CN109270265A - The lethal immune cell function assessment kit of human peripheral blood and appraisal procedure - Google Patents
The lethal immune cell function assessment kit of human peripheral blood and appraisal procedure Download PDFInfo
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Abstract
The present invention provides a kind of lethal immune cell function assessment kit of human peripheral blood and appraisal procedure.The lethal immune cell function assessment kit of human peripheral blood of the invention includes: anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody, anti-human CD94 antibody, anti-human NKP30 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ anti-δ, anti-human 2 antibody of V δ, and the above antibody carries fluorescein label.The lethal immune cell function assessment kit of human peripheral blood of the invention can be used for carrying out comprehensive assessment to the immune function of immunocyte lethal in human peripheral blood, easy to use, safe.
Description
Technical field
The present invention relates to medical fields more particularly to a kind of lethal immune cell function of human peripheral blood to assess kit
And appraisal procedure.
Background technique
Lymphocyte (lymphocyte) is one kind of leucocyte, is the smallest leucocyte of volume, is generated by lymphoid organ,
It is the important cells ingredient of immune response function.Lymphocyte is a kind of cell line with Immune discrimination function, by it
The difference that migration, surface molecular and function occurs, can be divided into T lymphocyte (also known as T cell), bone-marrow-derived lymphocyte (also known as B cell)
With natural kill (NK) cell.T cell and B cell are all Antigen-specific lymphocytes, their initial source be it is identical,
It both is from hematopoietic tissue.T lymphocyte is with blood circulation to thymus gland, maturation under the action ofs thymin etc., and B cell is in marrow
Middle differentiation and maturation.
After by antigenic stimulus, T lymphocyte is converted into lymphoblast, then is divided into sensitized T lymphocyte, participates in
Cellular immunity, immune function are mainly anti-intracellular infection, oncocyte and variant cell etc.；And bone-marrow-derived lymphocyte is first to be converted into
Plasmablast, then it is divided into thick liquid cell, it generates and immunoglobulin,exocrine (antibody), participation humoral immunity, function is to generate
Antibody, offers antigen and secretory cell intrinsic factor participates in immunological regulation；NK cell does not depend on antigenic stimulus and spontaneously plays
Cellulotoxic effect has the function of killing target cell.
Lethal immunocyte is can directly to play antitumor, anti-infectious immunocyte, mainly includes CD8+T thin
Born of the same parents, NK cell and gamma delta T cells three categories, the functional status variation of these immunocytes is for monitoring immunity of organisms, clinic
The validity of associated treatment and the influence etc. treated to immune function have highly important Clinical significance of MG.
Currently, the immune function of the lethal immunocyte of comprehensive assessment human body is not all capable of in clinical and third party testing agency
The method of energy.
Summary of the invention
The purpose of the present invention, which first consists in, provides a kind of lethal immune cell function assessment kit of human peripheral blood, energy
Flow cytometer is enough combined to be used to carry out comprehensive assessment, user to the immune function of immunocyte lethal in human peripheral blood
Just, safety.
The object of the invention is also to provide a kind of lethal immune cell function appraisal procedures of human peripheral blood, to human body
The immune function of lethal immunocyte carries out comprehensive assessment in peripheral blood.
In order to achieve the above object, present invention firstly provides a kind of lethal immune cell functions of human peripheral blood to assess reagent
Box, comprising: resist with fluorescein-labeled anti-CD3antibody, with fluorescein-labeled anti-human CD8 antibody, with fluorescein-labeled
Human CD 45 RA antibody, with fluorescein-labeled anti-human CCR7 antibody, with fluorescein-labeled anti-human CD28 antibody, band fluorescein mark
Note anti-human CD38 antibody, with fluorescein-labeled anti-human CD57 antibody, with fluorescein-labeled anti-human HLA-DR antibodies, with glimmering
Light element label anti-human PD-1 antibody, with fluorescein-labeled anti-human CD56 antibody, with fluorescein-labeled anti-human CD94 antibody,
With fluorescein-labeled anti-human NKP30 antibody, with fluorescein-labeled anti-human NKP46 antibody, with fluorescein-labeled anti-human
NKG2D antibody, with fluorescein-labeled anti-human KIR antibody, with fluorescein-labeled anti-human γ anti-δ, with fluorescein-labeled
Anti-human 2 antibody of V δ.
Optionally, the anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human
CD28 antibody, anti-human CD38 antibody, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody,
Anti-human CD94 antibody, anti-human NKP30 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ δ are anti-
Body, anti-human 2 antibody of V δ are monoclonal antibody.
Optionally, the anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human
CD28 antibody, anti-human CD38 antibody, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody,
Anti-human CD94 antibody, anti-human NKP30 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ δ are anti-
Any one in body, anti-human 2 antibody of V δ is pulvis or liquid preparation.
Optionally, the anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human
CD28 antibody, anti-human CD38 antibody, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody,
Anti-human CD94 antibody, anti-human NKP30 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ δ are anti-
Body, anti-human 2 antibody of V δ are all made of the encapsulation of antibody reagent pipe, and the antibody reagent pipe is light transmittance in 10% brown plastics below
Pipe.
Preferably, the light transmittance of the antibody reagent pipe is zero, i.e., completely opaque.
Optionally, when the antibody contained in the antibody reagent pipe is liquid preparation, antibody in the antibody reagent pipe
Volume is 0.5ml-5ml.
Optionally, the anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human
CD28 antibody, anti-human CD38 antibody, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody,
Anti-human CD94 antibody, anti-human NKP30 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ δ are anti-
Body, anti-human 2 antibody of V δ are liquid preparation.
Optionally, the anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human
CD28 antibody, anti-human CD38 antibody, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody,
Anti-human CD94 antibody, anti-human NKP30 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ δ are anti-
At least one of body, anti-human 2 antibody of V δ are pulvis, and the lethal immune cell function assessment kit of human peripheral blood is also
Including phosphate buffer.
Optionally, the anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human
CD28 antibody, anti-human CD38 antibody, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody,
Anti-human CD94 antibody, anti-human NKP30 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ δ are anti-
Body, anti-human 2 antibody of V δ fluorescein label selected from PerCP-Cy5.5, BV510, FITC, AF647, PE-Cy7, BV421, PE,
AF647、APC-H7、BB515。
The present invention also provides a kind of lethal immune cell function appraisal procedures of human peripheral blood, include the following steps:
Step 1 provides human peripheral blood as described above lethal immune cell function assessment kit；
Peripheral blood mononuclear cells is extracted from 2ml-10ml human peripheral blood；
Step 2, when all antibody in the human peripheral blood lethal immune cell function assessment kit are liquid
When body preparation, takes 1 μ l-5 μ l respectively from all antibody, all mixed with the peripheral blood mononuclear cells, at 2 DEG C -6
It is protected from light incubation 10 minutes to 30 minutes under the conditions of DEG C, obtains cell detection sample；
When at least a kind of antibody is pulvis in the lethal immune cell function assessment kit of the human peripheral blood,
Pulvis is dissolved as by antibody-solutions using phosphate buffer first, takes 1 μ l- respectively from the antibody of all liq form later
5 μ l, are all mixed with the peripheral blood mononuclear cells, and incubation 10 minutes to 30 minutes is protected from light under the conditions of 2 DEG C -6 DEG C,
Obtain cell detection sample；
The concentration of antibody is 0.1-2mg/ml in the liquid preparation；The pulvis obtains after mixing with phosphate buffer
Antibody-solutions in antibody concentration be 0.1-2mg/ml；
Step 3 tests and analyzes the cell detection sample using flow cytometer.
Beneficial effects of the present invention:
The lethal immune cell function assessment kit of human peripheral blood of the invention can be used in conjunction with flow cytometer
Comprehensive assessment is carried out to the immune function of immunocyte lethal in human peripheral blood, can be used for human immunity health control evaluation
And in the panimmunities functional assessment project such as immune function assessment of infections relating patient.Kit user of the invention
Just, and it is safe to the human body.The lethal immune cell function appraisal procedure of human peripheral blood of the invention can be to human peripheral blood
In lethal immunocyte immune function carry out comprehensive assessment, operating procedure is simple, safe operation process.
Interior first time establishes the immune function to immunocyte lethal in human peripheral blood to the present invention at the international level
The method for carrying out comprehensive assessment is carried out using function of the panimmunity functional parameter to immunocyte lethal in human peripheral blood
Comprehensive assessment prompts detected person to use when the immune function for detecting lethal immunocyte in human peripheral blood is too low
The intervention means such as immunization therapy promote the immune function of itself, avoid the occurrence of various diseases relevant to immune function,
Effectively promote the health status of human body.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of the scope of the invention.
Fig. 1 is illustrated using the lethal immune cell function appraisal procedure of human peripheral blood of the invention to human peripheral blood
The partial results that middle CD8+T cell mass obtains after being tested and analyzed.
Specific embodiment
Term as used herein:
" by ... preparation " it is synonymous with "comprising".Term "comprising" used herein, " comprising ", " having ", " containing "
Or its any other deformation, it is intended that cover non-exclusionism includes.For example, composition, step, method comprising listed elements,
Product or device are not necessarily limited to those elements, but may include not expressly listed other elements or such composition, step
Suddenly, method, product or the intrinsic element of device.
Conjunction " by ... form " exclude any element that do not point out, step or component.If in claim,
This phrase will make claim closed, so that it is not included the material in addition to the material of those descriptions, but relative
Except customary impurities.When phrase " by ... form " be rather than immediately following theme in the clause that appears in claim main body after
When, only it is limited to element described in the clause；Other elements be not excluded the claim as a whole it
Outside.
Equivalent, concentration or other values or parameter are excellent with range, preferred scope or a series of upper limit preferred values and lower limit
When the Range Representation that choosing value limits, this should be understood as specifically disclosing by any range limit or preferred value and any range
Any pairing of lower limit or preferred value is formed by all ranges, regardless of whether the range separately discloses.For example, when open
When range " 1~5 ", described range should be interpreted as including range " 1~4 ", " 1~3 ", " 1~2 ", " 1~2 and 4~
5 ", " 1~3 and 5 " etc..When numberical range is described herein, unless otherwise stated, otherwise the range is intended to include its end
Value and all integers and score in the range.
" mass parts " refer to the basic measurement unit for indicating the mass ratio relationship of multiple components, and 1 part can indicate arbitrary list
Position quality, can such as be expressed as 1g, may also indicate that 2.689g etc..If we say that the mass parts of component A are a parts, the matter of B component
Measuring part is b parts, then it represents that the quality of component A and the mass ratio a:b of B component.Alternatively, indicating that the quality of component A is aK, B group
The quality divided is bK (K is arbitrary number, indicates multiplying factor).It can not misread, unlike mass fraction, all components
The sum of mass parts be not limited to 100 parts of limitation.
"and/or" is used to indicate that one of illustrated situation or both may to occur, for example, A and/or B includes (A
And B) and (A or B)；
In addition, indefinite article "an" before element of the present invention or component and "one" quantitative requirement to element or component
(i.e. frequency of occurrence) unrestriction.Therefore "one" or "an" should be read as including one or at least one, and odd number
The element or component of form also include plural form, unless the obvious purport of the quantity refers to singular.
Present invention firstly provides a kind of lethal immune cell functions of human peripheral blood to assess kit, comprising: band fluorescence
Element label anti-CD3antibody, with fluorescein-labeled anti-human CD8 antibody, with fluorescein-labeled anti-human CD 45 RA antibody, band
Fluorescein-labeled anti-human CCR7 antibody resists with fluorescein-labeled anti-human CD28 antibody, with fluorescein-labeled anti-human CD38
Body, with fluorescein-labeled anti-human CD57 antibody, with fluorescein-labeled anti-human HLA-DR antibodies, with fluorescein-labeled anti-human
PD-1 antibody, with fluorescein-labeled anti-human CD56 antibody, with fluorescein-labeled anti-human CD94 antibody, with fluorescein-labeled
Anti-human NKP30 antibody, with fluorescein-labeled anti-human NKP46 antibody, with fluorescein-labeled anti-human NKG2D antibody, band fluorescence
Element label anti-human KIR antibody, with fluorescein-labeled anti-human γ anti-δ, with fluorescein-labeled anti-human 2 antibody of V δ.
The lethal immune cell function assessment kit of human peripheral blood of the invention can be used in conjunction with flow cytometer
Lethal immunocyte in human peripheral blood is tested and analyzed, 32 immune functions are obtained, described 32 immune
Functional parameter includes:
Initialize ratio, the terminal differentiation CD8+T cell (Terminal of CD8+T cell (Naive CD8+T cells)
Differentiation CD8+T cells) ratio, center remember CD8+T cell (Central memory CD8+T
Cells ratio, the ratio of responsiveness memory CD8+T cell (Effective memory CD8+T cells), failure CD8+)
The ratio of T cell (Exhaustion of CD8+T cells), function interdiction CD8+T cell (Inhibitory CD8+T
Cells ratio, the ratio of functionality CD8+T cell (Potential functional CD8+T cells), total memory)
Property CD8+T cell (Total memory CD8+T cells) ratio, return nest Memorability CD8+T cell
The ratio of (Homingmemory CD8+T cells), terminal aging CD8+T cell (Terminally senescent CD8+T
Cells ratio), the ratio of NK T cell (NKT cells), the ratio of NK cell (NK cells), prematurity NK cell
The ratio of (Immature NK cells), the ratio of maturation NK cell (Mature NK cells), prematurity/maturation NK ratio
Value, the ratio of early function blocking property NK cell (Early inhibition of NK cells), advanced stage function interdiction NK
The ratio of cell (Late inhibitory NK cells), the ratio of activated NK (Activated NK cells), often
The ratio of the lethal NK cell (Conventional killer NK cells) of rule, virus infection specific killing NK cell
Ratio, the ratio of gamma delta T cells (gamma delta T cells), the ratio of 1 positive T cell of V δ, the ratio of 2 positive T cell of V δ, V δ 1+/
The ratio of V δ 2+, the ratio of 2 positive T cell of functionality V δ, the ratio of function inhibitio V δ 2+T cell, conventional lethal V δ 2
The ratio of positive T cell, the ratio of 2 positive T cell of virus infection specific killing V δ, 1 positive T cell of functionality V δ ratio
Example, the ratio of 1 positive T cell of function inhibitio V δ, ratio, the virus infection of conventional lethal 1 positive T cell of V δ are specifically killed
The ratio of 1 positive T cell of wound property V δ.
The ratio for initializing CD8+T cell is that CD8+T cell number and CD8+T are initialized in the human peripheral blood of same volume
The ratio of total number of cells.Initializing CD8+T cell is one of important cells group of cellular immunotherapy, represents CD8+T cell
Reserve capabillity.The term of reference for initializing the ratio of CD8+T cell is 35.4%~53.2%.
The ratio of terminal differentiation CD8+T cell be same volume human peripheral blood in terminal differentiation CD8+T cell number with
The ratio of CD8+T total number of cells.Terminal differentiation CD8+T cell is the cd8 cell without differentiation more new function.Terminal differentiation CD8+T
The term of reference of the ratio of cell is 21.2%~40.3%.
Center remember CD8+T cell ratio be same volume human peripheral blood in center remember CD8+T cell number with
The ratio of CD8+T total number of cells.Center memory CD8+T cell is adjusted to virus, antigen subinfection or the sensitive cell of stimulation again
Dynamic tachysynthesis response can stablize proliferation in secondary lymphatic organ and be divided into effector cell under antigenic stimulus.Center memory
The term of reference of the ratio of CD8+T cell is 4.8%~6.1%.
The ratio that responsiveness remembers CD8+T cell is that responsiveness remembers CD8+T cell in the human peripheral blood of same volume
Several ratios with CD8+T total number of cells.Responsiveness, which remembers CD8+T cell, has anti-infective, anti-tumor function, releasable a variety of anti-
Virocyte cerebroysin and to mutation cell have killing ability.The term of reference of ratio of responsiveness memory CD8+T cell is
33.1%~56.3%.
The ratio of failure CD8+T cell is failure CD8+T cell number and CD8+T cell in the human peripheral blood of same volume
The ratio of sum.Failure CD8+T cell number is associated with a variety of viral infections, if failure CD8+T cytosis, shows
CD8+T cell is anti-infective, antitumor and removing target cell ability reduces.The term of reference of the ratio of failure CD8+T cell is
30%~55%.
The ratio of function interdiction CD8+T cell is function interdiction CD8+T cell in the human peripheral blood of same volume
Several ratios with CD8+T total number of cells.Function interdiction CD8+T is the CD8+T cell of function inhibitio, especially with it is antitumor
Ability is related, is maintaining body's immunity stable state and is establishing in immune tolerance to play an important role, can be used as
The immune detection index of autoimmune disease, AIDS and immune deficiency, ratio increase, then immunological effect effect is pressed down
System.The term of reference of the ratio of function interdiction CD8+T cell is 24.4 ± 7.6%.
The ratio of functional CD8+T cell is functionality CD8+T cell number and CD8+T in the human peripheral blood of same volume
The ratio of total number of cells.Functional CD8+T cell is to kill sick germ infection with the active CD8+T cell of immune function
Target cell and tumour cell, important effector function is played in antitumor, anti-infective and allograft rejection.Function
Property CD8+T cell ratio term of reference be 52.5%-70%.
The ratio of total Memorability CD8+T cell is Memorability CD8+T cell total in the human peripheral blood of same volume
Several ratios with CD8+T total number of cells.Total Memorability CD8+T cell is can be stimulated again by antigen and quickly activate and play straight
Connect the subgroup of killing tumor cell or germ infection cell.The term of reference of the ratio of total Memorability CD8+T cell is
12.9%~25.8%.
The ratio for returning nest Memorability CD8+T cell is that nest Memorability CD8+T is returned in the human peripheral blood of same volume
The ratio of cell number and total Memorability CD8+T cell number.Returning nest Memorability CD8+T cell is that can move to second level leaching
Bar organ receives antigenic stimulus and re-activation is effector cell, is the important indicator of the treatment and prognosis of HIV infection patient.It returns
The term of reference of the ratio of nest Memorability CD8+T cell is 73% ± 10.3%.
The ratio of terminal aging CD8+T cell be same volume human peripheral blood in terminal aging CD8+T cell number with
The ratio of CD8+T total number of cells.Terminal aging CD8+T cell has the characteristics that low proliferation, easy apoptosis, and ratio raising is exempted from it
The decline of epidemic disease effect is related.The term of reference of the ratio of terminal aging CD8+T cell is 5.45%~20.48%.
The ratio of NK T cell is the ratio of NK T cell number and total number of lymphocytes in the human peripheral blood of same volume.
NK T cell is with the active one kind NK cell of direct killing.The term of reference of the ratio of NK T cell be 11.71%~
33.1%.
The ratio of NK cell is the ratio of NK cell number and total number of lymphocytes in the human peripheral blood of same volume.NK is thin
Born of the same parents are the important immunocytes of human body, with antitumor, viral infection resisting and immunological regulation have it is close contact, also participate in super quick
The generation of reaction and autoimmune disease can identify target cell, killing medium.The term of reference of the ratio of NK cell is
6.26%~37.16%.
The ratio of prematurity NK cell is prematurity NK cell number and NK total number of cells in the human peripheral blood of same volume
Ratio.Prematurity NK cell is a subgroup of NK cell, and mature NK cell ratio, to the killing ability of target cell compared with
It is low, but most can efficiently generate cell factor such as IFN-gamma.The term of reference of the ratio of prematurity NK cell be 5.6~
27.8%.
The ratio of mature NK cell is the ratio of maturation NK cell number and NK total number of cells in the human peripheral blood of same volume
Value.Mature NK cell has powerful killing activity from immature NK cell differentiation, can direct killing rake cell.Mature NK
The term of reference of the ratio of cell is 43~82%.
Prematurity/maturation NK ratio is the ratio of prematurity NK cell number and maturation NK cell number.The ratio is with the state of an illness
Development and curative effect have close ties, and ratio increases generally related to unsatisfactory curative effect or disease progression.The term of reference of the ratio is
0.068~0.72.
The ratio of early function blocking property NK cell is that early function blocking property NK is thin in the human peripheral blood of same volume
The ratio of born of the same parents' number and NK total number of cells.Early function blocking property NK cell can reinforce the killing ability of NK cell.Early function
The term of reference of the ratio of blocking property NK cell is 36.3%~52.7%.
The ratio of advanced stage function interdiction NK cell is that the human peripheral blood middle and advanced stage function interdiction NK of same volume is thin
The ratio of born of the same parents' number and NK total number of cells.Advanced stage function interdiction NK cell is modulability NK cell.Advanced stage, function interdiction NK was thin
The term of reference of the ratio of born of the same parents is 22%~29%.
The ratio of activated NK is the ratio of activated NK number and NK total number of cells in the human peripheral blood of same volume
Value.Activated NK is that have antitumor, antiviral functions functional NK cells.The term of reference of the ratio of activated NK
It is 25%~53%.
The ratio of conventional lethal NK cell is lethal NK cell number conventional in the human peripheral blood of same volume
With the ratio of NK total number of cells.Conventional lethal NK cell is the effector cell of antineoplastic immune, mainly to tumour cell and
The cell of germ infection plays killing or scavenging effect.The term of reference of the ratio of conventional lethal NK cell is 38.5%~
64%.
The ratio of virus infection specific killing NK cell is that virus infection is specifically killed in the human peripheral blood of same volume
The ratio of wound property NK cell number and NK total number of cells.Virus infection specific killing NK cell is directly related with virus infection, disease
The ratio of poison infection specific killing NK cell reduces related to virus infection, tumour progression etc..Virus infection specific killing
The term of reference of the ratio of NK cell is 9.93%~80.85%.
The ratio of gamma delta T cells is the ratio of gamma delta T cells number and T cell sum in the human peripheral blood of same volume.γδ
T cell is the first line of defence for participating in immunity of organism, has certain non-specific lethal effect, and anti-swollen with wide spectrum
Tumor, anti-infectious function.The term of reference of the ratio of gamma delta T cells is 0.5%~8.2%.
The ratio of 1 positive T cell of V δ is that 1 positive T cell number of V δ and gamma delta T cells are total in the human peripheral blood of same volume
Several ratio.1 positive T cell of V δ is the γ delta cell subgroup with immunosupress, regulatory function.The ratio of 1 positive T cell of V δ
Term of reference be 8.2%~24%.
The ratio of 2 positive T cell of V δ is that 2 positive T cell number of V δ and gamma delta T cells are total in the human peripheral blood of same volume
Several ratio.2 positive T cell of V δ is mainly to play antitumor, anti-infectious function gamma delta T cells subgroup.2 positive T cell of V δ
The term of reference of ratio is 46.8%~72%.
The ratio of V δ 1+/V δ 2+ is the ratio of 1 positive T cell number of V δ and 2 positive T cell number of V δ.The ratio and gamma delta T are thin
The functional status of born of the same parents' subgroup is directly related, and ratio increases the function reduction for generally showing gamma delta T cells subgroup.1 positive T cell of V δ
Several term of reference with the ratio of 2 positive T cell of V δ are 0.12~0.51.
The ratio of functional 2 positive T cell of V δ is 2 positive T cell number of functionality V δ in the human peripheral blood of same volume
With the ratio of gamma delta T cells sum.The ratio with antitumor, anti-infective function is directly related.The ratio of functional 2 positive T cell of V δ
The term of reference of example is 94.6%~99.8%.
The ratio of 2 positive T cell of function inhibitio V δ is that function inhibitio V δ 2 is positive in the human peripheral blood of same volume
The ratio of T cell number and 2 gamma delta T cells of V δ sum.2 positive T cell of function inhibitio V δ can control cell transition activation, swell
Ratio raising shows that anti-tumor function is suppressed in tumor patient.The term of reference of the ratio of function inhibitio V δ 2+T cell is
6.4%~38.9%.
The ratio of conventional lethal 2 positive T cell of V δ is lethal V δ 2 conventional in the human peripheral blood of same volume
The ratio of positive T cell number and 2 gamma delta T cells of V δ sum.Conventional lethal 2 positive T cell of V δ can play antitumor, anti-
Infection effect.The term of reference of the ratio of conventional lethal 2 positive T cell of V δ is 0~1%.
The ratio of 2 positive T cell of virus infection specific killing V δ is virus infection in the human peripheral blood of same volume
The ratio of 2 positive T cell number of specific killing V δ and 2 gamma delta T cells of V δ sum.2 positive T of virus infection specific killing V δ is thin
Born of the same parents are directly related with virus infection, and the ratio of 2 positive T cell of virus infection specific killing V δ reduces and virus infection, tumour
The correlations such as progress, and influence the activation of cell.The term of reference of the ratio of 2 positive T cell of virus infection specific killing V δ is 0
~0.8%.
The ratio of functional 1 positive T cell of V δ is in the human peripheral blood of same volume be same volume human peripheral
The ratio of functionality V δ 1 positive T cell and 1 gamma delta T cells of V δ sum in blood.Functional 1 positive T cell of V δ is with antitumor
The Vdelta1 cell mass of function.The term of reference of the ratio of functional 1 positive T cell of V δ is 94.6%~99.8%.
The ratio of 1 positive T cell of function inhibitio V δ is that function inhibitio V δ 1 is positive in the human peripheral blood of same volume
The ratio of T cell number and 1 gamma delta T cells of V δ sum.1 positive T cell of function inhibitio V δ has function inhibitio
Vdelta1 cell mass inhibits cell hyperactivity.The term of reference of the ratio of 1 positive T cell of function inhibitio V δ is 8.4%
~38.9%.
The ratio of conventional lethal 1 positive T cell of V δ is lethal V δ 1 conventional in the human peripheral blood of same volume
The ratio of positive T cell number and 1 gamma delta T cells of V δ sum.Conventional lethal 1 positive T cell of V δ be can play it is antitumor, anti-
The Vdelta1 cell mass of infection effect.The term of reference of the ratio of conventional lethal 1 positive T cell of V δ is 0~1%.
The ratio of 1 positive T cell of virus infection specific killing V δ is human peripheral in the human peripheral blood of same volume
The ratio of 1 positive T cell number of virus infection specific killing V δ and 1 gamma delta T cells of V δ sum in blood.Virus infection specific killing
Property V 1 positive T cell of δ it is directly related with virus infection, ratio reduce it is related to virus infection, tumour progression etc..Virus infection is special
The term of reference of the ratio of different lethal 1 positive T cell of V δ is 0~9.6%.
Specifically, the term of reference mentioned in the application refers to the normal range (NR) of Healthy People.
Above-mentioned 32 immune functions can reflect the immune function shape of the lethal immunocyte of human peripheral blood comprehensively
State, by analyzing above 32 immune functions, if it is determined that immunologic hypofunction (such as the functional cell of human body
Tail off, inhibitory cells tail off, failure cell increases), then prompt may need to carry out immunologic intervention, such as pass through drug, function
Can the method for food or biological therapy improve the immune function of body.
To obtain above-mentioned 32 immune functions, it is necessary first to extract peripheral blood mononuclear cells from human peripheral blood
(PBMC), taken respectively from all antibody of this kit later a certain amount of, all mixed with peripheral blood mononuclear cells,
It is incubated for, obtains cell detection sample, cell detection sample is detected using flow cytometer later, utilizes flow cytometer
After included analysis software or third-party analysis software are for statistical analysis to the analysis result of various immunocytes, in acquisition
State 32 immune functions.
Specifically, the cell sign of each immunocyte group are as follows:
Initialize CD8+T cell: CD3+CD8+CCR7+CD45RA+；
Terminal differentiation CD8+T cell: CD3+CD8+CCR7-CD45RA+；
Remember CD8+T cell: CD3+CD8+CCR7+CD45RA- in center；
Responsiveness remembers CD8+T cell: CD3+CD8+CCR7-CD45RA-；
Failure CD8+T cell: CD3+CD8+CD28-；
Function interdiction CD8+T cell: CD3+CD8+PD-1+；
Functional CD8+T cell: CD3+CD8+CD28+；
Terminal aging CD8+T cell: CD3+CD8+CD28-CD57+；
Total Memorability CD8+T cell: CD3+CD8+HLADR+；
Return nest Memorability CD8+T cell: CD3+CD8+HLADR+CD38+；
NKT cell: CD3+CD56+；
NK cell: CD3-CD56+；
Prematurity NK cell: CD3-CD56+bright；
Mature NK cell: CD3-CD56+dim；
Early function blocking property NK cell: CD3-CD56+CD94+KIR-；
Advanced stage function interdiction NK cell: CD3-CD56+CD94-KIR+；
Activated NK: CD3-CD56+NKG2D+；
Conventional lethal NK cell: CD3-CD56+NKP30+；
Virus infection specific killing NK cell: CD3-CD56+NKP46+；
Gamma delta T cells: CD3+ γ δ+；
2 positive T cell of V δ: CD3+ γ δ+V δ 2+；
Functional 2 positive T cell of V δ: CD3+ γ δ+V δ 2+NKG2D+；
2 positive T cell of inhibition V δ: CD3+ γ δ+V δ 2+PD1+；
Conventional lethal 2 positive T cell of V δ: CD3+ γ δ+V δ 2+P30+；
2 positive T cell of virus infection specific killing V δ: CD3+ γ δ+V δ 2+P46+；
Functional 1 positive T cell of V δ: CD3+ γ δ+V δ 1+NKG2D+；
1 positive T cell of inhibition V δ: CD3+ γ δ+V δ 1+PD1+；
Conventional lethal 1 positive T cell of V δ: CD3+ γ δ+V δ 1+P30+；
1 positive T cell of virus infection specific killing V δ: CD3+ γ δ+V δ 1+P46+；
Wherein, CD56+bright indicates that CD56 membrane protein molecule expression quantity is high, and CD56+dim indicates CD56 membrane protein molecule
Expression quantity is low.
In above-mentioned reaction result, antibody is subsequent+cell is represented to the reaction of the antibody as the positive, antibody is subsequent-and generation
Table cell is feminine gender to the reaction of the antibody, since combined reaction result of each cell subsets to all antibody is different,
Flow cytometer can be screened and be analyzed to each cell subsets according to different reaction results.
Specifically, the counting mode of 1 positive T cell of V δ is that the quantity of gamma delta T cells subtracts the quantity of 2 positive T cell of V δ.
Preferably, the anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human
CD28 antibody, anti-human CD38 antibody, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody,
Anti-human CD94 antibody, anti-human NKP30 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ δ are anti-
Body, anti-human 2 antibody of V δ are monoclonal antibody.This is because the specificity of monoclonal antibody is good, fluorescence intensity after dyeing with
Antigenic expression is linear relationship, and the difference between monoclonal antibody difference production batch is smaller.Although polyclonal antibody
It can produce stronger signal, but poor specificity, fluorescence intensity and antigen levels are not linear relationships after dyeing, and more grams
It is widely different between grand antibody difference production batch, thus during flow cytometer detection using monoclonal antibody can guarantee it is higher
Detection accuracy.
Optionally, the anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human
CD28 antibody, anti-human CD38 antibody, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody,
Anti-human CD94 antibody, anti-human NKP30 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ δ are anti-
Any one in body, anti-human 2 antibody of V δ is pulvis or liquid preparation.
The pulvis needs to be added appropriate phosphate buffer (PBS) before use and is dissolved, and the liquid preparation is not
It needs to dissolve, directly use.The pulvis can be placed under the conditions of 4 DEG C with liquid preparation and save, and pulvis can also be placed in -20 DEG C
Under the conditions of save.
Specifically, in the liquid preparation antibody concentration be 0.1-2mg/ml (such as 0.1mg/ml, 0.5mg/ml,
1mg/ml、1.5mg/ml、2mg/ml)。
Optionally, the ingredient of the liquid preparation includes antibody and phosphate buffer.
Specifically, the pulvis is in use, be mixed to get antibody-solutions for the pulvis and phosphate buffer, it is described anti-
The concentration of antibody is 0.1-2mg/ml (such as 0.1mg/ml, 0.5mg/ml, 1mg/ml, 1.5mg/ml, 2mg/ml) in liquid solution.
Optionally, the anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human
CD28 antibody, anti-human CD38 antibody, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody,
Anti-human CD94 antibody, anti-human NKP30 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ δ are anti-
Body, anti-human 2 antibody of V δ are all made of the encapsulation of antibody reagent pipe.
Preferably, the antibody reagent pipe is light transmittance in 10% brown plastic tube below；It is furthermore preferred that the antibody
The light transmittance of Reagent Tube is zero, i.e., completely opaque.
Optionally, when the antibody contained in the antibody reagent pipe is liquid preparation, antibody in the antibody reagent pipe
Volume is 0.5ml-5ml, such as 0.5ml, 1ml, 2ml, 3ml, 4ml, 5ml etc., the use time of the antibody in the antibody reagent pipe
Number is 50-500 times.
Optionally, the anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human
CD28 antibody, anti-human CD38 antibody, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody,
Anti-human CD94 antibody, anti-human NKP30 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ δ are anti-
Body, anti-human 2 antibody of V δ are liquid preparation, and liquid preparation uses more convenient dissolution before capable of saving pulvis use
Step, and it is avoided that caused detection error problem when pulvis dissolution is uneven.
, it is understood that although the antibody of powder form uses comparatively laborious, the step of increasing dissolution is needed, but
It is for the antibody compared to liquid forms, the shelf-life of the antibody of powder form is longer.
When the anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human CD28 are anti-
It is body, anti-human CD38 antibody, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody, anti-human
CD94 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ anti-δ, resists anti-human NKP30 antibody
When at least one of 2 antibody of people V δ is pulvis, the lethal immune cell function assessment kit of human peripheral blood is also wrapped
Phosphate buffer (PBS) is included, the phosphate buffer can be placed in reagent bottle.By providing phosphate buffer, to make
User has saved the time for preparing phosphate buffer, keeps the use of antibody more convenient.
Optionally, the anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human
CD28 antibody, anti-human CD38 antibody, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody,
Anti-human CD94 antibody, anti-human NKP30 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ δ are anti-
Body, anti-human 2 antibody of V δ fluorescein label selected from PerCP-Cy5.5, BV510, FITC, AF647, PE-Cy7, BV421, PE,
AF647、APC-H7、BB515。
The present invention also provides a kind of lethal immune cell function appraisal procedures of human peripheral blood, include the following steps:
Step 1 provides human peripheral blood described above lethal immune cell function assessment kit；
Peripheral blood mononuclear cells is extracted from 2ml-10ml (such as 2ml, 4ml, 6ml, 8ml, 10ml) human peripheral blood
(PBMC)。
Since the particular content of the lethal immune cell function assessment kit of the human peripheral blood has been carried out above
Detailed description, therefore details are not described herein again.
Optionally, the method that peripheral blood mononuclear cells (PBMC) is extracted from human peripheral blood includes the following steps:
1) whole blood sample of 2ml-10ml human peripheral blood is added in centrifuge tube；
2) isometric phosphate buffer (PBS) is added in whole blood sample, PBS+ whole blood sample is fluctuated mixed
Uniformly, sample diluent is obtained；
3) new 15ml centrifuge tube is taken, FICOLL liquid and sample diluent is added, it is usually dilute according to FICOLL liquid and sample
Release the ratio addition of the volume ratio 1:1 of liquid；
4) centrifuge tube is tilted 45 °, sample diluent is carefully added into centrifuge tube with 1000 μ L pipette tips are adherent；
5) 20min is centrifuged with 2000rpm revolving speed；
6) a little upper layer yellow serum is sopped up using 1000 μ l pipette tips, rear white confluent monolayer cells to the new centrifuge tube of suction (cannot
It is drawn onto red blood cell)；
7) isometric phosphate buffer (PBS) is taken to be centrifuged 15min with 1500rpm revolving speed；
8) PBS is outwelled, centrifuge tube is upside down on paper and is absorbed water；
9) 3ml erythrocyte cracked liquid is added in centrifuge tube, cracks 5min after blowing and beating uniformly, during which turns upside down frequently mixed
It is even；
10) 5min is centrifuged with 1000rpm revolving speed；
11) erythrocyte cracked liquid is outwelled, centrifuge tube is upside down on paper and is absorbed water；
12) 5ml PBS is added, 5min is centrifuged with 1000rpm revolving speed；
13) PBS is outwelled, white precipitate is peripheral blood mononuclear cells (PBMC).
Step 2, when all antibody in the human peripheral blood lethal immune cell function assessment kit are liquid
When body preparation, take 1 μ l-5 μ l (such as 1 μ l, 2 μ l, 3 μ l, 4 μ l, 5 μ l) respectively from all antibody, all with the peripheral blood
Mononuclearcell is mixed, and is protected from light incubation under the conditions of 2 DEG C -6 DEG C (such as 2 DEG C, 3 DEG C, 4 DEG C, 5 DEG C, 6 DEG C, preferably 4 DEG C)
10 minutes to 30 minutes (such as 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes), obtain cell detection sample；
When at least a kind of antibody is pulvis in the lethal immune cell function assessment kit of the human peripheral blood,
Pulvis is dissolved as by antibody-solutions using appropriate phosphate buffer (PBS) first, later from the antibody of all liq form
1 μ l-5 μ l (such as 1 μ l, 2 μ l, 3 μ l, 4 μ l, 5 μ l) is taken respectively, is all mixed with the peripheral blood mononuclear cells, 2
Incubation 10 minutes to 30 minutes (such as 10 is protected from light under the conditions of DEG C -6 DEG C (such as 2 DEG C, 3 DEG C, 4 DEG C, 5 DEG C, 6 DEG C, preferably 4 DEG C)
Minute, 15 minutes, 20 minutes, 25 minutes, 30 minutes), obtain cell detection sample.
Specifically, the condition being protected from light refers to intensity of illumination in 0.2lux environment below.
Specifically, the concentration of antibody is 0.1-2mg/ml in the liquid preparation；The pulvis and phosphate buffer are mixed
The concentration of antibody is 0.1-2mg/ml in the antibody-solutions obtained after conjunction.
Step 3 tests and analyzes the cell detection sample using flow cytometer.
Fig. 1 is illustrated using the lethal immune cell function appraisal procedure of human peripheral blood of the invention to human peripheral blood
In lethal immunocyte tested and analyzed after obtained partial results.
CD3+CD8+ above Fig. 1 indicates all cells in Fig. 1 to the CD3 antibody response positive and to CD8 antibody
Reacting positive, these cells are CD8+T cell mass；It is additionally provided in Fig. 1 by being mutually perpendicular to staggered horizontal line and ordinate forms
Cross；
Firstly, the CCR7 to the longitudinal axis is analyzed, using the horizontal line in cross as line of demarcation, CCR7 is shown above horizontal line
+ as a result, horizontal line below be shown CCR7- result；
Secondly, the CD45RA to horizontal axis is analyzed, using the ordinate in cross as line of demarcation, it is shown on the left of ordinate
CD45RA-'s as a result, be shown the result of CD45RA+ on the right side of ordinate；
In other words, it is divided with cross, it is CD3+CD8+CD45RA- that upper left region, which is reaction result, in Fig. 1
The distributed areas of CD8+T cell are remembered in the region of CCR7+, i.e. center, and the digital display centre memory CD8+T cell in Fig. 1 exists
Accounting (i.e. the ratio of center memory CD8+T cell) in CD8+T cell mass is 0.39%；
The region in upper right side is the region that reaction result is CD3+CD8+CD45RA+CCR7+ in Fig. 1, i.e. initialization CD8+T
The distributed areas of cell, accounting of the number display initialization CD8+T cell in CD8+T cell mass in Fig. 1 (initialize
The ratio of CD8+T cell) it is 5.38%；
The region of lower left is the region that reaction result is CD3+CD8+CD45RA-CCR7- in Fig. 1, i.e. responsiveness is remembered
The distributed areas of CD8+T cell, accounting of the digital demonstration effect memory CD8+T cell in CD8+T cell mass in Fig. 1
(i.e. the ratio of responsiveness memory CD8+T cell) is 16.6%；
The region of lower right is the region that reaction result is CD3+CD8+CD45RA+CCR7-, i.e. terminal differentiation CD8 in Fig. 1
The distributed areas of+T cell, accounting (i.e. terminal of the number display terminal differentiation CD8+T cell in CD8+T cell mass in Fig. 1
Break up the ratio of CD8+T cell) it is 77.6%.
In conclusion the lethal immune cell function assessment kit of human peripheral blood of the invention can be thin in conjunction with streaming
Born of the same parents' instrument is used to carry out comprehensive assessment to the immune function of immunocyte lethal in human peripheral blood, can be used for human immunity health
In the panimmunities functional assessment projects such as the immune function of administrative evaluation and infections relating patient assessment.Kit of the invention
It is easy to use and safe to the human body.The lethal immune cell function appraisal procedure of human peripheral blood of the invention can be to human body
The immune function of lethal immunocyte carries out comprehensive assessment in peripheral blood, and operating procedure is simple, safe operation process.
The numberical range of each technological parameter as involved in the present invention can not all embody in the above-described embodiments,
As long as but those skilled in the art's envisioned any numerical value fallen into the above-mentioned numberical range completely can be implemented this
Invention also includes any combination of occurrence in several numberical ranges certainly.Herein, due to space considerations, be omitted to
Out in certain one or more numberical range occurrence embodiment, this disclosure for being not to be construed as technical solution of the present invention do not fill
Point.
The Applicant declares that the present invention is explained by the above embodiments detailed process equipment and process flow of the invention,
But the present invention is not limited to the above detailed process equipment and process flow, that is, it is above-mentioned detailed not mean that the present invention must rely on
Process equipment and process flow could be implemented.It should be clear to those skilled in the art, any improvement in the present invention,
Addition, concrete mode selection of equivalence replacement and auxiliary element to each raw material of product of the present invention etc., fall in protection of the invention
In range.
Claims (10)
1. a kind of lethal immune cell function of human peripheral blood assesses kit characterized by comprising band fluorescein marks
Anti-CD3antibody, with fluorescein-labeled anti-human CD8 antibody, with fluorescein-labeled anti-human CD 45 RA antibody, band fluorescein
Label anti-human CCR7 antibody, with fluorescein-labeled anti-human CD28 antibody, with fluorescein-labeled anti-human CD38 antibody, with glimmering
The anti-human CD57 antibody of light element label resists with fluorescein-labeled anti-human HLA-DR antibodies, with fluorescein-labeled anti-human PD-1
Body, with fluorescein-labeled anti-human CD56 antibody, with fluorescein-labeled anti-human CD94 antibody, with fluorescein-labeled anti-human
NKP30 antibody, with fluorescein-labeled anti-human NKP46 antibody, with fluorescein-labeled anti-human NKG2D antibody, band fluorescein mark
Note anti-human KIR antibody, with fluorescein-labeled anti-human γ anti-δ, with fluorescein-labeled anti-human 2 antibody of V δ.
2. the lethal immune cell function of human peripheral blood as described in claim 1 assesses kit, which is characterized in that described
Anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human CD28 antibody, anti-human CD38 are anti-
It is body, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody, anti-human CD94 antibody, anti-human
NKP30 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ anti-δ, anti-human 2 antibody of V δ are
Monoclonal antibody.
3. the lethal immune cell function of human peripheral blood as described in claim 1 assesses kit, which is characterized in that described
Anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human CD28 antibody, anti-human CD38 are anti-
It is body, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody, anti-human CD94 antibody, anti-human
NKP30 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ anti-δ, in anti-human 2 antibody of V δ
Any one is pulvis or liquid preparation.
4. the lethal immune cell function of human peripheral blood as claimed in claim 3 assesses kit, which is characterized in that described
Anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human CD28 antibody, anti-human CD38 are anti-
It is body, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody, anti-human CD94 antibody, anti-human
NKP30 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ anti-δ, anti-human 2 antibody of V δ are adopted
It is encapsulated with antibody reagent pipe, the antibody reagent pipe is light transmittance in 10% brown plastic tube below.
5. the lethal immune cell function of human peripheral blood as claimed in claim 4 assesses kit, which is characterized in that described
The light transmittance of antibody reagent pipe is zero, i.e., completely opaque.
6. the lethal immune cell function of human peripheral blood as claimed in claim 4 assesses kit, which is characterized in that described
When the antibody contained in antibody reagent pipe is liquid preparation, the volume of antibody is 0.5ml-5ml in the antibody reagent pipe.
7. the lethal immune cell function of human peripheral blood as claimed in claim 3 assesses kit, which is characterized in that described
Anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human CD28 antibody, anti-human CD38 are anti-
It is body, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody, anti-human CD94 antibody, anti-human
NKP30 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ anti-δ, anti-human 2 antibody of V δ are
Liquid preparation.
8. the lethal immune cell function of human peripheral blood as claimed in claim 3 assesses kit, which is characterized in that described
Anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human CD28 antibody, anti-human CD38 are anti-
It is body, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody, anti-human CD94 antibody, anti-human
NKP30 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ anti-δ, in anti-human 2 antibody of V δ
At least one is pulvis, and the lethal immune cell function assessment kit of human peripheral blood further includes phosphate buffer.
9. the lethal immune cell function of human peripheral blood as described in claim 1 assesses kit, which is characterized in that described
Anti-CD3antibody, anti-human CD8 antibody, anti-human CD 45 RA antibody, anti-human CCR7 antibody, anti-human CD28 antibody, anti-human CD38 are anti-
It is body, anti-human CD57 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human CD56 antibody, anti-human CD94 antibody, anti-human
NKP30 antibody, anti-human NKP46 antibody, anti-human NKG2D antibody, anti-human KIR antibody, anti-human γ anti-δ, anti-human 2 antibody of V δ it is glimmering
Light element label is selected from PerCP-Cy5.5, BV510, FITC, AF647, PE-Cy7, BV421, PE, AF647, APC-H7, BB515.
10. a kind of lethal immune cell function appraisal procedure of human peripheral blood, which comprises the steps of:
The lethal immunocyte function of human peripheral blood of step 1, offer as described in any one of claim 1 to claim 9
Kit can be assessed；
Peripheral blood mononuclear cells is extracted from 2ml-10ml human peripheral blood；
Step 2, when all antibody in the human peripheral blood lethal immune cell function assessment kit are liquid system
When agent, takes 1 μ l-5 μ l respectively from all antibody, all mixed with the peripheral blood mononuclear cells, in 2 DEG C of -6 DEG C of items
It is protected from light incubation 10 minutes to 30 minutes under part, obtains cell detection sample；
When at least a kind of antibody is pulvis in the lethal immune cell function assessment kit of the human peripheral blood, first
Pulvis is dissolved as by antibody-solutions using phosphate buffer, takes 1 μ l-5 μ l respectively from the antibody of all liq form later,
It is all mixed with the peripheral blood mononuclear cells, incubation 10 minutes to 30 minutes is protected from light under the conditions of 2 DEG C -6 DEG C, is obtained
Cell detection sample；
The concentration of antibody is 0.1-2mg/ml in the liquid preparation；The pulvis obtains anti-after mixing with phosphate buffer
The concentration of antibody is 0.1-2mg/ml in liquid solution；
Step 3 tests and analyzes the cell detection sample using flow cytometer.
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