CN107677823A - A kind of protein chip kit and its detection method for being used to detect tumour antigen - Google Patents
A kind of protein chip kit and its detection method for being used to detect tumour antigen Download PDFInfo
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- CN107677823A CN107677823A CN201610635953.4A CN201610635953A CN107677823A CN 107677823 A CN107677823 A CN 107677823A CN 201610635953 A CN201610635953 A CN 201610635953A CN 107677823 A CN107677823 A CN 107677823A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The present invention relates to the horizontal detection field of tumour antigen, more particularly to a kind of protein chip kit and its detection method for being used to detect tumour antigen.A kind of protein chip kit for being used to detect tumour antigen, including the material of antibody one and the display antibody one, and/or the material of antibody two and the display antibody two；The antigen of antibody one is TKTL1, and the antigen of antibody two is DNASE1L1；Protein chip kit also includes carrying aldehyde group modified slide.The TKTL1 (transketolase sample 1) and DNASE1L1 (sample 1 of deoxyribonuclease 1) that the present invention is directed in blood using protein chip technology are detected, only need to use micro sample, in the short period of time, it can be obtained by required data, sensitivity and better reliability simultaneously, operation are more simple.
Description
Technical field
The present invention relates to the horizontal detection field of tumour antigen, in particular to a kind of egg for being used to detect tumour antigen
White chip agent box and its detection method.
Background technology
Monocytes/Macrophages are the important components of human immune system's cell.Human peripheral's haemocyte can be main
It is divided into cytode (predominantly erythrocyte) and karyocyte (leucocyte etc.).Monocyte accounts for karyocyte sum
5% or so, it is the important component of antigen presenting cell in immune system (APC cells) by medullary system hematopoietic progenitor cell.
Its maturation in human recycle system is broken up and realizes that immunologic function can be roughly divided into following steps：List in capillary
Nucleus → through tissue → discovery heterologous material (bacterium, pathogen, tumour cell etc.) outside vascular wall intravasation, differentiation
Macrophage → phagocytosis for maturation simultaneously digests, by antigen submission to specific cell surface molecule → flow back via lymphatic vessel
Lethal immunocyte is activated to peripheral blood → completion antigen submission.As the external pathogen invasion of fight and understand that itself is non-
The sentry of normal cell mechanism, macrophage are the important steps that immune system forms immune response, and effect is great.In normal person
There is the cell towards anti-tumor (immortalizing, improper apoptosis) development to produce all the time in vivo, normal immunity of organism machine
System can be removed it in time by said process.But with the evolution of tumour cell, generate the machine that protected from immune kills
System, or the decline of body overall immune power, immunologic escape is formed, finally form tumor mass in patient's body.As can be seen here
No matter phagocytosis of the macrophage for anti-tumor cell in healthy population or cancer patient's body, be all not exist all the time
Carry out.Can be me for the observation of tumour source material in Monocytes/Macrophages (including protein, accounting, lipid etc.)
Provide a monitoring in-vivo tumour occur, the new perspective of development, its application prospect is necessarily very extensive.
Important component of the Monocytes/Macrophages as karyocyte, prior art is generally by its surface antigen molecules
What CD14 and CD16 was detected, it is currently available the antibody of maturation.Main clinical practice concentrates on haemocyte composition inspection
Survey with hematopoietic system cancer (such as：Grain/monocytic leukemia) in terms of detection in, provide important references for clinical diagnosis.
From the point of view of current patent and literature search, the monitoring of tumour antigen and entity tumor is contacted and need further to study.
In view of this, it is special to propose the present invention.
The content of the invention
The first object of the present invention is to provide a kind of protein chip kit for being used to detect tumour antigen, the kit
TKTL1 (transketolase-sample 1) and DNASE1L1 (deoxyribonuclease 1- samples 1) in blood is directed to using protein chip technology
Detected, and then know the level of tumour antigen in blood.
The second object of the present invention is to provide a kind of detection method of tumour antigen, and this method uses protein chip technology
Detected, it is only necessary to use micro sample, in the short period of time, it is possible to obtain required data, while sensitivity
And better reliability, operation are more simple.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
A kind of protein chip kit for being used to detect tumour antigen, including the thing of antibody one and the display antibody one
Matter,
And/or
The material of antibody two and the display antibody two；
The antigen of the antibody one is TKTL1, and the antigen of the antibody two is DNASE1L1；
The protein chip kit also includes carrying aldehyde group modified slide.
The difference of tumour cell and normal somatic cell, it is main at two aspects：
1. metabolism：Tumour cell is often shown pair because its propagation is fast and is not regulated and controled by normal cell-cycle
The demand of the formula of hungering and thirst of energy matter (glucose etc.), but due to the variation of its metabolic mechanism, metabolism particularly energy generation
Thank to efficiency and be often far below normal somatic cell, utilized for example, fully effective metabolism can not be carried out to glucose, but a kind of wave
Take the consumption of formula.And enzyme material of the tumour cell required in this metabolic process, its expression quantity are also deposited with normal somatic cell
In huge difference, the break-through point that can be detected as tumour cell.
2. Apoptosis：Another feature that tumour cell is different from normal somatic cell is exactly to immortalize, and tumour cell is
Not by normal cell-cycle regulation and control.Apoptosis Mechanism, which has had, more clearly to be studied, and tumour cell not apoptosis, that must
The expression of the right factor, semiochemicals or the enzyme for causing regulating cell apoptosis either to be played a key effect in apoptosis process
There is obvious exception in amount, and this can also serve as the break-through point of tumour cell detection.
TKTL1 (transketolase-sample 1) and DNASE1L1 (deoxyribonuclease 1- samples 1) correspond respectively to 2 points of the above, its
Catabolite in macrophage is two target checking matters of detection technique of the present invention, is briefly discussed below：
TKTL1：Non-oxide approach in main regulation glycometabolism approach, the high expression in kinds of tumors, its expression quantity with
Developing stage, invasion and attack and the transfer and prognosis of tumour have substantial connection.Nutrition arrangement and follow-up treatment to tumor patient
Etc. have important suggesting effect.
DNASE1L1：Played an important role in apoptosis process, but in tumour cell, normal Apoptosis mechanism loses
Effect, DNASE1L1 activity is suppressed, but its great expression and is accumulated in tumour cell.Its expression quantity, which can be used as, differentiates tumour
The index of cell or Apoptosis mechanism abnormal cell.
The present invention is detected for TKTL1 and DNASE1L1 using protein chip technology, to judge that tumour resists in blood
Former level.
Further, fluorescence labeling of the material of the display antibody one for mark on the antibody one, it is described
Show fluorescence labeling of the material of the antibody two for mark on the antibody two；
Or
The antibody one is the antibody one in two kinds of different genera sources, and the material of the display antibody one is with glimmering
Signal and with any antibody one pairing secondary antibody；The antibody two is the antibody two in two kinds of different genera sources, described
The material for showing the antibody two is secondary antibody that is with fluorescence labeling and being matched with any antibody two.
Wherein, the secondary antibody of the secondary antibody of antibody one and antibody two can be it is identical can also be different.
Further, antibody one is anti-TKTL1, and the antibody two is anti-DNASE1L1.
Anti-TKTL1 and anti-DNASE1L1 is commercially available prod, and both antibody can originate from various animals, such as conventional
Mouse, rat, rabbit, monkey etc..As anti-TKTL1 can be bought from sigma, article No. HPA000505, rabbit
polyclonal；Anti-DNASE1L1 can be bought from sigma, article No. HPA072635, rabbit polyclonal；Accordingly
Ground, secondary antibody can be PE polyclonal antibody of the purchase from abcam donkey anti-rabbits, and its article No. is ab7007.
It is described above the same, secondary antibody can also be dispensed in the present invention, this is by by anti-TKTL1 and anti-
DNASE1L1 carries out fluorescence labeling realization.
Further, the protein chip kit also includes lysate A, lysate B, confining liquid, cell washing buffer
Any one or a few in liquid；
The lysate A contains each composition of following concentration：
10±0.5mM Tris-HCl(pH 7.4-7.6)、10±0.5mM NaH2PO4, 130 ± 5mM NaCl, 1% ±
0.05%Triton X-100 and 10 ± 0.5mM Na4P2O7；
The lysate B contains each composition of following concentration：
150 ± 5mM NaCl, 0.1% ± 0.005%Triton X-100,1 ± 0.05mM EDTA, 20 ± 0.1mM
HEPES and 10% ± 0.5% glycerine；
The PBS that it is 0.8%-1.2%BSA containing percentage by volume that the confining liquid, which is,.
Further, the fluorescence labeling is any of PE, CY5, CY7, per-CP and TRITC.
Fluorescence labeling is for the content of quantitatively corresponding antibody.
Present invention also offers a kind of detection method of tumour antigen, and blood is detected using above-mentioned protein chip kit
In leucocyte lysate in TKTL1 and DNASE1L1 know the information of tumour antigen.
This method is detected using protein chip technology, it is only necessary to uses micro sample, in the short period of time, just
Required data, while sensitivity and better reliability can be obtained, operation is more simple.
Further, the leucocyte lysate is prepared by the following method：
Blood sample is taken, using described lysate A splitting erythrocytes, removes all the components in addition to leucocyte；
Obtained leucocyte uses described lysate B to be cracked, and obtains leucocyte lysate.
The present invention removes all the components in addition to leucocyte by by the erythrocyte splitting of blood sample, obtains white
Cell, all the components of removal described herein in addition to leucocyte are also only to try to remove.Obtained leucocyte, which cracks, to be treated
Survey thing.Leucocyte can swallow tumour cell, by carrying out specific detection, increase detection to the leucocyte lysate in blood
Accuracy and specificity.
Preferably, the lysate A and the blood sample volume ratio are 15-20:1, the time of cracking is 10-
20min。
Empirical tests, lysate A can specificity by erythrocyte splitting, provide good basis for the removal of red blood cell.Pass through
The lysate A of special ratios is added in blood, and limits specific pyrolysis time, sufficiently to be cracked red blood cell.
Further, all the components in addition to leucocyte are removed by the way of centrifugation, the condition of the centrifugation is：750-
850g, 4-6min, supernatant is removed after centrifugation.
The step is different from the sedimentation degree of uncracked leucocyte according to the composition after cracking and is separated.Through testing,
The condition of above-mentioned centrifugation can reach more preferable separating effect.
In order that the purity of obtained leucocyte is more preferable, the composition that other in blood remain is removed, it is preferable that after centrifugation
The precipitation arrived is washed 2-3 times using PBS.
Further, the lysate B and the blood sample volume ratio are 4-6:1, the time of cracking is 8-
15min。
Empirical tests, lysate B can crack leucocyte, and good basis is provided to obtain the lysate of leucocyte.Pass through
The lysate B of special ratios is added, and limits specific pyrolysis time, sufficiently to crack leucocyte.
Further, the TKTL1 in the leucocyte lysate in described protein chip kit detection blood and
DNASE1L1's comprises the following steps that：
(a), the antibody one and the antibody two are separately fixed on the slide, using in claim 3
Confining liquid carries out Seal treatment；
(b), the leucocyte lysate is added separately to the load glass that on the slide of the fixation of antibody one and antibody two is fixed
On piece, after incubation, washed with PBS；
(c) antibody one with fluorescence labeling or the antibody two with fluorescence labeling, are added on corresponding slide, is kept away
After light is incubated, washed with PBS；
Or
Added on corresponding slide with the antibody one in step (a) different genera source or with step (a) different genera
The antibody two in source, after incubation, is washed with PBS, and the phase with fluorescence labeling is then added on corresponding slide
The secondary antibody of the secondary antibody for the antibody one answered or the corresponding antibody two with fluorescence labeling, after lucifuge is incubated, washed with PBS
Wash；
(d), detected using Fluorescence Scanner.
When detection is related to secondary antibody, in order to prevent secondary antibody and the antibody binding in step (a), sealed first
Processing is closed, then the antibody by the antibody one used twice and antibody two from different genera source, it is fine to ensure that detection
Stability and veracity.
Wherein, in step (a), the antibody one and the antibody two are separately fixed on the slide, are by antibody
One and antibody two be added separately on different slides or the different position of slide, the concentration of antibody one and antibody two is
1-10 μ g/mL, the volume of addition is generally 2-10 μ L, and after the completion of addition, 4-10 DEG C overnight, then discards molten on slide
Liquid, lavation buffer solution are washed 2-3 times, are dried；During being somebody's turn to do, it is according to the cross-link on antibody and slide, realizes anti-
The fixation of body.
In step (b), the addition of leucocyte lysate is generally：Obtained leucocyte lysate is diluted into 8-15
Times, then with the coated microballoon of antibody by volume 1:0.5-1.5 is added.
Addition depends on step (b) with fluorescence or without the antibody one and antibody two of fluorescence in step (c)
The antigen of middle addition, for sufficient antigen and antibody binding, the antibody one of addition and antibody two are somewhat excessive in step (c),
Excessive antibody removes by washing, and after addition, the concentration of antibody one and antibody two is 0.08-0.15 μ g/mL.
Similarly, the addition of secondary antibody is then according to the addition of corresponding primary antibody in step (c).Preferably, in step (c)
In, the final concentration of the secondary antibody of the secondary antibody of the antibody one with fluorescence labeling and the antibody two with fluorescence labeling is 0.08-
0.15μg/mL。
Aldehyde group modified slide is carried by commercially available purchase in the present invention, it is such as commercially available from U.S. TeleChem
The SuperAldehyde slides of company.
The present invention is also done while TKTL1 and DNASE1L1 in detecting leucocyte lysate with cell internal reference GAPDH
The ratio of control, destination protein detected value and internal reference is GCI indexes, by introducing cell internal reference, to control in detection process
Error, so as to increase the degree of accuracy of detection.
It should be noted that the TKTL1 and DNASE1L1 in leucocyte lysate of the present invention divide in whole detection process
Do not detected.In addition, the fluorescence that the wavelength used in multifunctional protein chip analysis detection of platform marks according to primary antibody or secondary antibody
Species selects.
Compared with prior art, beneficial effects of the present invention are：
(1) the invention provides the protein chip kit of detection tumour antigen, the kit to use protein chip technology
Detected, and then known for the TKTL1 (transketolase-sample 1) in blood and DNASE1L1 (deoxyribonuclease 1- samples 1)
The level of tumour antigen, can be applied to physical examination in blood, and early warning is provided for tumor patient.
(2) present invention also offers the detection method of tumour antigen, detected relative to the dicing method and PCR of tumor tissues
Means, this method are detected using protein chip technology, it is only necessary to use micro sample, in the short period of time, so that it may
To obtain a large amount of required data, while sensitivity and better reliability, operation are more simple and convenient.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition suggested according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, it is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
A kind of detection method of tumour antigen, comprises the following steps：
Prepare following solution：
Lysate A：10±0.5mM Tris-HCl(pH 7.5)、10±0.5mM NaH2PO4, 130 ± 5mM NaCl, 1%
± 0.05%Triton X-100 and 10 ± 0.5mM Na4P2O7；
Lysate B：150 ± 5mM NaCl, 0.1% ± 0.005%Triton X-100,1 ± 0.05mMEDTA, 20 ±
0.1mM HEPES and 10% ± 0.5% glycerine；
The PBS that it is 0.8%BSA containing percentage by volume that confining liquid, which is,；
1. taking the μ L of blood sample 100, add 2mL lysate A, the concussion that is vortexed mixes, and is incubated 15min, splitting erythrocyte；
2.800g centrifuges 5min, removes supernatant, slowly outwells supernatant, and residual liquid is drawn with paper, and be vortexed concussion cell；
3. adding 2ml PBS washings, it is vortexed and mixes, 800g centrifugation 5min, removes supernatant, wash 3 times, cell is resuspended after washing；
4. adding 500 μ l lysate B, leucocyte, pyrolysis time 10min, as measuring samples are cracked；
5. coating：Antibody anti-TKTL1 (mouse polyclonal) and anti-DNASE1L1 (mouse
Polyclonal) it is added separately on slide, after 4 DEG C are stayed overnight, discards the solution on slide, washed 3 times with PBS, with containing 1%
BSA PBS closing slides, 25 DEG C of closing 1h；
6. sample-adding：Measuring samples are separately added on slide, are put 37 DEG C and are incubated 1 hour, then PBS washings 3 times；Do simultaneously
Blank control (being not added with measuring samples), negative control (measuring samples are changed to lysate B) and positive control are (corresponding by measuring samples
Replace with TKTL1 albumen or DNASE1L1 albumen)；
7. anti-TKTL1 (rabbit polyclonal, sigma, article No. are added on corresponding slide
HPA000505) and anti-DNASE1L1 antibody (rabbit polyclonal, sigma, article No. HPA072635), 37 DEG C, incubate
Educate 20min；
8. add 2mL PBS to wash 2 times；
9. it is separately added into 1：Anti-rabbit-PE antibody (donkeypolyclonal, abcam, the article No. of 5000 dilutions
Ab7007), lucifuge is incubated 10min；
10. add 2mL PBS to wash 2 times, Fluorescence Scanner detection；
Instrument title：VersArray ChipReader 5um system(Bio-Rad)；
11. result interpretation method：Control, destination protein detected value and internal reference GAPDH ratio are used as by the use of cell internal reference GAPDH
Value is GCI indexes.
Embodiment 2
A kind of detection method of tumour antigen, comprises the following steps：
Prepare following solution：
Lysate A：10±0.5mM Tris-HCl(pH 7.4)、10±0.5mM NaH2PO4, 130 ± 5mM NaCl, 1%
± 0.05%Triton X-100 and 10 ± 0.5mM Na4P2O7；
Lysate B：150 ± 5mM NaCl, 0.1% ± 0.005%Triton X-100,1 ± 0.05mMEDTA, 20 ±
0.1mM HEPES and 10% ± 0.5% glycerine；
The PBS that it is 1%BSA containing percentage by volume that confining liquid, which is,；
1. taking the μ L of blood sample 200, add 3mL lysate A, the concussion that is vortexed mixes, and is incubated 10min, splitting erythrocyte；
2.750g centrifuges 6min, removes supernatant, slowly outwells supernatant, and residual liquid is drawn with paper, and be vortexed concussion cell；
3. adding 3ml PBS washings, it is vortexed and mixes, 750g centrifugation 5min, removes supernatant, wash 2 times, cell is resuspended after washing；
4. adding 800 μ l lysate B, leucocyte, pyrolysis time 8min, as measuring samples are cracked；
5. coating：Antibody anti-TKTL1 (mouse polyclonal) and anti-DNASE1L1 (mouse
Polyclonal) it is added separately on slide, after 4 DEG C are stayed overnight, discards the solution on slide, washed 3 times with PBS, with containing 1%
BSA PBS closing slides, 25 DEG C of closing 1h；
6. measuring samples are separately added on the slide of coated antibody, put 37 DEG C and be incubated 1 hour, then PBS washings 3
It is secondary；Doing blank control (being not added with measuring samples), negative control (measuring samples are changed to lysate B) and positive control simultaneously (will treat
Sample product replace with TKTL1 albumen or DNASE1L1 albumen accordingly)；
7. anti-TKTL1 (rabbit polyclonal, sigma, article No. are added on corresponding slide
HPA000505) and anti-DNASE1L1 antibody (rabbit polyclonal, sigma, article No. HPA072635), 37 DEG C, incubate
Educate 15min；
8. add 2mL PBS to wash 2 times；
9. it is separately added into 1：Anti-rabbit-PE antibody (donkeypolyclonal, abcam, the article No. of 5000 dilutions
Ab7007), lucifuge is incubated 15min；
10. add 2mL PBS to wash 2 times, Fluorescence Scanner detection；
Instrument title：VersArray ChipReader 5um system(Bio-Rad)；
11. result interpretation method：Control, destination protein detected value and internal reference GAPDH ratio are used as by the use of cell internal reference GAPDH
Value is GCI indexes.
Embodiment 3
A kind of detection method of tumour antigen, comprises the following steps：
Prepare following solution：
Lysate A：10±0.5mM Tris-HCl(pH 7.6)、10±0.5mM NaH2PO4, 130 ± 5mM NaCl, 1%
± 0.05%Triton X-100 and 10 ± 0.5mM Na4P2O7；
Lysate B：150 ± 5mM NaCl, 0.1% ± 0.005%Triton X-100,1 ± 0.05mMEDTA, 20 ±
0.1mM HEPES and 10% ± 0.5% glycerine；
The PBS that it is 1.2%BSA containing percentage by volume that confining liquid, which is,；
1. taking the μ L of blood sample 100, add 2mL lysate A, the concussion that is vortexed mixes, and is incubated 20min, splitting erythrocyte；
2.850g centrifuges 4min, removes supernatant, slowly outwells supernatant, and residual liquid is drawn with paper, and be vortexed concussion cell；
3. adding 2ml PBS washings, it is vortexed and mixes, 850g centrifugation 5min, removes supernatant, wash 3 times, cell is resuspended after washing；
4. adding 400 μ l lysate B, leucocyte, pyrolysis time 15min, as measuring samples are cracked；
5. coating：Antibody anti-TKTL1 (mouse polyclonal) and anti-DNASE1L1 (mouse
Polyclonal) it is added separately on slide, after 4 DEG C are stayed overnight, discards the solution on slide, washed 2 times with PBS, with containing 1%
BSA PBS closing slides, 25 DEG C of closing 1h；
6. sample-adding：Measuring samples are separately added on the slide of coated antibody, are put 37 DEG C and are incubated 1 hour, then PBS is washed
Wash 3 times；Doing blank control (being not added with measuring samples), negative control (measuring samples are changed to lysate B) and positive control simultaneously (will
Measuring samples replace with TKTL1 albumen or DNASE1L1 albumen accordingly)；
7. anti-TKTL1 (rabbit polyclonal, sigma, article No. are added on corresponding slide
HPA000505) and anti-DNASE1L1 antibody (rabbit polyclonal, sigma, article No. HPA072635), 37 DEG C, incubate
Educate 30min；
8. add 2mL PBS to wash 2 times；
9. it is separately added into 1：Anti-rabbit-PE antibody (donkeypolyclonal, abcam, the article No. of 5000 dilutions
Ab7007), lucifuge is incubated 10min；
10. add 2mL PBS to wash 3 times, Fluorescence Scanner detection；
Instrument title：VersArray ChipReader 5um system(Bio-Rad)；
11. result interpretation method：Control, destination protein detected value and internal reference GAPDH ratio are used as by the use of cell internal reference GAPDH
Value is GCI indexes.
Embodiment 4
A kind of detection method of tumour antigen, comprises the following steps：
Prepare following solution：
Lysate A：10±0.5mM Tris-HCl(pH 7.5)、10±0.5mM NaH2PO4, 130 ± 5mM NaCl, 1%
± 0.05%Triton X-100 and 10 ± 0.5mM Na4P2O7；
Lysate B：150 ± 5mM NaCl, 0.1% ± 0.005%Triton X-100,1 ± 0.05mMEDTA, 20 ±
0.1mM HEPES and 10% ± 0.5% glycerine；
The PBS that it is 1%BSA containing percentage by volume that confining liquid, which is,；
1. taking the μ L of blood sample 100, add 2mL lysate A, the concussion that is vortexed mixes, and is incubated 15min, splitting erythrocyte；
2.800g centrifuges 5min, removes supernatant, slowly outwells supernatant, and residual liquid is drawn with paper, and be vortexed concussion cell；
3. adding 2ml PBS washings, it is vortexed and mixes, 800g centrifugation 5min, removes supernatant, wash 3 times, cell is resuspended after washing；
4. adding 500 μ l lysate B, leucocyte, pyrolysis time 10min, as measuring samples are cracked；
5. coating：Antibody anti-TKTL1 (mouse polyclonal) and anti-DNASE1L1 (mouse
Polyclonal) it is added separately on slide, after 4 DEG C are stayed overnight, discards the solution on slide, washed 3 times with PBS, with containing 1%
BSA PBS closing slides, 25 DEG C of closing 1h；
6. measuring samples are separately added on the slide of coated antibody, put 37 DEG C and be incubated 1 hour, then PBS washings 3
It is secondary；Doing blank control (being not added with measuring samples), negative control (measuring samples are changed to lysate B) and positive control simultaneously (will treat
Sample product replace with TKTL1 albumen or DNASE1L1 albumen accordingly)；
7. the anti-TKTL1 with the fluorescence labeling or anti-with fluorescence labeling is added on corresponding slide
DNASE1L1 antibody, 37 DEG C, lucifuge is incubated 20min；
8. add 2mL PBS to wash 2 times, Fluorescence Scanner detection；
Instrument title：VersArray ChipReader 5um system(Bio-Rad)；
9. result interpretation method：Control, destination protein detected value and internal reference GAPDH ratio are used as by the use of cell internal reference GAPDH
Value is GCI indexes.
Test example 1
The blood sample of one phase of the cancer patient made a definite diagnosis is taken to be detected respectively using embodiment 1-4 method, the blood sample is divided equally
For 21 parts, each embodiment carries out parallel test 3, while sets control group 1-3, and each control group carries out parallel test 3；
Control group 1：Blood serum deprivation, the PBS solution that the obtained μ L of haemocyte 100 add 2mL to contain 1%SDS, mix, in 4 DEG C of cracking
10min, obtained lysate is as measuring samples；Control group 2：Blood serum deprivation, the obtained μ L of haemocyte 100 add 2mL to contain
1%Triton X-100 PBS solution, mix, crack 10min in 4 DEG C, obtained lysate is as measuring samples；Control group
3：The serum of blood is taken as measuring samples；Detecting step follow-up control group 1-3 is the same as embodiment 2.Then the fluorescence that will be obtained
Signal carries out counting statistics, respectively obtains TKTL1 and DNASE1L1 GCI indexes, the GCI indexes of three samples of parallel test
Standard deviation it is specifically as shown in table 1.
The testing result of table 1
| Group | TKTL1 standard deviations | DNASE1L1 standard deviations |
| Embodiment 1 | 0.018 | 0.025 |
| Embodiment 2 | 0.012 | 0.019 |
| Embodiment 3 | 0.017 | 0.027 |
| Embodiment 4 | 0.010 | 0.021 |
| Control group 1 | 0.275 | 0.287 |
| Control group 2 | 0.284 | 0.295 |
| Control group 3 | 0.009 | 0.013 |
As it can be seen from table 1 being detected using kit provided by the invention, each embodiment detects same sample
TKTL1 and DNASE1L1 GCI standard of indexs deviation is within 0.03；And the lysate of haemocyte is used in control group 1-2
As sample to be tested, because impurity is more, obtained data are unstable, and deviation is big；And it is used as in control group 3 using serum and treats test sample
This, although deviation is small, target protein content is seldom in serum, is substantially not detectable.In addition, the present invention is also to other samples
This detection is carried out, the standard deviation of identical sample is within 0.03.It can be seen that kit detection stability provided by the invention
Good, sensitivity is high.
Test example 2
The blood sample (sample 1), the blood sample (sample 2) of one phase of cancer patient, cancer of the early stage benign tumour patient made a definite diagnosis is taken respectively
Implementation is respectively adopted in the blood sample (sample 5) of the blood sample (sample 3) of the second stage of patient, the blood sample (sample 4) of three phase of cancer patient and normal person
The method of example 2 is detected, while the different multiple of each Sample Dilution is detected, obtained TKTL1 and DNASE1L1
GCI indexes analyzed, draw：After dilution, TKTL1 GCI indexes and DNASE1L1 GCI indexes still have sample
There are good sensitivity and the degree of accuracy；Also, the numerical value between the sample within 4 times of the different sample stostes made a definite diagnosis and dilution
It is respectively provided with significant difference.In addition, large quantities of experiments also demonstrate that the numerical value between the different samples made a definite diagnosis has significant difference.
In addition, the different blood sample of patients made a definite diagnosis are detected using the method in embodiment 1,3-4, obtained result and reality
It is consistent to apply the result of example 2.
Found through lot of experiments, detected sample detects respective strengths according to wherein TKTL1 and DNASE1L1 change
The specificity fluorescent of change, testing result is reliable and stable, and technical support is provided for the auxiliary detection of tumour.Specifically, it is of the invention
There can be the application of following aspect：
The level of blood testing tumour antigen to Different Individual, different individuals can be：Health and different times
Tumor patient；By large-scale experiment, obtained largely not by obtained TKTL1 and DNASE1L1 the GCI indexes to convert respectively
Tumour antigen with individual is horizontal, and then can set a particular value, to distinguish different patients.Due in the sample of measure
TKTL1 and DNASE1L1 numerical value caused by many factors, therefore, during subsequent detection, according to the blood to human body
The level of middle tumour antigen, if being above certain numerical value, need it is further detected, and then can just judge whether with swollen
Knurl.The present invention aids in predicting that individual suffers from tumor risk by determining GCI indexes, and technical support is provided for tumour early warning.
Although illustrate and describing the present invention with specific embodiment, but will be appreciated that without departing substantially from the present invention's
Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of protein chip kit for being used to detect tumour antigen, it is characterised in that including described in antibody one and display
The material of antibody one,
And/or
The material of antibody two and the display antibody two；
The antigen of the antibody one is TKTL1, and the antigen of the antibody two is DNASE1L1；
The protein chip kit also includes carrying aldehyde group modified slide.
2. protein chip kit according to claim 1, it is characterised in that the material of the display antibody one is
The fluorescence labeling on the antibody one is marked, the material of the display antibody two is that mark is glimmering on the antibody two
Signal；
Or
The antibody one is the antibody one in two kinds of different genera sources, and the material of the display antibody one is with fluorescence mark
Secondary antibody that is remembering and being matched with any antibody one；The antibody two is the antibody two in two kinds of different genera sources, the display
The material of the antibody two is secondary antibody that is with fluorescence labeling and being matched with any antibody two；
Preferably, antibody one is anti-TKTL1, and the antibody two is anti-DNASE1L1.
3. protein chip kit according to claim 1, it is characterised in that the protein chip kit also includes splitting
Solve any one or a few in liquid A, lysate B, confining liquid, Cell Wash Buffer；
The lysate A contains each composition of following concentration：
10±0.5mM Tris-HCl(pH 7.4-7.6)、10±0.5mM NaH2PO4, 130 ± 5mM NaCl, 1% ± 0.05%
Triton X-100 and 10 ± 0.5mM Na4P2O7；
The lysate B contains each composition of following concentration：
150 ± 5mM NaCl, 0.1% ± 0.005%Triton X-100,1 ± 0.05mM EDTA, 20 ± 0.1mM HEPES with
And 10% ± 0.5% glycerine；
The PBS that it is 0.8%-1.2%BSA containing percentage by volume that the confining liquid, which is,.
4. according to the protein chip kit described in claim any one of 1-3, it is characterised in that the fluorescence labeling be PE,
Any of CY5, CY7, per-CP and TRITC.
5. a kind of detection method of tumour antigen, it is characterised in that tried using the protein chip described in claim any one of 1-4
TKTL1 in leucocyte lysate and DNASE1L1 in agent box detection blood know the information of tumour antigen.
6. the detection method of tumour antigen according to claim 5, it is characterised in that the leucocyte lysate by with
It is prepared by lower section method：
Blood sample is taken, using described lysate A splitting erythrocytes, removes all the components in addition to leucocyte；
Obtained leucocyte uses described lysate B to be cracked, and obtains leucocyte lysate；
Preferably, the lysate A and the blood sample volume ratio are 15-20:1, the time of cracking is 10-20min.
7. the detection method of tumour antigen according to claim 6, it is characterised in that removed by the way of centrifugation except white
Extracellular all the components, the condition of the centrifugation are：750-850g, 4-6min, supernatant is removed after centrifugation；
Preferably, the precipitation obtained after centrifugation is washed 2-3 times using PBS.
8. the detection method of tumour antigen according to claim 6, it is characterised in that the lysate B and the blood
The volume ratio of sample is 4-6:1, the time of cracking is 8-15min.
9. the detection method of the tumour antigen according to claim any one of 5-8, it is characterised in that described protein chip
TKTL1 in leucocyte lysate and DNASE1L1 in kit detection blood are comprised the following steps that：
(a), the antibody one and the antibody two are separately fixed on the slide, using the closing in claim 3
Liquid carries out Seal treatment；
(b), the leucocyte lysate is added separately on the slide of the fixation of antibody one and on the slide that antibody two is fixed,
After incubation, washed with PBS；
(c) antibody one with fluorescence labeling or the antibody two with fluorescence labeling, are added on corresponding slide, lucifuge is incubated
After educating, washed with PBS；
Or
Added on corresponding slide with the antibody one in step (a) different genera source or with step (a) different genera source
Antibody two, after incubation, washed, then added on corresponding slide corresponding with fluorescence labeling with PBS
The secondary antibody of the secondary antibody of antibody one or corresponding antibody two with fluorescence labeling, after lucifuge is incubated, washed with PBS；
(d), detected using Fluorescence Scanner.
10. the detection method of tumour antigen according to claim 5, it is characterised in that also set up cell internal reference GAPDH works
To compare, the information of the tumour antigen is judged according to destination protein detected value and cell internal reference GAPDH detected values.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1338634A (en) * | 2001-09-29 | 2002-03-06 | 上海晶泰生物技术有限公司 | Protein chip for diagnosing early malignant tumor |
| CN1726393A (en) * | 2002-12-18 | 2006-01-25 | 约翰内斯·科伊 | Compounds and methods for detection of carcinomas and their precursor lesions |
| CN101142486A (en) * | 2005-03-07 | 2008-03-12 | 约翰内斯·科伊 | Therapeutic and diagnostic uses of tktl1 and inhibitors and activators thereof |
| CN101628224A (en) * | 2009-08-14 | 2010-01-20 | 陕西北美基因股份有限公司 | Magnetic silicon dioxide microspheres modified with diethylaminoethyl and preparation method and application thereof |
| CN102251046A (en) * | 2011-07-29 | 2011-11-23 | 王荣 | Method for detecting tumor mutant gene in blood |
| WO2013126872A1 (en) * | 2012-02-24 | 2013-08-29 | Wayne State University | Anti-cancer therapeutic strategy to overcome cancer resistance and to enable tailoring treatment to patients |
| CN103571826A (en) * | 2013-11-23 | 2014-02-12 | 河北联合大学 | Efficient extraction method for whole blood genome DNA (deoxyribonucleic acid) |
| CN103789298A (en) * | 2013-12-30 | 2014-05-14 | 湖南圣湘生物科技有限公司 | Erythrocyte lysis buffer and application thereof |
-
2016
- 2016-08-02 CN CN201610635953.4A patent/CN107677823A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1338634A (en) * | 2001-09-29 | 2002-03-06 | 上海晶泰生物技术有限公司 | Protein chip for diagnosing early malignant tumor |
| CN1726393A (en) * | 2002-12-18 | 2006-01-25 | 约翰内斯·科伊 | Compounds and methods for detection of carcinomas and their precursor lesions |
| CN101142486A (en) * | 2005-03-07 | 2008-03-12 | 约翰内斯·科伊 | Therapeutic and diagnostic uses of tktl1 and inhibitors and activators thereof |
| CN101628224A (en) * | 2009-08-14 | 2010-01-20 | 陕西北美基因股份有限公司 | Magnetic silicon dioxide microspheres modified with diethylaminoethyl and preparation method and application thereof |
| CN102251046A (en) * | 2011-07-29 | 2011-11-23 | 王荣 | Method for detecting tumor mutant gene in blood |
| WO2013126872A1 (en) * | 2012-02-24 | 2013-08-29 | Wayne State University | Anti-cancer therapeutic strategy to overcome cancer resistance and to enable tailoring treatment to patients |
| CN103571826A (en) * | 2013-11-23 | 2014-02-12 | 河北联合大学 | Efficient extraction method for whole blood genome DNA (deoxyribonucleic acid) |
| CN103789298A (en) * | 2013-12-30 | 2014-05-14 | 湖南圣湘生物科技有限公司 | Erythrocyte lysis buffer and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| MARTIN GRIMM 等: "A biomarker based detection and characterization of carcinomas exploiting two fundamental biophysical mechanisms in mammalian cells", 《BMC CANCER》 * |
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