CN107091924A - The chain lymphoproliferative syndrome diagnostic kits of X and its application - Google Patents
The chain lymphoproliferative syndrome diagnostic kits of X and its application Download PDFInfo
- Publication number
- CN107091924A CN107091924A CN201710437610.1A CN201710437610A CN107091924A CN 107091924 A CN107091924 A CN 107091924A CN 201710437610 A CN201710437610 A CN 201710437610A CN 107091924 A CN107091924 A CN 107091924A
- Authority
- CN
- China
- Prior art keywords
- antibody
- human
- xiap
- incubated
- streaming
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to medical diagnostic techniqu field, in particular to a kind of chain lymphoproliferative syndrome diagnostic kits of X and its application.The kit includes：Anti-human CD3, CD56 streaming fluorescence antibody, anti-human XIAP antibody and its isotype control Ab and the corresponding Western Blot secondary antibodies of the anti-human XIAP antibody and streaming fluorescence secondary antibody.The method of XIAP protein abundances in detection T and NK cells based on the diagnostic kit, detection is quick, and flux is big, and the degree of accuracy is high, and human factor influence is smaller.
Description
Technical field
The present invention relates to medical diagnostic techniqu field, diagnosed in particular to a kind of chain lymphoproliferative syndromes of X-
Kit and its application.
Background technology
Studies on inhibitor of apoptosis proteins (inhibitor of apoptosis protain, IAPs) is found in shaft-like disease first
Poison, host cell is dead caused by suppressing virus infection, and XIAP (X chain lock IAPs, X-linked
Inhibitor of apoptosis protain) it is wherein important member, it is positioned at Xq25, cDNA total length 2540bp, N-terminal
Mainly there are 3 baculoviral IAP repetitive sequence BIR1-BIR3, and BIR is about containing 70 amino acid, its special zinc finger knot
Structure is the interaction that XIAP participates in protein-protein, protein and DNA.While the special connection bridge between BIR structures
Linker, can be combined with Caspase, suppress Caspase activity, and then suppress Apoptosis.
The chain lymphoproliferative syndromes of X- (X-linked Lymph Proliferative Disease, XLP) are
A kind of rare, often lethal PID, can be by Epstein-Barr virus (Epstein-Barr virus, EBV)
Infection-induced, shows as explosive infectious mononucleosis, dysgammaglobulinemia and lymphoproliferative disease
And lymthoma.The chain lymphoproliferative syndromes of X- include XLP-1 and XLP-2, with the heredity of x linked recessive mode, are more common in man
Property.This disease is main by coding lymph signal anakmetomeres GAP-associated protein GAP (XIAP), the chain survivins of X- (XIAP) and IL-
The mutation of T cell kinases (ITK) gene of 2 inductions causes, and gene sequencing is the foundation for making a definite diagnosis XLP；XIAP、XIAP、ITK
Albumen expression can also as examination XLP means.Family history is the primary subjective index for needing to consider, other diagnostic criteria
EBNA antibody tests after clinical manifestation, ebv infection including infant etc..
XLP-2 and BIRC4 genes (expression XIAP albumen) are relevant.When BIRC4 genes are defective, then XIAP eggs are shown
White missing, causes lymphocyte excessive Apoptosis, causes the dysimmunity to EBV.
Presently, there are the selection of the detection method, wherein target cell of a variety of XIAP albumen, confirmation as a result, the problems such as be skill
The key problem in art field, is to determine whether testing result is preferably crucial.Still lack the operating process of unified standard at present.
In view of this, it is special to propose the present invention.
The content of the invention
It is an object of the invention to provide a kind of chain lymphoproliferative syndrome diagnostic kits of X- and use the reagent
Box detects the detection method of XIAP protein abundances, described detection method can solve that prior art detection speed is slow, precision is relatively low,
The problems such as accuracy is poor, testing result is easily influenceed by the subjective factor of tester.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
A kind of chain lymphoproliferative syndrome diagnostic kits of X-, the kit includes：
Anti-human CD3, CD56 streaming fluorescence antibody, anti-human XIAP antibody and its isotype control Ab and described anti-human
The corresponding Western Blot secondary antibodies of XIAP antibody and streaming fluorescence secondary antibody.
It is preferred that, the corresponding streaming fluorescence secondary antibody of anti-human XIAP antibody is anti-mouse IgG1 streaming fluorescence antibodies.
It is preferred that, anti-human CD3, CD56 streaming fluorescence antibody, anti-human XIAP antibody and mouse IgG1 albumen are purchased from respectively
4abio companies, eBioscience companies, BD companies and Abcam companies, article No. is respectively FHF003-100,17-0566-42, #
610763 and #ab18443；
It is preferred that, the streaming fluorescence secondary antibody of anti-human XIAP antibody is purchased from BD companies, and article No. is #550083；
It is preferred that, kit as described above, the fluorescence labeling of streaming fluorescence antibody institute band includes：FITC、Alexa
350th, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminacrine, BODIPY
630/650th, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5- carboxyl -4 ',
5 '-two chloro- 2 ', 7 '-dimethoxyfluoresceins, 5- carboxyls -2 ', 4 ', 5 ', 7 '-tetrachlorofluorescein, CF, 5- carboxylics
Base rhodamine, 6- carboxyrhodamines, 6- carboxyls tetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, CY5.5, Cy7,
6-FAM, dansyl Cl, fluorescein, HEX, 6-JOE, NBD (7- nitro benzo -2- oxa-s -1,3- diazole), Oregon Green
488th, Oregon Green 500, Oregon Green514, Pacific Blue, phthalic acid, terephthalic acid (TPA), isophthalic
The solid purple of dioctyl phthalate, cresols, cresols royal purple, brilliant cresyl blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, Huang are fast
Purine, succinylfluoresceins, rare earth metal cryptate, the three pairs of pyridine radicals diamines europiums, europium cryptate or chelate, two
The blue dyestuff of amine, dicyanin, La Jolla, allophycocyanin, allococyanin B, phycocyanin C, phycocyanin R, sulphur
Amine, algae red green white, phycoerythrin R, REG, rhodamine are green, rhodamine isothiocyanates, red rhodamine, ROX, TAMRA, TET,
It is a variety of in the different mercaptan of tetramethylrhodamine, tetramethylrhodamine and texas Red.
It is preferred that, kit as described above, the fluorescence labeling that the anti-human CD3, CD56, streaming fluorescence secondary antibody are carried
It is followed successively by FITC, APC, PE.
It is preferred that, kit as described above also includes anti-coagulants, cell fixative, cell rupture of membranes in the kit
One or more in agent, buffer solution, protein lysate；
It is furthermore preferred that one or more of the anti-coagulants in EDTA, heparin, citrate and oxalates；
It is furthermore preferred that the cell fixative is purchased from eBioscience, article No. #00-5521-00；
It is furthermore preferred that the cell rupture of membranes agent is purchased from eBioscience, article No. #00-8333-56；
It is furthermore preferred that the formula of the streaming buffer solution is：Contain 1.8%~2.2%FBS and 1.8mM~2.2mM
EDTA 1 × PBS solution；
It is furthermore preferred that the protein lysate is RIPA.
As above the kit described in any one prepare diagnosis the chain lymphoproliferative syndromes of X- product in should
With；
It is preferred that, as above the kit described in any one is preparing the 2 chain lymphoproliferative syndromes of type X- of diagnosis
Application in product.
It is preferred that, application as described above, using when treat the pre-treatment step of sample this progress and include：
1), separate the PMNC in sample to be checked and and count adjustment cell concentration；
2), to step 1) in cell in simultaneously add anti-human CD3, CD56 streaming fluorescence antibody and be incubated；
3), centrifuge, precipitation is incubated altogether with cell fixative；
4), centrifuge, precipitation is incubated altogether with the agent of cell rupture of membranes；
5), centrifuge, washed after precipitation and be resuspended with streaming buffer solution；
6) sample after resuspension, is divided into two groups, anti-human XIAP antibody and its isotype control Ab is separately added into and is incubated；
7), centrifuge, washed after precipitation and be resuspended with streaming buffer solution；
8), often pipe adds the corresponding streaming fluorescence secondary antibody of anti-human XIAP antibody and is incubated；
9), centrifuge, washed after precipitation and be resuspended with streaming buffer solution；And use flow cytomery；
And/or；
A), separate the PMNC in sample to be checked and collect albumen with protein lysate cracking；
B), albumen is carried out using the anti-human XIAP antibody and the corresponding secondary antibody of the anti-human XIAP antibody
Western Blot are tested, and read the gray value of gained band；
Wherein step 1)~9) with step a)~b) do not have sequencing.
It is preferred that, application as described above, in step 1) in, the cell concentration is 3.5~4.5 × 106/ml；May be used also
To select 4 × 106/ml。
It is preferred that, application as described above, in step 2) in, incubation conditions are：
Room temperature lucifuge is incubated 15~25min；It is also an option that being incubated 20min.
It is preferred that, application as described above, in step 2) in, the concentration of CD3, CD56 antibody is respectively 8~12 μ l/
100 μ l, 4~6 μ l/100 μ l；
It is preferred that, in step 6) in, the antibody concentration of anti-human XIAP antibody and its isotype control Ab is 0.5~0.7 μ
l/100μl；
It is preferred that, in step 8) in, the antibody concentration of the corresponding streaming fluorescence secondary antibody of anti-human XIAP antibody is 1.8~2.2 μ
l/100μl。
It is preferred that, application as described above, in step b), anti-human XIAP antibody presses 0.5~1.5：1000 additions, room temperature
Lower shake is incubated 2~6 DEG C of 8~16h of incubation after 1~3h；
It is furthermore preferred that in step b), anti-human XIAP antibody presses 1：1000 addition, at room temperature shake be incubated 2h after 4 DEG C incubate
Educate 10~14h.
A kind of method that XIAP protein abundances in T and NK cells are detected using kit as described above, including：
1), separate the PMNC in sample to be checked and and count adjustment cell concentration；
2), to step 1) in cell in simultaneously add anti-human CD3, CD56 streaming fluorescence antibody and be incubated；
3), centrifuge, precipitation is incubated altogether with cell fixative；
4), centrifuge, precipitation is incubated altogether with the agent of cell rupture of membranes；
5), centrifuge, washed after precipitation and be resuspended with streaming buffer solution；
6) sample after resuspension, is divided into two groups, anti-human XIAP antibody and its isotype control Ab is separately added into and is incubated；
7), centrifuge, washed after precipitation and be resuspended with streaming buffer solution；
8), often pipe adds the corresponding streaming fluorescence secondary antibody of anti-human XIAP antibody and is incubated；
9), centrifuge, washed after precipitation and be resuspended with streaming buffer solution, and detected with flow cytometer；
And/or；
A), separate the PMNC in sample to be checked and collect albumen with protein lysate cracking；
B), albumen is carried out using the anti-human XIAP antibody and the corresponding secondary antibody of the anti-human XIAP antibody
Western Blot are tested, and read the gray value of gained band；
Wherein step 1)~9) with step a)~b) do not have sequencing.
Methods described is non-diagnostic purposes.
Flow cytometry (Flow Cytometry, FCM) be one kind on functional level to unicellular or other biological grain
The sub detection means for carrying out quantitative analysis and sorting, it can be with up to ten thousand cells of high speed analysis, and energy is while from a cell
Measure multiple parameters.The amplitude of variation of XIAP protein expression abundance is detected using flow cytometry, then coordinates WB to test
Verified, can quickly, precision, evaluate exactly to XIAP protein expression levels.
CD3 (cluster of differentiation 3 break up cluster 3), acceptor existed only in T cell surface, thus can
Mark as T cell；
CD56 (cluster of differentiation 56 break up cluster 56) is also known as N-CAM (N-
CAM), it is widely present in the cell of most neuroectodermal origin, tissue and tumour, including retinoblastoma,
Medulloblastoma, astrocytoma, neuroblastoma, small cell carcinoma.Also tumour, nature derived from some mesoderms are expressed in
Kill cell and NK lymthomas.CD56 is used as the mark of NK cells in the present invention.
It is preferred that, method as described above, in step 1) in, the cell concentration is 3.5~4.5 × 106/ml；May be used also
To select 4 × 106/ml。
It is preferred that, method as described above, in step 2) in, incubation conditions are：
Room temperature lucifuge is incubated 15~25min；It is also an option that being incubated 20min.
It is preferred that, method as described above, in step 2) in, the concentration of CD3, CD56 antibody is respectively 8~12 μ l/
100 μ l, 4~6 μ l/100 μ l；
It is preferred that, in step 6) in, the antibody concentration of anti-human XIAP antibody and its isotype control Ab is 0.5~0.7 μ
l/100μl；
It is preferred that, in step 8) in, the antibody concentration of the corresponding streaming fluorescence secondary antibody of anti-human XIAP antibody is 1.8~2.2 μ
l/100μl。
It is preferred that, method as described above, in step b), anti-human XIAP antibody presses 0.5~1.5：1000 additions, room temperature
Lower shake is incubated 2~6 DEG C of 8~16h of incubation after 1~3h；
It is furthermore preferred that in step b), anti-human XIAP antibody presses 1：1000 addition, at room temperature shake be incubated 2h after 4 DEG C incubate
Educate 10~14h.
Compared with prior art, beneficial effects of the present invention are：
1), in detection CTL and the NK cell based on the chain lymphoproliferative syndrome diagnostic kits of X- that the present invention is provided
The method of XIAP protein abundances, detection is quick, and flux is big, and the degree of accuracy is high, and human factor influence is smaller.
2), the method that the present invention establishes one kind fluidic cell born of the same parents instrument (FACS) quick detection XIAP protein expressions, after
The expression quantity of XIAP albumen is confirmed with Western Blot.Entered by the details in the key technology flows such as convection type detection and WB
Row is preferred to be limited, and establishes the technical regulation of stable specification.
Brief description of the drawings
, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art
The accompanying drawing used required in embodiment or description of the prior art is briefly described, it should be apparent that, in describing below
Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid
Put, other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 is the Flow cytometry result of embodiment 3；
Fig. 2 is that embodiment 3Western Blot verify testing result.
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 advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, be
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
The expression of XIAP albumen in S11, Flow cytometry NK and T cell
1. ethylenediamine tetra-acetic acid (Ethylenediamine-tetraaceticacid, EDTA) or liquaemin (Heparin
Sodium) anti-freezing venous whole 2ml, Ficoll density-gradient centrifugation methods separating peripheral blood mononuclear cells (PBMC), and count,
It is 3.5 × 10 to adjust cell concentration6/ ml suspensions are standby.
2. each Guan Zhongjia 200ul diluting cells, this pipe is generally as sample tube.Often pipe plus 16ul CD3-FITC,
12ul CD56-APC, after room temperature lucifuge is incubated 15 minutes, often pipe plus 2ml PBS, 1200 turns, centrifugation in 5 minutes, abandon supernatant.
3. fix：4 × fixative (eBioscience, #00-5521-00) is diluted to 1 ×, often pipe plus 2ml, concussion are mixed
Even, 4 degree of lucifuges were fixed after 50 minutes, and 1200 turns, 5 minutes centrifuge, and abandon supernatant.
4. rupture of membranes：10 × rupture of membranes agent (eBioscience, #00-8333-56) is diluted to 1 ×, per Guan Xianjia 1ml ruptures of membranes
Agent, concussion is mixed, and 1200 turns, 5 minutes centrifuge, and abandon supernatant.Then per Guan Zaijia 2ml rupture of membranes agent, concussion is mixed, and 4 degree of lucifuges are broken
Film 40 minutes, 1200 turns, 5 minutes centrifuge, and abandon supernatant.
5. often pipe plus 2ml streamings buffer solution (2.0%FBS and 2.0mM EDTA 1 × PBS solution), are mixed, 1200 turns, 5
Minute centrifugation, abandons supernatant.
6. control tube is divided：Sample cell is often managed plus 200ul streaming buffer solutions, is mixed, and draws 100ul to corresponding control tube.
7. the anti-human μ l of XIAP antibody (BD, #610763) 0.5 are added in sample tube, control tube adds the μ l of mouse IgG1 0.5
(Abcam, #ab18443).Room temperature lucifuge is incubated 15min
8. often pipe plus 2ml streaming buffer solutions, 1200 turns, centrifugation in 5 minutes, abandons supernatant.
10. often pipe adds the anti-mouse IgG1-PE (BD, #550083) of 1.8ul, and room temperature lucifuge is incubated 20 minutes
Often pipe plus 2ml streaming buffer solutions, 1200 turns, centrifugation in 5 minutes, abandon supernatant.
Often pipe adds 100ul streaming buffer solutions, upper machine testing.
S12, Western Blot confirm the expression of XIAP albumen
1. EDTA or liquaemin anti-freezing venous whole 2ml, Ficoll density-gradient centrifugation method the separation single core of peripheral blood are thin
Born of the same parents (PBMC).
2. the protein lysate (RIPA) of 100ul precoolings is added, is incubated 30 minutes on ice
3. 4 DEG C, 12000rpm centrifugations 15min takes supernatant
4. protein concentration (BCA kits) is determined
5. albumen boils sample：According to albumen：5 × loading buffer=4:After 1 mixing, 100 DEG C of water-baths 5 minutes
6. 12%SDS-PAGE glue is prepared
7. loading and electrophoresis：Sequentially add albumen Marker and well-done albumen sample to be analyzed (per hole 10ug).One
As concentrate glue 70V, separation gel 120V, electrophoresis until bromine phenol dye front under gel end, that is, stop.
8. transferring film：Pvdf membrane, constant current 300mA, 60 minutes.
9. close：Film is placed in etui, 5% skimmed milk power (TBST) is added, closes 2 on decolorization swinging table at room temperature
Hour.
10. it is directly added into primary antibody (the mouse anti-human XIAP, 1 prepared:1000, BD, #610763) (with 5% skimmed milk power/TBST
It is diluted to debita spissitudo), 4 DEG C of refrigerator overnights after being moved 2 hours on decolorization swinging table at room temperature.
Film is taken out, film is washed with TBST on decolorization swinging table at room temperature, totally three times, every time 10 minutes
Add secondary antibody (the rabbit-anti mouse, 1 prepared:2000, Abcam, #ab6728) (diluted with 5% skimmed milk power/TBST
To debita spissitudo), shaken 1 hour on decolorization swinging table at room temperature.
Film is taken out, film is washed with TBST on decolorization swinging table at room temperature, totally three times, every time 10 minutes
The liquid of giving out light (Thermo, #34577) prepared is dropped in the albumen side of film, the two is fully contacted
Fluorescence imaging analysis instrument program is set, and runs development.
Embodiment 2
The expression of XIAP albumen in S21, Flow cytometry NK and T cell
1. ethylenediamine tetra-acetic acid (Ethylenediamine-tetraaceticacid, EDTA) or liquaemin (Heparin
Sodium) anti-freezing venous whole 2ml, Ficoll density-gradient centrifugation methods separating peripheral blood mononuclear cells (PBMC), and count,
It is 4.5 × 10 to adjust cell concentration6/ ml suspensions are standby.
2. each Guan Zhongjia 200ul diluting cells, this pipe is generally as sample tube.Often pipe plus 24ul CD3-FITC,
8ul CD56-APC, room temperature lucifuge be incubated 25 minutes after, often pipe plus 2ml streamings buffer solution (2.0%FBS and 2.0mM EDTA's
1 × PBS solution), 1200 turns, 5 minutes centrifuge, and abandon supernatant.
3. fix：4 × fixative (eBioscience, #00-5521-00) is diluted to 1 ×, often pipe plus 2ml, concussion are mixed
Even, 4 degree of lucifuges were fixed after 50 minutes, and 1200 turns, 5 minutes centrifuge, and abandon supernatant.
4. rupture of membranes：10 × rupture of membranes agent (eBioscience, #00-8333-56) is diluted to 1 ×, per Guan Xianjia 1ml ruptures of membranes
Agent, concussion is mixed, and 1200 turns, 5 minutes centrifuge, and abandon supernatant.Then per Guan Zaijia 2ml rupture of membranes agent, concussion is mixed, and 4 degree of lucifuges are broken
Film 40 minutes, 1200 turns, 5 minutes centrifuge, and abandon supernatant.
5. often pipe plus 2ml streaming buffer solutions, mixing, 1200 turns, 5 minutes centrifuge, and abandon supernatant.
6. control tube is divided：Sample cell is often managed plus 200ul streaming buffer solutions, is mixed, and draws 100ul to corresponding control tube.
7. the μ l of anti-human XIAP (BD, #610763) 0.7 are added in sample tube, control tube adds mouse IgG10.7 μ l (Abcam, #
ab18443).Room temperature lucifuge is incubated 25min
8. often pipe plus 2ml streaming buffer solutions, 1200 turns, centrifugation in 5 minutes, abandons supernatant.
10. often pipe adds the anti-mouse IgG1-PE (BD, #550083) of 2.2ul, and room temperature lucifuge is incubated 20 minutes
Often pipe plus 2ml streaming buffer solutions, 1200 turns, centrifugation in 5 minutes, abandon supernatant.
Often pipe adds 100ul streaming buffer solutions, upper machine testing.
S22, Western Blot confirm the same S12 of expression of XIAP albumen.
Embodiment 3
The expression of XIAP albumen in S31, Flow cytometry NK and T cell
1. ethylenediamine tetra-acetic acid (Ethylenediamine-tetraaceticacid, EDTA) or liquaemin (Heparin
Sodium) anti-freezing venous whole 2ml, Ficoll density-gradient centrifugation methods separating peripheral blood mononuclear cells (PBMC), and count,
It is 4.0 × 10 to adjust cell concentration6/ ml suspensions are standby.
2. each Guan Zhongjia 200ul diluting cells, this pipe is generally as sample tube.Often pipe plus 20ul CD3-FITC,
10ul CD56-APC, after room temperature lucifuge is incubated 20 minutes, often pipe plus 2ml PBS, 1200 turns, centrifugation in 5 minutes, abandon supernatant.
3. fix：4 × fixative (eBioscience, #00-5521-00) is diluted to 1 ×, often pipe plus 2ml, concussion are mixed
Even, 4 degree of lucifuges were fixed after 50 minutes, and 1200 turns, 5 minutes centrifuge, and abandon supernatant.
4. rupture of membranes：10 × rupture of membranes agent (eBioscience, #00-8333-56) is diluted to 1 ×, per Guan Xianjia 1ml ruptures of membranes
Agent, concussion is mixed, and 1200 turns, 5 minutes centrifuge, and abandon supernatant.Then per Guan Zaijia 2ml rupture of membranes agent, concussion is mixed, and 4 degree of lucifuges are broken
Film 40 minutes, 1200 turns, 5 minutes centrifuge, and abandon supernatant.
5. often pipe plus 2ml streamings buffer solution (2.0%FBS and 2.0mM EDTA 1 × PBS solution), are mixed, 1200 turns, 5
Minute centrifugation, abandons supernatant.
6. control tube is divided：Sample cell is often managed plus 200ul streaming buffer solutions, is mixed, and draws 100ul to corresponding control tube.
7. the anti-human μ l of XIAP antibody (BD, #610763) 0.67 are added in sample tube, control tube adds the μ l of mouse IgG1 0.67
(Abcam, #ab18443).Room temperature lucifuge is incubated 20 minutes
8. often pipe plus 2ml streaming buffer solutions, 1200 turns, centrifugation in 5 minutes, abandons supernatant.
10. often pipe adds the anti-mouse IgG1-PE (BD, #550083) of 2ul, and room temperature lucifuge is incubated 20 minutes
Often pipe plus 2ml streaming buffer solutions, 1200 turns, centrifugation in 5 minutes, abandon supernatant.
Often pipe adds 100ul streaming buffer solutions, upper machine testing.
S32, Western Blot confirm the expression of XIAP albumen
Same S12.
The XIAP protein expression levels of XLP-1 patient and normal person are detected with the method for embodiment 3, fluidic cell
Fig. 1 is shown in art detection, and Western Blot results are shown in Fig. 2.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
The present invention is described in detail with reference to foregoing embodiments for pipe, but it will be understood by those within the art that：Its
The technical scheme described in foregoing embodiments can still be modified, or to which part or all technical characteristic
Carry out equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention skill
The scope of art scheme.
Claims (10)
1. a kind of chain lymphoproliferative syndrome diagnostic kits of X-, it is characterised in that the kit includes：
Anti-human CD3, CD56 streaming fluorescence antibody, anti-human XIAP antibody and its isotype control Ab and the anti-human XIAP
The corresponding Western Blot secondary antibodies of antibody and streaming fluorescence secondary antibody.
2. kit according to claim 1, it is characterised in that the anti-human CD3, CD56, streaming fluorescence secondary antibody institute band
Some fluorescence labelings are followed successively by FITC, APC, PE.
3. kit according to claim 1 or 2, it is characterised in that also include anti-coagulants, cell in the kit solid
Determine the one or more in agent, the agent of cell rupture of membranes, buffer solution, protein lysate.
4. kit according to claim 3, it is characterised in that the formula of the streaming buffer solution is：Containing 1.8%~
2.2%FBS and 1.8mM~2.2mM EDTA 1 × PBS solution.
5. the kit described in any one of Claims 1 to 4 is preparing the product of the diagnosis chain lymphoproliferative syndromes of X-
In application.
6. the side of XIAP protein abundances in the kit detection T and NK cells described in a kind of 1~4 any one of usage right requirement
Method, it is characterised in that including：
1) separates PMNC in sample to be checked and and counts adjustment cell concentration；
2) is to step 1) in cell in add anti-human CD3, CD56 streaming fluorescence antibody simultaneously and be incubated；
3) is centrifuged, and precipitation is incubated altogether with cell fixative；
4) is centrifuged, and precipitation is incubated altogether with the agent of cell rupture of membranes；
5) is centrifuged, and is washed after precipitation and is resuspended with streaming buffer solution；
6) sample after resuspension is divided into two groups by, is separately added into anti-human XIAP antibody and its isotype control Ab and is incubated；
7) is centrifuged, and is washed after precipitation and is resuspended with streaming buffer solution；
8), which is often managed, adds the corresponding streaming fluorescence secondary antibody of anti-human XIAP antibody and is incubated；
9) is centrifuged, and is washed after precipitation and is resuspended with streaming buffer solution, and is detected with flow cytometer；
And/or；
A) separates the PMNC in sample to be checked and collects albumen with protein lysate cracking；
B) albumen is carried out Western by using the anti-human XIAP antibody and the corresponding secondary antibody of the anti-human XIAP antibody
Blot is tested, and reads the gray value or fluorescent value of gained band；
Wherein step 1)~9) with step a)~b) do not have sequencing.
7. method according to claim 6, it is characterised in that in step 1) in, the cell concentration is 3.5~4.5 ×
106/ml。
8. method according to claim 6, it is characterised in that in step 2) in, incubation conditions are：
Room temperature lucifuge is incubated 15~25min.
9. method according to claim 6, it is characterised in that in step 2) in, the concentration difference of CD3, CD56 antibody
For 8~12 μ l/100 μ l, 4~6 μ l/100 μ l；
It is preferred that, in step 6) in, the antibody concentration of anti-human XIAP antibody and its isotype control Ab is 0.5~0.7 μ l/
100μl；
It is preferred that, in step 8) in, the antibody concentration of the corresponding streaming fluorescence secondary antibody of anti-human XIAP antibody is 1.8~2.2 μ l/
100μl。
10. method according to claim 6, it is characterised in that in step b), anti-human XIAP antibody presses 0.5~1.5：
1000 additions, shake be incubated 2~6 DEG C of 8~16h of incubation after 1~3h at room temperature.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710437610.1A CN107091924A (en) | 2017-06-12 | 2017-06-12 | The chain lymphoproliferative syndrome diagnostic kits of X and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710437610.1A CN107091924A (en) | 2017-06-12 | 2017-06-12 | The chain lymphoproliferative syndrome diagnostic kits of X and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107091924A true CN107091924A (en) | 2017-08-25 |
Family
ID=59639287
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710437610.1A Pending CN107091924A (en) | 2017-06-12 | 2017-06-12 | The chain lymphoproliferative syndrome diagnostic kits of X and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107091924A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107576790A (en) * | 2017-11-03 | 2018-01-12 | 深圳市巴德生物科技有限公司 | A kind of drying human lymphocyte CD4 surface antigen quality-control products and preparation method thereof |
| CN108956573A (en) * | 2018-08-02 | 2018-12-07 | 重庆医科大学附属儿童医院 | It is a kind of detect XIAP albumen kit and its application |
| CN111527406A (en) * | 2018-12-01 | 2020-08-11 | 铭道创新（北京）医疗技术有限公司 | Preparation method of lymphocyte sample for flow cytometry analysis |
-
2017
- 2017-06-12 CN CN201710437610.1A patent/CN107091924A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| J.SAYOS ET AL: "The X-linked lymphoproliferative-disease gene product SAP regulates signals induced through the co-receptor SLAM", 《NATURE》 * |
| KENTARO SHINOZAKI ET AL，: "Activation-dependent Tcell expression of the X-linked lymphoproliferative disease gene product SLAM-associated protein and its assessment for patient detection", 《INTERNATIONAL IMMUNOLOGY》 * |
| REBECCA A.MARSH: "Using flow cytometry to screenpatients for X-linked lymphoproliferative disease due to SAP deficiency and XIAP deficiency", 《JOURNAL OF IMMUNOLOGICAL METHODS》 * |
| REBECCAA.MARSH ET AL: "A Rapid Flow Cytometric Screening Test for X-Linked Lymphoproliferative Disease due to XIAP Deficiency", 《CLINICAL CYTOMETRY SOCIETY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107576790A (en) * | 2017-11-03 | 2018-01-12 | 深圳市巴德生物科技有限公司 | A kind of drying human lymphocyte CD4 surface antigen quality-control products and preparation method thereof |
| CN108956573A (en) * | 2018-08-02 | 2018-12-07 | 重庆医科大学附属儿童医院 | It is a kind of detect XIAP albumen kit and its application |
| CN111527406A (en) * | 2018-12-01 | 2020-08-11 | 铭道创新（北京）医疗技术有限公司 | Preparation method of lymphocyte sample for flow cytometry analysis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11199549B2 (en) | MEl'hods and means for diagnosing spondylarthritis using autoantibody markers | |
| Ott et al. | Clinical usefulness of microarray‐based IgE detection in children with suspected food allergy | |
| JP5946218B2 (en) | Assay for JC virus antibody | |
| CN109142755A (en) | It is a kind of diagnose early stage esophageal squamous cell carcinoma four kinds of autoantibody combined detection kits and application | |
| CN107091924A (en) | The chain lymphoproliferative syndrome diagnostic kits of X and its application | |
| CN106680503B (en) | The screening and identification method and its application of a kind of ECH1 autoantibodies | |
| CN107045062B (en) | Detect colloidal gold immuno-chromatography test paper strip, the kit and preparation method thereof of human neutrophil genatinase associated lipocalin | |
| WO2024001044A1 (en) | Biomarker combination related to lung cancer, kit containing same, and use thereof | |
| US20210190780A1 (en) | Serum thymidine kinase 1 detection kit based on automatic chemiluminescence analyzer | |
| CN105911291A (en) | Urine trace protein detection kit and detection method thereof | |
| Favaloro | Appropriate laboratory assessment as a critical facet in the proper diagnosis and classification of von Willebrand disorder | |
| CN110133272A (en) | Method for the excretion body in astroglia source to be enriched with or detected from biological fluid | |
| CN107037219A (en) | The chain lymphoproliferative syndrome diagnostic kits of X and its application | |
| CN111349683A (en) | Application of basophils of granulocyte group as allergic disease diagnosis marker | |
| CN104272113A (en) | Method for characterising plasma cell associated diseases | |
| JP7220474B2 (en) | A Rapid, On-Demand Heparin-Induced Thrombocytopenia Functional Assay | |
| Yi et al. | COVID-19: advance in laboratory diagnostic strategy and technology | |
| CN106680515A (en) | Polymolecular marker composition used for lung cancer diagnosis | |
| KR100495241B1 (en) | A kit for diagnosing bronchial asthma and chronic rhinitis comprising mammal cytokeratin 18 protein | |
| EP3835308A1 (en) | Synthetic peptide for detecting hiv-1 | |
| WO2017204295A1 (en) | Gastrointestinal cancer determination method | |
| CN102959398B (en) | Marker containing HPaR as active ingredient for diagnosing lung cancer | |
| CN113552338A (en) | Allergen specificity IgE antibody detection kit and preparation method thereof | |
| CN109804234B (en) | Blood unit test kit | |
| AU2011215826B2 (en) | Compositions and methods for predicting cardiovascular events |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170825 |
|
| RJ01 | Rejection of invention patent application after publication |