve
First by concentration be 0.05-30ng/ml various concentration tumor markers standard items sample respectively with buffer, visit
After needle 1 mixes, it is placed in constant temperature in incubator and is protected from light, add probe 2 and mix well, be placed in constant temperature in incubator and be protected from light,
Add terminate liquid to mix well, which is placed in the fluorescence intensity of 2 maximum emission wavelength of detection probe on flow cytometer, is passed through
The fluorescence intensity for encoding wavelength determines coding microball, determines the type of tested tumor markers；It is reacted on record flow cytometer
System is in the fluorescence intensity of 2 maximum emission wavelength of probe, and there are F when tumor markers, F when tumor markers are not present0, with
(F-F0) standard curve drawn respectively to different tumor markers concentration, fit equation is that taking for concentration and fluorescence intensity is common
Value after logarithm；
Five, sample to be tested detects
The fluorescence signal that sample to be tested is detected by fluorescence resonance energy transfer system, by the fluorescence signal value of sample to be tested
It brings standard curve into, obtains the content of variety classes tumor markers in sample to be tested.
As a preference of the present invention, the method for preparing fluorescence-encoded micro-beads in step A are as follows: take carboxylic polystyrene micro-
Ball cleans carboxylic polystyrene microsphere with swelling solution, and the carboxylic polystyrene microsphere after cleaning is divided into several parts, point
The quantum dot of launch wavelength 565nm is added not into coding microball quantum dot and launch wavelength 605nm is encoded, and hair is added
The quantum dot of the long 645nm of ejected wave provides energy for fluorescent dye fluorescence resonance energy transfer, the coding microball after quantum dot is added
It is incubated at room temperature in swelling system respectively, after the reaction was completed centrifugal treating, obtains quantum point coding microball, the quantum dot that will be obtained
Coding microball cleaning, is dried in vacuo to get the fluorescent microsphere of different quantum-dot codings is arrived centrifugation.
As a preference of the present invention, in step B marked by streptavidin coding microball method are as follows: take step A not same amount
Carboxylic polystyrene fluorescent microsphere after son point coding, is dissolved in morpholino b acid buffer, while it is affine that strepto- is added
Element, carbodiimide hydrochloride and N- hydroxysuccinimide ester, react after mixing, are centrifuged later, and strepto- can be obtained in cleaning
The fluorescence-encoded micro-beads of Avidin modification.
As a preference of the present invention, with the method for biotin labeling capture antibody in step C are as follows: dissolved and lived with anhydrous DMF
The biotin of change is incubated at room temperature after capture antibody is added, and is dialysed, is removed with capture antisera overnight of the PBS solution to biotin labeling
Unreacted biotin is removed, the capture antibody of biotin labeling can be obtained.
As a preference of the present invention, the method that fluorochrome label detects antibody 1 in step 2 are as follows: filled with PBS buffer solution
Divide dissolution fluorescein APC, be bisected into 4 parts, is separately added into CEA, SCC, CYFRA21-1, NSE detection antibody 1, while carbon is added
Diimmonium salt hydrochlorate and N- hydroxysuccinimide ester, incubation at room temperature, carry out elution separation by purification column, obtain APC mark
The detection antibody of note, i.e. probe 2.
As a preference of the present invention, the terminate liquid in the step 3 is the PBS buffer solution containing 0.1%SDS.
As present invention further optimization, coding microball in the step A is four kinds, be denoted as coding microball 1,2,3,
4；The quantum dot of 5 μ L launch wavelength 565nm, the quantum dot of 5 μ L launch wavelength 645nm are added in coding microball 1；Coding microball 2
The middle amount that the quantum dot of 3 μ L launch wavelength 565nm, the quantum dot of 7 μ L launch wavelength 605nm, 5 μ L launch wavelength 645nm is added
Sub- point；The quantum dot of 7 μ L launch wavelength 565nm, the quantum dot of 3 μ L launch wavelength 605nm, 5 μ L hair are added in coding microball 3
The quantum dot of the long 645nm of ejected wave；Quantum dot, the 5 μ L launch wavelength 645nm of 5 μ L launch wavelength 605nm are added in coding microball 4
Quantum dot；Capture antibody in step C is the capture antibody 1 of CEA, SCC, CYFRA21-1, NSE；Coding microball 1,2,3,4
Respectively with the capture antibody coupling of CEA, SCC, CYFRA21-1, NSE, probe 1 is obtained.
As it is of the invention still more preferably, the swelling solution be 5% chloroform and 95% n-butanol mixed liquor.
As it is of the invention still more preferably, the quantum dot be CdSe quantum dot.
It is of the invention a little and the utility model has the advantages that
(1) method provided by the invention is detected based on flow cytometry, easy to operate, quickly, is not needed to anti-
The antibody being not connected in system is answered to be separated；And different coding microballs and antibody coupling, it can be used for based on fluorescence resonance energy
The multicomponent biological analysis of markers system for measuring transfer, has the characteristics that high throughput, can detect more person-portions simultaneously.
(2) present invention has high sensitivity, high accuracy, high specificity, required blood volume compared with traditional detection means
It is few, quickly the advantages that detection.Iconography detection and pathology detection often more difficult discovery early stage in existing lesion detection approach
Case.And since the growth of malignant tumour is extremely complex multi-pathogenesis process, an index, sensitivity and standard are only detected
True property is not high, is easy missing inspection, and method of the invention can realize multicomponent tumor markers joint-detection, can detect 4 simultaneously
Kind lung cancer associated tumors marker, substantially increases the sensitivity and accuracy of detection, compensates for existing lesion detection approach
The deficiency of single Indexs measure.
(3) method non-invasive provided by the invention, detection safety is simple and easy to do, adopts venous blood 2ml, isolates serum
It can be detected, and without any radioactive pollution, there is no any injury to patient.
Detailed description of the invention
Fig. 1 is fluorescence resonance energy transfering system figure of the present invention.
Fig. 2 is the canonical plotting of CEA of the present invention.
Fig. 3 is the canonical plotting of SCC of the present invention.
Fig. 4 is the canonical plotting of CYFRA21-1 of the present invention.
Fig. 5 is the canonical plotting of NSE of the present invention.
Fig. 6 is the flow cytometry analysis figure of quantum point coding microball prepared by the present invention.
Specific embodiment
The present invention is described in further details with reference to the accompanying drawings and detailed description, but it is to the present invention that this, which is not,
Limitation, those skilled in the art's basic thought according to the present invention, various modifications may be made and replacement, without departing from
Basic ideas of the invention, are all within the scope of the present invention.
Technical solution of the present invention is specific as follows:
One, the preparation of probe 1
A, the preparation of coding microball
CdSe quantum dot is embedded with swelling method:
Carboxylic polystyrene microsphere 100mg, swelling solution (5% chloroform, 95% n-butanol) 1mL is taken to clean carboxylated polyphenyl
Ethylene microballoon is divided into four parts twice, by the carboxylic polystyrene microsphere after cleaning, is denoted as coding microball 1,2,3,4, point
The quantum dot of launch wavelength 565nm is added not into coding microball 1,2,3,4 quantum dot and launch wavelength 605nm is compiled
Code, the quantum dot that launch wavelength 645nm is added provide energy for APC fluorescence resonance energy transfer；
Wherein, the quantum dot of 5 μ L launch wavelength 565nm, the quantum of 5 μ L launch wavelength 645nm are added in coding microball 1
Point；
The quantum dot of 3 μ L launch wavelength 565nm, quantum dot, the 5 μ L of 7 μ L launch wavelength 605nm are added in coding microball 2
The quantum dot of launch wavelength 645nm；
The quantum dot of 7 μ L launch wavelength 565nm, quantum dot, the 5 μ L of 3 μ L launch wavelength 605nm are added in coding microball 3
The quantum dot of launch wavelength 645nm；
The quantum dot of 5 μ L launch wavelength 605nm, the quantum dot of 5 μ L launch wavelength 645nm are added in coding microball 4；
Coding microball 1,2,3,4 after quantum dot is added is incubated at room temperature 30min in swelling system respectively；After the reaction was completed
It is centrifuged 1min under the conditions of 3000rpm/min, obtains quantum point coding microball 1,2,3,4；By obtained quantum point coding microball according to
Secondary to use 1mL cleaning solution, 1mL washes of absolute alcohol, cleaning solution is mixed liquor (50% n-butanol, 50% second of n-butanol and ethyl alcohol
Alcohol), 1min is centrifuged after cleaning under the conditions of 3000rpm/min, aforesaid operations are repeated 2 times, and 37 DEG C of vacuum drying for 24 hours, obtain later
To the fluorescent microsphere of four kinds of different quantum-dot codings.
B, marked by streptavidin coding microball
Carboxylic polystyrene fluorescent microsphere 20mg after taking step A difference quantum-dot coding is dissolved in the morpholine second of 1mL
In sulfonate buffer (0.1mol/L, pH=6.0), while 1mg Streptavidin is added, 0.4mg carbodiimide hydrochloride (EDC)
And 0.6mg N- hydroxysuccinimide ester (NHS), 30min is reacted after mixing, and 1 min is centrifuged under the conditions of 3000rpm/min,
Then 3 fluorescence-encoded micro-beads that Streptavidin modification can be obtained are cleaned using deionized water.
C, biotin labeling captures antibody 1
It is specific as follows: to use 1ml with biotin N- hydroxysuccinimide ester (Bio-NHS) the mark capturing antibody 1 of activation
Anhydrous DMF dissolves the biotin N- hydroxysuccinimide ester of 10mg activation, takes 10ul that 800ul (200ug) capture antibody 1 is added
After be incubated at room temperature 1h, with 100mL PBS solution (0.1mol/L, pH=7.0) to the capture antibody of biotin labeling in 4 DEG C of conditions
Under be dialyzed overnight, remove unreacted biotin N- hydroxysuccinimide ester, the capture antibody of biotin labeling can be obtained
2。
D, coding microball and antibody coupling
Due to high affinity effect between biotin and Streptavidin, Streptavidin can be modified fluorescence-encoded
The capture antibody 2 of microballoon and biotin labeling is combined by affinity interaction, and the compound in conjunction with after becomes probe 1, probe 1
Antigen capture can be carried out, to establish marker detection immunoassay method；Coding microball 1,2,3,4 therein respectively with cancer
Embryonal antigen (CEA), squamous cell carcinoma-related antigen (SCC), cytokeratin 19 fragment (CYFRA21-1), neuronal specificity
The capture antibody 1 of four kinds of tumor markers of enolase (NSE) is coupled, be denoted as respectively CEA- probe 1, SCC- probe 1,
CYFRA21-1- probe 1, NSE- probe 1.
Two, the preparation of probe 2
With 4ml PBS buffer solution (0.1mol/L, pH=7.0) sufficiently dissolution 4mg fluorescein APC, 4 parts are bisected into, respectively
CEA, SCC, CYFRA21-1, NSE that 0.2mg is added detect antibody 1, while 0.4mg carbodiimide hydrochloride (EDC) is added
And 0.6mg N- hydroxysuccinimide ester (NHS), it is incubated at room temperature 1h, it is pure by SephacrylS-300 HR molecular exclusion
Change column and carry out elution separation, elution rate 1ml/min obtains the detection antibody 2 of APC label, i.e. probe 2, is denoted as CEA- respectively
Probe 2, SCC- probe 2, CYFRA21-1- probe 2, NSE- probe 2.
Three, prepared by fluorescence resonance energy transfer system
Fluorescence resonance energy transfering system is as shown in Figure 1, wherein 488nm is excitation wavelength, while excitation-emission wavelength is
The wavelength of the quantum dot of 565nm, 605nm and 645nm, 565nm and 605nm are as coding wavelength, at 565nm and 605nm
Different fluorescence intensities can determine coding microball 1,2,3,4；If there are CEA, SCC, CYFRA21-1, NSE waitings in sample
When surveying substance, then the detection antibody that CEA, SCC, CYFRA21-1, NSE will mark coding microball (probe 1) and APC
(probe 2) combines, to be respectively formed probe 1-CEA- probe 2, probe 1-SCC- probe 2, probe 1-CYFRA21-
The fluorescence resonance energy transfer system of 1- probe 2, probe 1-NSE- probe 2；At this point, what the quantum dot as donor was issued
The light of 645nm will be absorbed by APC, and APC issues the fluorescence of 680nm after absorption, detect coding microball 1,2,3,4 respectively at this time
The fluorescence intensity of 680nm can obtain the concentration of CEA, SCC, CYFRA21-1, NSE respectively；
Fluorescence resonance energy transfer system of the present invention carries out the reaction system of multicomponent biological marker detection are as follows:
Four, working curve
First by the standard items sample CEA (0.5-500ng/ml) of various concentration, SCC (0.5-100ng/ml), CYFRA21-1
(0.5-100ng/ml), NSE (0.5-250ng/ml) are mixed with buffer, probe 1 (CEA, SCC, CYFRA21-1, NSE) respectively
It after conjunction, is placed in constant temperature in 37 DEG C of incubators and is protected from light 15min, it is abundant to add probe 2 (CEA, SCC, CYFRA21-1, NSE)
It mixes, is placed in constant temperature in 37 DEG C of incubators and is protected from light 15min, add terminate liquid to mix well, which is placed in flow cytometer
Fluorescence intensity at upper detection 680nm, the specific value of standard items sample are as shown in table 1 below.
Each master sample value (concentration unit ng/mL) of 1 Specification Curve of Increasing of table
| Title | Concentration 1 | Concentration 2 | Concentration 3 | Concentration 4 | Concentration 5 | Concentration 6 | Concentration 7 |
| CEA | 0 | 0.5 | 5.0 | 40 | 100 | 250 | 500 |
| SCC | 0 | 0.5 | 2 | 8 | 20 | 50 | 100 |
| CYFRA21-1 | 0 | 0.5 | 2 | 8 | 20 | 50 | 100 |
| NSE | 0 | 0.5 | 5 | 20 | 50 | 125 | 250 |
Coding (the fluidic cell of different quantum point coding microballs of microsphere is determined by different fluorescence at 565 and 605
Instrument analysis chart is shown in Fig. 6, and part A is coding microball 4 in Fig. 6, and B is code segment microballoon 3, and C portion is coding microball 1, and the part D is
Coding microball 2), to determine the type of tested tumor markers, it is maximum in probe 2 to record reaction system on flow cytometer
The fluorescence intensity of launch wavelength (at 680nm), there are F when CEA1, F when CEA is not present1 0, there are F when SCC2, CEA is not present
When F2 0, there are F when CYFRA21-13, F when CYFRA21-1 is not present3 0, there are F when NSE4, F when NSE is not present4 0, with (F-F0)
Standard curve is drawn to CEA, SCC, CYFRA21-1, NSE concentration respectively, as a result sees Fig. 2 to Fig. 5, fit equation is concentration and glimmering
The value of luminous intensity taken after common logarithm.
Five, sample to be tested detects
By 2 fluorescence resonance energy transfer system of probe 1-CEA, SCC, CYFRA21-1, NSE- probe, detect to test sample
This fluorescence signal brings the fluorescence intensity level of sample to be tested into standard curve as shown in table 2, in you can get it sample to be tested
The content of CEA, SCC, CYFRA21-1, NSE.
The content table of CEA, SCC, CYFRA21-1, NSE in 2 sample to be tested of table
| Title | Sample 1a | Concentrationb(ng/ml) | Sample 2 | Concentration (ng/ml) |
| CEA | 1123 | 7.48 | 5327 | 42.56 |
| SCC | 438 | 1.18 | 3364 | 10.33 |
| CYFRA21-1 | 874 | 4.16 | 5709 | 34.12 |
| NSE | 1753 | 10.86 | 10183 | 100.25 |
A: fluorescence intensity at the 680nm of test sample
B: fluorescence intensity is taken and brings fit equation into respectively after logarithm and calculates, calculated result carries out index again
Concentration value can be obtained in calculating.
With for CAE, fluorescence intensity 1123 is illustrated:
The fit equation of CEA is y=2.267+0.896x
Lg (1123)=3.05, i.e. 3.05=2.267+0.896x acquire x=0.874
So the concentration of CEA is 100.874=7.48 (ng/ml).
Claims (9)
1. the method for the joint-detection lung cancer tumor marker based on flow cytometry, which is characterized in that specifically include following step
It is rapid:
One, the preparation of probe 1
A, different types of fluorescence-encoded micro-beads, the number of the type of the fluorescence-encoded micro-beads and tumor markers to be detected are prepared
It measures corresponding；
B, respectively with the fluorescence-encoded micro-beads of marked by streptavidin step A preparation；
C, antibody is captured with biotin labeling, the capture antibody is the capture antibody of tumor markers to be detected；
D, by the capture antibody coupling of the coding microball of step B marked by streptavidin and step C biotin labeling, different tumours
Marker is combined with different types of fluorescence-encoded micro-beads, identifies tumor-marker by the fluorescence intensity of fluorescence-encoded micro-beads
Object；
Two, the preparation of probe 2
Antibody is detected with fluorochrome label, the detection antibody and capture antibody are pairing antibody；
Three, fluorescence resonance energy transfer system carries out the reaction system of multicomponent biological marker detection are as follows:
It, will be by corresponding fluorescence when being added in sample or standard items there are when tumor markers in above-mentioned reaction system
Coding microball capture, probe 1 are combined with probe 2, form fluorescence resonance energy transfer system；At this point, passing through coding wave
Long fluorescence intensity determines coding microball, determines the type of tested tumor markers；By the coding microball that will test in probe 2
The fluorescence intensity of maximum emission wavelength brings working curve into, the concentration of you can get it variety classes tumor markers；
Four, working curve
First by concentration be 0.05-30ng/ml various concentration tumor markers standard items sample respectively with buffer, probe 1
It after mixing, is placed in constant temperature in incubator and is protected from light, add probe 2 and mix well, be placed in constant temperature in incubator and be protected from light, add end
Only liquid mixes well, which is placed in the fluorescence intensity of 2 maximum emission wavelength of detection probe on flow cytometer, passes through coding
The fluorescence intensity of wavelength determines coding microball, determines the type of tested tumor markers；Record reaction system on flow cytometer
In the fluorescence intensity of 2 maximum emission wavelength of probe, there are F when tumor markers, F when tumor markers are not present0, with (F-F0)
Standard curve drawn respectively to different tumor markers concentration, fit equation be concentration and fluorescence intensity take common logarithm after
Value；
Five, sample to be tested detects
The fluorescence signal that sample to be tested is detected by fluorescence resonance energy transfer system, the fluorescence signal value of sample to be tested is brought into
Standard curve obtains the content of variety classes tumor markers in sample to be tested.
2. the method for the joint-detection lung cancer tumor marker described in claim 1 based on flow cytometry, which is characterized in that
The method of fluorescence-encoded micro-beads is prepared in step A are as follows: take carboxylic polystyrene microsphere, clean carboxylated polyphenyl second with swelling solution
Carboxylic polystyrene microsphere after cleaning is divided into several parts, launch wavelength is added into coding microball respectively by alkene microballoon
The quantum dot of 565nm and the quantum dot of launch wavelength 605nm are encoded, and the quantum dot that launch wavelength 645nm is added is fluorescence
Dye fluorescence Resonance energy transfer provides energy, and the coding microball after quantum dot is added is incubated at room temperature in swelling system respectively,
Centrifugal treating after the reaction was completed obtains quantum point coding microball, and the cleaning of obtained quantum point coding microball, centrifugation, vacuum are done
It is dry to get arrive different quantum-dot codings fluorescent microsphere.
3. the method for the joint-detection lung cancer tumor marker described in claim 1 based on flow cytometry, which is characterized in that
The method of marked by streptavidin coding microball in step B are as follows: the carboxylic polystyrene after taking step A difference quantum-dot coding
Fluorescent microsphere is dissolved in morpholino b acid buffer, while Streptavidin is added, carbodiimide hydrochloride and N- hydroxyl
Succimide ester is reacted after mixing, is centrifuged later, and the fluorescence-encoded micro-beads of Streptavidin modification can be obtained in cleaning.
4. the method for the joint-detection lung cancer tumor marker described in claim 1 based on flow cytometry, which is characterized in that
With the method for biotin labeling capture antibody in step C are as follows: with the biotin of anhydrous DMF dissolution activation, be added after capturing antibody
Incubation at room temperature is dialysed with capture antisera overnight of the PBS solution to biotin labeling, removes unreacted biotin, can be obtained
The capture antibody of biotin labeling.
5. the method for the joint-detection lung cancer tumor marker described in claim 1 based on flow cytometry, which is characterized in that
The method of fluorochrome label detection antibody 1 in step 2 are as follows: fluorescein APC is sufficiently dissolved with PBS buffer solution, is bisected into 4 parts,
It is separately added into CEA, SCC, CYFRA21-1, NSE detection antibody 1, while carbodiimide hydrochloride and N- hydroxyl fourth two is added
Imide ester, incubation at room temperature, carries out elution separation by purification column, obtains the detection antibody of APC label, i.e. probe 2.
6. the method for the joint-detection lung cancer tumor marker described in claim 1 based on flow cytometry, which is characterized in that
Terminate liquid in the step 3 is the PBS buffer solution containing 0.1%SDS.
7. the method for the joint-detection lung cancer tumor marker as claimed in claim 2 based on flow cytometry, which is characterized in that
Coding microball in the step A is four kinds, is denoted as coding microball 1,2,3,4；5 μ L launch wavelengths are added in coding microball 1
The quantum dot of the quantum dot of 565nm, 5 μ L launch wavelength 645nm；The quantum of 3 μ L launch wavelength 565nm is added in coding microball 2
Point, the quantum dot of 7 μ L launch wavelength 605nm, 5 μ L launch wavelength 645nm quantum dot；7 μ L transmitted waves are added in coding microball 3
The quantum dot of long 565nm, the quantum dot of 3 μ L launch wavelength 605nm, 5 μ L launch wavelength 645nm quantum dot；In coding microball 4
The quantum dot of 5 μ L launch wavelength 605nm, the quantum dot of 5 μ L launch wavelength 645nm is added；Capture antibody in step C be CEA,
The capture antibody 1 of SCC, CYFRA21-1, NSE；The capture with CEA, SCC, CYFRA21-1, NSE respectively of coding microball 1,2,3,4
Antibody coupling obtains probe 1.
8. the method for the joint-detection lung cancer tumor marker as claimed in claim 2 based on flow cytometry, which is characterized in that
Swelling solution is the mixed liquor of 5% chloroform and 95% n-butanol.
9. the method for the joint-detection lung cancer tumor marker as claimed in claim 2 based on flow cytometry, which is characterized in that
The quantum dot is CdSe quantum dot.
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