CN109406775A - Autoimmune disease patient's immune function assesses kit and appraisal procedure - Google Patents
Autoimmune disease patient's immune function assesses kit and appraisal procedure Download PDFInfo
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Abstract
The present invention provides a kind of autoimmune disease patient's immune function assessment kit and appraisal procedure.Autoimmune disease patient's immune function assessment kit of the invention includes: anti-human CD3, CD4, CD8, CD19, CD21, CD24, CD25, CD27, CD28, CD38, CD57, CD127, CD45RA, CXCR3, CXCR5, CCR4, CCR6, CCR7, HLA-DR, PD-1, IgD, IgM antibody, and the above antibody carries fluorescein label.The kit can be used in carrying out comprehensive assessment to the immune cell function of autoimmune disease patient, easy to use, safe.
Description
Technical field
The present invention relates to medical field more particularly to a kind of autoimmune disease patient's immune function assessment kit and
Appraisal procedure.
Background technique
The generation of the immune function of human immunocyte and various diseases, progress have a directly related property, such as tumour
Occur, development process is exactly because of the result that human autoimmune's cell function is lowered or is suppressed for a long time.And autoimmunity
Property disease is generally related to immune cell function abnormal activation, leads to immune cells attack autologous tissue or organ, causes chronic
The autoimmune diseases such as diseases associated with inflammation, such as rheumatism.But domestic and international Research Literature is made a general survey of, it finds international at present
On have not been used in the side of special assessment autoimmune disease periphery immune cell function in generation, development and therapeutic process
Method.Clinical, third party testing agency evaluation immune function of human body method also rests on the quantity of cell, ratio both at home and abroad at present
Equal conventional indexs.For example, the blood routine of hospital simply shows the quantitative range and ratio of leucocyte, as CD3 cell quantity,
Ratio, quantity, ratio of CD4+T cell etc..The participation that these data carry out clinical diagnosis to doctor has little significance.Currently, packet
Include the method without can really evaluate immunocyte function itself and quality such as clinical and third party testing agency.
Summary of the invention
The purpose of the present invention, which first consists in, provides a kind of autoimmune disease patient's immune function assessment kit, can
It is used to carry out the immune cell function of autoimmune disease patient comprehensive assessment, easy to use, peace in conjunction with flow cytometer
Entirely.
The object of the invention is also to provide a kind of autoimmune disease patient immune function appraisal procedures, can be to certainly
The function of the immunocyte of body immunological diseases patient carries out comprehensive assessment.
In order to achieve the above object, present invention firstly provides a kind of autoimmune disease patient's immune functions to assess reagent
Box, comprising: resist with fluorescein-labeled anti-CD3antibody, with fluorescein-labeled anti-human CD4 antibody, with fluorescein-labeled
People CD8 antibody is marked with fluorescein-labeled anti human CD 19 antibody, with fluorescein-labeled anti-human CD21 antibody, band fluorescein
Anti-human CD24 antibody, with fluorescein-labeled anti-human CD25 antibody, with fluorescein-labeled anti-human CD27 antibody, band fluorescein
Label anti-human CD28 antibody, with fluorescein-labeled anti-human CD38 antibody, with fluorescein-labeled anti-human CD57 antibody, with glimmering
Light element label anti-human CD127 antibody, with fluorescein-labeled anti-human CD 45 RA antibody, with fluorescein-labeled anti-human CXCR3
Antibody resists with fluorescein-labeled anti-human CXCR5 antibody, with fluorescein-labeled anti-human CCR4 antibody, with fluorescein-labeled
People CCR6 antibody, with fluorescein-labeled anti-human CCR7 antibody, with fluorescein-labeled anti-human HLA-DR antibodies, band fluorescein mark
Note anti-human PD-1 antibody, with fluorescein-labeled anti-human IgD antibody, with fluorescein-labeled anti-human IgM antibodies.
Optionally, the anti-CD3antibody, anti-human CD4 antibody, anti-human CD8 antibody, anti human CD 19 antibody, anti-human CD21
It is antibody, anti-human CD24 antibody, anti-human CD25 antibody, anti-human CD27 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human
CD57 antibody, anti-human CD127 antibody, anti-human CD 45 RA antibody, anti-human CXCR3 antibody, anti-human CXCR5 antibody, anti-human CCR4 are anti-
Body, anti-human CCR6 antibody, anti-human CCR7 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human IgD antibody, anti-human IgM
Antibody is monoclonal antibody.
Optionally, the anti-CD3antibody, anti-human CD4 antibody, anti-human CD8 antibody, anti human CD 19 antibody, anti-human CD21
It is antibody, anti-human CD24 antibody, anti-human CD25 antibody, anti-human CD27 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human
CD57 antibody, anti-human CD127 antibody, anti-human CD 45 RA antibody, anti-human CXCR3 antibody, anti-human CXCR5 antibody, anti-human CCR4 are anti-
Body, anti-human CCR6 antibody, anti-human CCR7 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human IgD antibody, anti-human IgM
Any one in antibody is pulvis or liquid preparation.
Optionally, the anti-CD3antibody, anti-human CD4 antibody, anti-human CD8 antibody, anti human CD 19 antibody, anti-human CD21
It is antibody, anti-human CD24 antibody, anti-human CD25 antibody, anti-human CD27 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human
CD57 antibody, anti-human CD127 antibody, anti-human CD 45 RA antibody, anti-human CXCR3 antibody, anti-human CXCR5 antibody, anti-human CCR4 are anti-
Body, anti-human CCR6 antibody, anti-human CCR7 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human IgD antibody, anti-human IgM
Antibody is all made of the encapsulation of antibody reagent pipe, and the antibody reagent pipe is light transmittance in 10% brown plastic tube below.
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 CD4 antibody, anti-human CD8 antibody, anti human CD 19 antibody, anti-human CD21
It is antibody, anti-human CD24 antibody, anti-human CD25 antibody, anti-human CD27 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human
CD57 antibody, anti-human CD127 antibody, anti-human CD 45 RA antibody, anti-human CXCR3 antibody, anti-human CXCR5 antibody, anti-human CCR4 are anti-
Body, anti-human CCR6 antibody, anti-human CCR7 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human IgD antibody, anti-human IgM
Antibody is liquid preparation.
Optionally, the anti-CD3antibody, anti-human CD4 antibody, anti-human CD8 antibody, anti human CD 19 antibody, anti-human CD21
It is antibody, anti-human CD24 antibody, anti-human CD25 antibody, anti-human CD27 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human
CD57 antibody, anti-human CD127 antibody, anti-human CD 45 RA antibody, anti-human CXCR3 antibody, anti-human CXCR5 antibody, anti-human CCR4 are anti-
Body, anti-human CCR6 antibody, anti-human CCR7 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human IgD antibody, anti-human IgM
At least one of antibody is pulvis, and autoimmune disease patient's immune function assessment kit further includes that phosphate is slow
Fliud flushing.
Optionally, the anti-CD3antibody, anti-human CD4 antibody, anti-human CD8 antibody, anti human CD 19 antibody, anti-human CD21
It is antibody, anti-human CD24 antibody, anti-human CD25 antibody, anti-human CD27 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human
CD57 antibody, anti-human CD127 antibody, anti-human CD 45 RA antibody, anti-human CXCR3 antibody, anti-human CXCR5 antibody, anti-human CCR4 are anti-
Body, anti-human CCR6 antibody, anti-human CCR7 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human IgD antibody, anti-human IgM
The fluorescein label of antibody is selected from PerCP-Cy5.5, BV510, FITC, AF647, PE-Cy7, BV421, PE, AF647, APC-
H7、BB515。
The present invention also provides a kind of autoimmune disease patient immune function appraisal procedures, include the following steps:
Step 1 provides autoimmune disease patient's immune function assessment kit as described above；
Peripheral blood mononuclear cells is extracted from 2ml-10ml human peripheral blood；
Step 2, when autoimmune disease patient's immune function assessment kit in all antibody be liquid
When 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 DEG C
Under the conditions of be protected from light incubation 10 minutes to 30 minutes, obtain cell detection sample；
It is first when at least a kind of antibody is pulvis in autoimmune disease patient's immune function assessment kit
Pulvis is first dissolved as by antibody-solutions using phosphate buffer, takes 1 μ l-5 μ respectively from the antibody of all liq form later
L is 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, is obtained
To 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:
Autoimmune disease patient's immune function assessment kit of the invention can in conjunction with flow cytometer for pair
The immune cell function of autoimmune disease patient carries out comprehensive assessment, easy to use and safe to the human body.It is of the invention from
Body immunological diseases patient immune function appraisal procedure can carry out the function of the immunocyte of autoimmune disease patient
Comprehensive assessment, operating procedure is simple, safe operation process.
The present invention interior immune cell function progress established for the first time to autoimmune disease patient at the international level
The method of comprehensive assessment, using panimmunity functional parameter to the function of the immunocyte in autoimmune disease peripheral blood in patients
It can be carried out comprehensive assessment, these indexs can pass through than more comprehensively reflecting the immune functional state of autoimmune disorders
Carrying out analysis to the above immune function may determine that the immune functional state of autoimmune disorders, if it is determined that patient
Immunocyte overactivity then prompts the patient that may need immunosuppressive therapy intervention, such as passes through drug or the side of biological therapy
Method achievees the purpose that balance body's immunity.The immune functional state that this kit can be used for autoimmune disorders is commented
Estimate, the monitoring to immune cell function is especially realized during treatment or medication.
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.
Since the function of autoimmune disease and CD4+T cell is out of proportion, CD8+T cell function is out of proportion and B
Cellular malfunction etc. is closely related, therefore present invention is generally directed to CD4+T cell, CD8+T cell and B cells to carry out function
Detection.
Present invention firstly provides a kind of autoimmune disease patient's immune functions to assess kit, comprising: band fluorescein
The anti-CD3antibody of label, with fluorescein-labeled anti-human CD4 antibody, with fluorescein-labeled anti-human CD8 antibody, band fluorescence
Element label anti human CD 19 antibody, with fluorescein-labeled anti-human CD21 antibody, with fluorescein-labeled anti-human CD24 antibody, band
Fluorescein-labeled anti-human CD25 antibody resists with fluorescein-labeled anti-human CD27 antibody, with fluorescein-labeled anti-human CD28
Body, with fluorescein-labeled anti-human CD38 antibody, with fluorescein-labeled anti-human CD57 antibody, with fluorescein-labeled anti-human
CD127 antibody, with fluorescein-labeled anti-human CD 45 RA antibody, with fluorescein-labeled anti-human CXCR3 antibody, band fluorescein mark
Note anti-human CXCR5 antibody, with fluorescein-labeled anti-human CCR4 antibody, with fluorescein-labeled anti-human CCR6 antibody, with glimmering
The anti-human CCR7 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 IgD antibody, with fluorescein-labeled anti-human IgM antibodies.
Autoimmune disease patient's immune function assessment kit of the invention can in conjunction with flow cytometer for pair
The T cell and B cell of autoimmune disease patient tests and analyzes, and obtains 46 immune functions, and described 46 are exempted from
Epidemic disease functional parameter includes:
It is T cell toatl proportion, the ratio of helper T lymphocyte, the ratio of killer T cell, the ratio of double positive T cells, auxiliary
The ratio of helping property T cell and killer T cell, initialize the ratio of CD4+T cell, the ratio of terminal differentiation CD4+T cell, in
The heart remembers ratio, the ratio of failure CD4+T cell, functionality CD4 of the ratio of CD4+T cell, responsiveness memory CD4+T cell
The ratio of+T cell, the ratio of regulatory T cells, the ratio for initializing CD8+T cell, the ratio of terminal differentiation CD8+T cell,
Remember the ratio of CD8+T cell, the responsiveness memory ratio of CD8+T cell, the ratio of failure CD8+T cell, function resistance in center
The ratio of disconnected property CD8+T cell, the ratio of total Memorability CD8+T cell, returns nest note at the ratio of functionality CD8+T cell
The ratio of the property recalled CD8+T cell, the ratio of terminal aging CD8+T cell, the ratio of Th1 cell, the ratio of Th2 cell, Th17
The ratio of cell, the ratio (Th1/Th2) of Th1 cell and Th2 cell, Th17 cell and Th2 cell ratio (Th17/Th2),
The ratio (Th1+Th17/Th2) of Th1 cell+Th17 cell and Th2 cell, the ratio of Tfh cell, function inhibitio Tfh cell
Ratio, periphery auxiliary the ratio of Tfh cell, the ratio of Tfh1 cell, the ratio of Tfh2 cell, the ratio of Tfh17 cell,
The ratio of Tc1 cell (1 type CD8+T cell), the ratio of Tc2 cell (2 type CD8+T cell), (17 type CD8+T are thin for Tc17 cell
Born of the same parents) ratio, the ratio of B cell, memory non-conversion hysteria B cell (memory unswitched (MZ-like) B cells)
Ratio, the ratio of the B cell (classic memory switched B cells) of memory conversion, thick liquid cell
(Plasmablast) ratio of ratio, CD21 negative B cell (CD21low B cells), B progenitor cells (B
Cells ratio), preceding juvenile cell (B cells) ratio, the ratio of B10 cell (B10cells), memory
The ratio of property B cell (Memory B cells).
T cell toatl proportion is the ratio of T cell sum and total number of lymphocytes in the human peripheral blood of same volume；T is thin
The effect of born of the same parents is to play adaptive immunity function；The term of reference of T cell toatl proportion is 45.76%~77.45%.
The ratio of helper T lymphocyte is the ratio of helper T lymphocyte number and T cell sum in the human peripheral blood of same volume
Value；Helper T lymphocyte can play key effect in adaptive immunity, as antibody generates, the phagocytic function of macrophage, kills
Signal need to be transmitted by helper T lymphocyte by hurting lethal effect of cell etc.；The term of reference of the ratio of helper T lymphocyte be 40%~
67%.
The ratio of killer T cell is the ratio of killer T cell number and T cell sum in the human peripheral blood of same volume
Value；Killer T cell can effectively eliminate infected cell and kill to special antigen reactive target cell is generated
It goes out；The term of reference of the ratio of killer T cell is 31%~58%.
The ratio of double positive T cells is the ratio of double positive T cell numbers and T cell sum in the human peripheral blood of same volume
Value；Double positive T cells are mainly undifferentiated thymus T cells；The term of reference of the ratio of double positive T cells is 1%~5%.
The ratio (Th/Tc) of helper T lymphocyte and killer T cell is complementary T in the human peripheral blood of same volume
The ratio of cell number and killer T cell number；The term of reference of the ratio (Th/Tc) of helper T lymphocyte and killer T cell is
0.57%~2.44%, the ratio is significant in immune function assessment, when Th/Tc ratio exceeds above-mentioned term of reference,
Such as without acute infection or allergy, malignant tumour and autoimmune disease should be vigilant；Ratio is common in lower than above-mentioned term of reference
Malignant tumor patient, genetic immunodeficiency disease patient, AIDS patient apply immunosuppressor person.
The ratio for initializing CD4+T cell is that CD4+T cell number and CD4+T are initialized in the human peripheral blood of same volume
The ratio of total number of cells；Initialization CD4+T cell, which can be identified and be generated to the pathogen that immune system was never met, exempts from
Epidemic disease response generates reaction；The term of reference for initializing the ratio of CD4+T cell is 36.3% ± 15.3%.
The ratio of terminal differentiation CD4+T cell be same volume human peripheral blood in terminal differentiation CD4+T cell number with
The ratio of CD4+T total number of cells；Terminal differentiation CD4+T cell is the CD4+T cell without differentiation updating ability；Terminal differentiation CD4+
The term of reference of the ratio of T cell is 15.3% ± 1.5%.
Center remember CD4+T cell ratio be same volume human peripheral blood in center remember CD4+T cell number with
The ratio of CD4+T total number of cells；Center memory CD4+T cell has quick reaction to antigenic stimulus, and it is outstanding to generate effect to vaccine
It is important；The term of reference that the ratio of CD4+T cell is remembered at center is 33.8% ± 15.6%.
The ratio that responsiveness remembers CD4+T cell is that responsiveness remembers CD4+T cell in the human peripheral blood of same volume
Several ratios with CD4+T total number of cells；Responsiveness remember CD4+T cell have effector function, can all kinds of inflammation of quick release because
Son；The term of reference that responsiveness remembers the ratio of CD4+T cell is 27.6%~55.9%.
The ratio of failure CD4+T cell is failure CD4+T cell number and CD4+T cell in the human peripheral blood of same volume
The ratio of sum；Failure CD4+T cell number is associated with various autoimmune disease, such as type 1 diabetes, rheumatoid etc.；It declines
The term of reference for exhausting the ratio of CD4+T cell is 11.96% ± 8.60%.
The ratio of functional CD4+T cell is functionality CD4+T cell number and CD4+T in the human peripheral blood of same volume
The ratio of total number of cells；Functional CD4+T cell is can reactivity CD4+T cell；The reference of the ratio of functional CD4+T cell
Range is 89.56% ± 3.06%.
The ratio of regulatory T cells is regulatory T cells number and CD4+T total number of cells in the human peripheral blood of same volume
Ratio；Regulatory T cells are all played in tumour, immune deficiency, repulsion to the important adjusting factor for maintaining immunologic balance
Important function；The term of reference of the ratio of regulatory T cells is 4.14% ± 1.85%.
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；Initialize CD8+T cell
Ratio term of reference be 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；It is sensitive to virus infection that CD8+T cell is remembered at center；Remember the ratio of CD8+T cell in center
Term of reference is 4.61%~6.0%.
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 antiviral functions, releasable a variety of antiviral thin
Born of the same parents' cerebroysin and have killing ability；The term of reference that responsiveness remembers the ratio of 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；The reference model of the ratio of failure CD8+T cell
Enclose 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 16.8%~32%.
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.Secondary lymphoid can be moved to by returning nest Memorability CD8+T cell
Organ receives antigenic stimulus and re-activation is effector cell, is the important indicator of the treatment and prognosis of HIV infection patient.Return nest
Property Memorability CD8+T cell ratio term of reference be 62.7%~83.3%.
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 Th1 cell is the ratio of Th1 cell number and Th total number of cells in the human peripheral blood of same volume.Th1
Cell is also known as inflammatory Tcell, Major Secretory interleukin-22, interferon-γ, interferon-a, tumor necrosis factor etc., main to be situated between
The immune response of guided cell poison and local inflammation-related, auxiliary antibody generate, and participate in cellular immunity and delayed type hypersensitivity inflammation
Generation, delayed hypersensitivity T cell can be considered as.The participation of Th1 cell resists germ intracellular immune.The ratio mistake of Th1 cell
Height can cause organ specific autoimmune's disease.The term of reference of the ratio of Th1 cell is 14%~46%.
The ratio of Th2 cell is the ratio of Th2 cell number and Th total number of cells in the human peripheral blood of same volume.Th2
Cell Major Secretory IL-4,5,6 and 10 etc., major function is stimulation B cell proliferation and generates immunoglobulin G (IgG1)
It is related with humoral immunity with E (IgE) antibody.The participation of Th2 cell resists extracellular cause of disease parasite immunity.The ratio of Th2 cell is excessively high
It can cause allergic inflammation and asthma.The term of reference of the ratio of Th2 cell is 12%~25%.
The ratio of Th17 cell is the ratio of Th17 cell number and Th total number of cells in the human peripheral blood of same volume.
Th17 cell participation mediating inflammatory reaction (resisting extracellular bacterium, fungal infection), autoimmune disease, tumour and graft rejection etc.
Occurrence and development.Th17 cell mainly plays pro-inflammatory effect, and ratio is excessively high to cause diseases associated with inflammation (EAE, CIA etc.).Th17
The term of reference of the ratio of cell is 0.8%~8.3%.
The ratio (Th1/Th2) of Th1 cell and Th2 cell is Th1 cell number and Th2 in the human peripheral blood of same volume
The ratio of cell number；The term of reference of the ratio (Th1/Th2) of Th1 cell and Th2 cell is 0.71~3.33；Th1/Th2 ratio
Example, which increases, will promote cell immune response, enhance the activity of CD+8T cell, enhance disease in anti-tumor capacity and scavenger-cell
The ability of poison, but increase inflammatory reaction simultaneously, as hepatitis B virus infection can aggravate the damage of liver cell.Th1/Th2 ratio reduces
It will promote humoral immune reaction, but inhibit cell immune response, cytotoxic T lymphocyte (cytotoxicity
Tlymphocyte, CTL) cell activity decrease, hepatocellular injury mitigation.
The ratio (Th17/Th2) of Th17 cell and Th2 cell be in the human peripheral blood of same volume Th17 cell number with
The ratio of Th2 cell number；The term of reference of the ratio (Th17/Th2) of Th17 cell and Th2 cell is 0.04~0.6；Th17/
The raising of Th2 ratio occurs to diseases associated with inflammation, the tumour etc. including chronic inflammation, development is related.
The ratio (Th1+Th17/Th2) of Th1 cell+Th17 cell and Th2 cell is in the human peripheral blood of same volume
The ratio of Th1 cell+Th17 cell number and Th2 cell number；The ratio (Th17/Th2) of Th1 cell+Th17 cell and Th2 cell
Term of reference be 0.89~3.79；The diseases such as Th1+Th17/Th2 and autoimmune disease, tumour and graft-versus-host
Occurrence and development it is related.
The ratio of Tfh cell is the ratio of Tfh cell number and CD4+T total number of cells in the human peripheral blood of same volume.
Tfh cell secretes cytokine profiles and a variety of costimulatory molecules of expression complete the booster action that antibody is generated to B cell.Tfh
The ratio of cell is excessively high to cause antibody associated autoimmune disease.The ratio of Tfh cell is too low to cause immune deficiency.Th17
The term of reference of the ratio of cell is 23%~41.2%.
The ratio of function inhibitio Tfh cell be same volume human peripheral blood in function inhibitio Tfh cell number with
The ratio of CD4+T total number of cells.Function inhibitio Tfh cell has immune down regulation, and it is living to can inhibit Tfh cell transition
Change.The term of reference of the ratio of function inhibitio Tfh cell is 15.2%~36%.
The ratio of periphery auxiliary Tfh cell is periphery auxiliary Tfh cell number and CD4+T in the human peripheral blood of same volume
The ratio of total number of cells.Periphery auxiliary Tfh cell is with the T helper cell for inhibiting function.The ratio of periphery auxiliary Tfh cell
Term of reference be 22.6%~39.6%.
The ratio of Tfh1 cell is the ratio of Tfh1 cell number and Tfh total number of cells in the human peripheral blood of same volume.
Tfh1 cell does not play a major role in Help B Cells differentiation, poor related to the antibody response of influenza vaccines.Tfh1 cell exists
Quantity is reduced in the autoimmunity diseases such as spontaneous lymthoma.The term of reference of the ratio of Tfh1 cell is 19.2%~29.8%.
The ratio of Tfh2 cell is the ratio of Tfh2 cell number and Tfh total number of cells in the human peripheral blood of same volume.
Tfh2 cell more effectively can assist naive B cell to be differentiated to form plasmablast, non-suddenly in autoimmunity disease and high-level B cell
Quantity increases in the patients such as golden surprise lymthoma.The term of reference of the ratio of Tfh2 cell is 15.3%~23.6%.
The ratio of Tfh17 cell is the ratio of Tfh17 cell number and Tfh total number of cells in the human peripheral blood of same volume
Value.Tfh17 cell effectively can assist naive B cell to be differentiated to form plasmablast as Tfh2 cell.The function of Tfh17 cell
It can be similar with the function of Tfh2 cell.The term of reference of the ratio of Tfh17 cell is 31%~40%.
The ratio of Tc1 cell (1 type CD8+T cell) is Tc1 cell number and Tc cell in the human peripheral blood of same volume
The ratio of sum.Tc1 cell is 1 type cytotoxic T cell, can migrate to inflammation part, secrete INF- γ, kill target cell, reach
To the purpose of viral infection resisting.
The ratio of Tc2 cell (2 type CD8+T cell) is Tc2 cell number and Tc cell in the human peripheral blood of same volume
The ratio of sum.Tc2 cell is 2 type cytotoxic T cells, secretes IL4, IL5, IL10.Tc2 cell mainly with chronic infection, swollen
The chronic diseases such as tumor, autoimmune disease are related.This group of cytosis are related to the severity of disease and progress.
The ratio of Tc17 cell (17 type CD8+T cell) is that Tc17 cell number and Tc are thin in the human peripheral blood of same volume
The ratio of born of the same parents' sum.Tc17 cell is a group cytotoxic T cell for generating interleukin-17, is sent out in antitumor, anti-infectious immunity
Wave important function.Tc17 cell plays a significant role in anti-influenza A virus together with Tc1 cell.
The ratio of B cell is the ratio of B cell number and total number of lymphocytes in the human peripheral blood of same volume.B cell
It is main to generate antibody, critical function is played in humoral immunity.The term of reference of the ratio of B cell is 5~15%.
The ratio for remembering non-conversion hysteria B cell (memory unswitched (MZ-like) B cells) is same volume
Human peripheral blood in remember the ratio of non-conversion hysteria B cell number Yu B cell sum.Remembering non-conversion hysteria B cell is memory-type B
Cell precursors, quantity representative can be converted to memory-type B cell ability.Remember the reference model of the ratio of non-conversion hysteria B cell
Enclose is 3.8%~35.6%.
The ratio for the B cell (classic memory switched B cells) converted is remembered into the people of same volume
The B cell number of conversion and the ratio of B cell sum are remembered in peripheral body.The B cell of memory conversion belongs to switched memory
Type B cell, surface Ig are transformed into from IgD for IgG, IgA or IgE, can be further to Plasmablast (plasmablast)
Conversion, starts a large amount of secretory antibodies.The term of reference of the ratio for the B cell converted is remembered into 4.6%~35.5%.
The ratio of thick liquid cell (Plasmablast) is the human peripheral blood mesoplasmatocyte number and B cell sum of same volume
Ratio.Thick liquid cell by mature B cell through antigenic stimulus activate come, major function is secretory antibody, neutralization antigen.Thick liquid cell
Ratio term of reference be 0.3%~7.8%.
The ratio of CD21 negative B cell (CD21low B cells) is CD21 feminine gender B in the human peripheral blood of same volume
The ratio of cell number and B cell sum.CD21 negative B cell is a kind of B cell of memory-type, in chronic inflammations such as autoimmunity diseases
Quantity increases in disease patient.The term of reference of the ratio of CD21 negative B cell is 1.9%~19.3%.
B progenitor cells (B cells) ratio be same volume human peripheral blood in B progenitor cells number and B
The ratio of total number of cells.B progenitor cells are not yet exposed or the B cell of contact antigen, can be broken up by antigenic stimulus and be generated not
Same type B cell represents the reserve capabillity of B cell.The term of reference of the ratio of B progenitor cells is 17.9%~75.1%.
Preceding juvenile cell (B cells) ratio be same volume human peripheral blood in preceding juvenile cell
Several ratios with B cell sum.Its ability for being converted into juvenile cell of the proportional representation of preceding juvenile cell.
The ratio of B10 cell (B10cells) is B10 cell number and B cell sum in the human peripheral blood of same volume
Ratio.B10 cell is that a group plays immunological regulation and inhibits the B cell of function, IL-10 can be secreted, in tumor development process
In to immune response rise inhibiting effect, can prevent from being immunoreacted the damage to body itself in autoimmune disease.
The ratio of memory B cells (Memory B cells) is memory B cells in the human peripheral blood of same volume
Several ratios with B cell sum.Memory B cells are the important effect cells for generating immunological memory function, can be to antigen two
Secondary stimulation quick response generates lot of antibodies.The term of reference of the ratio of memory B cells is 11%~46.6%.
Specifically, the term of reference mentioned in the application refers to the normal range (NR) of Healthy People.
Above-mentioned 46 immune functions can than more comprehensively reflecting the immune functional state of autoimmune disorders,
It may determine that the immune functional state of autoimmune disorders by carrying out analysis to the above immune function, if it is determined that suffering from
The immunocyte overactivity of person then prompts the patient that may need immunosuppressive therapy intervention, such as passes through drug or biological therapy
Method achieve the purpose that balance body's immunity.This kit can be used for the immune function shape of autoimmune disorders
The monitoring to immune cell function is realized during state assessment etc., especially treatment or medication.
To obtain above-mentioned 46 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 40 immune functions.
Specifically, the cell sign of each immunocyte group are as follows:
The total group of T cell: CD3+；
Helper T lymphocyte: CD3+CD4+；
Killer T cell: CD3+CD8+；
Double positive T cells: CD3+CD4+CD8+；
Initialize CD4+T cell: CD3+CD4+CD45RA+CCR7+；
Terminal differentiation CD4+T cell: CD3+CD4+CD45RA+CCR7-；
Remember CD4+T cell: CD3+CD4+CD45RA-CCR7+ in center；
Responsiveness remembers CD4+T cell: CD3+CD4+CD45RA-CCR7-；
Failure CD4+T cell: CD3+CD4+CD28-；
Functional CD4+T cell: CD3+CD4+CD28+；
Regulatory T cells: CD3+CD4+CD25+CD127-；
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+；
Th1 cell: CD3+CD4+CXCR5-CXCR3+CCR4-；
Th2 cell: CD3+CD4+CXCR5-CXCR3-CCR4+；
Th17 cell: CD3+CD4+CXCR5-CXCR3-CCR4-CCR6+；
Tfh cell: CD3+CD4+CXCR5+；
Function inhibitio Tfh cell: CD3+CD4+CXCR5+PD-1+；
Periphery auxiliary Tfh cell: CD3+CD4+CXCR5-PD-1+；
Tfh1 cell: CD3+CD4+CXCR5+CXCR3+CCR4-；
Tfh2 cell: CD3+CD4+CXCR5+CXCR3-CCR4+；
Tfh17 cell: CD3+CD4+CXCR5+CXCR3-CCR4-CCR6+；
Tc1 cell: CD3+CD8+CXCR5-CXCR3+CCR4-；
Tc2 cell: CD3+CD8+CXCR5-CXCR3-CCR4+；
Tc17 cell: CD3+CD8+CXCR5-CXCR3-CCR4-CCR6+；
The total group of B cell: CD3-CD19+；
Remember non-conversion hysteria B cell: CD3-CD19+CD27+IgD+；
Remember the B cell of conversion: CD3-CD19+IgD-IgM-CD27+CD38low；
Thick liquid cell: CD3-CD19+IgD-IgM-CD27+CD38+；
CD21 negative B cell: CD3-CD19+IgD+CD27-CD38low CD21low；
B progenitor cells: CD3-CD19+CD27-IgD+；
Preceding juvenile cell: CD3-CD19+IgM-IgD-CD27-CD38+；
B10 cell: CD3-CD19+IgD+IgM+CD27+CD24+CD38-；
Memory B cells: CD3-CD19+IgD+IgM+CD27+CD38+CD24+.
Preferably, the anti-CD3antibody, anti-human CD4 antibody, anti-human CD8 antibody, anti human CD 19 antibody, anti-human CD21
It is antibody, anti-human CD24 antibody, anti-human CD25 antibody, anti-human CD27 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human
CD57 antibody, anti-human CD127 antibody, anti-human CD 45 RA antibody, anti-human CXCR3 antibody, anti-human CXCR5 antibody, anti-human CCR4 are anti-
Body, anti-human CCR6 antibody, anti-human CCR7 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human IgD antibody, anti-human IgM
Antibody is monoclonal antibody.This is because the specificity of monoclonal antibody is good, fluorescence intensity and antigen presentation water after dyeing
Flat is linear relationship, and the difference between monoclonal antibody difference production batch is smaller.Although polyclonal antibody can produce more
Strong signal, but poor specificity, fluorescence intensity and antigen levels are not linear relationships after dyeing, and polyclonal antibody is different
It is widely different between production batch, therefore can guarantee that higher detection is accurate using monoclonal antibody during flow cytometer detection
Degree.
Optionally, the anti-CD3antibody, anti-human CD4 antibody, anti-human CD8 antibody, anti human CD 19 antibody, anti-human CD21
It is antibody, anti-human CD24 antibody, anti-human CD25 antibody, anti-human CD27 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human
CD57 antibody, anti-human CD127 antibody, anti-human CD 45 RA antibody, anti-human CXCR3 antibody, anti-human CXCR5 antibody, anti-human CCR4 are anti-
Body, anti-human CCR6 antibody, anti-human CCR7 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human IgD antibody, anti-human IgM
Any one in antibody 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 CD4 antibody, anti-human CD8 antibody, anti human CD 19 antibody, anti-human CD21
It is antibody, anti-human CD24 antibody, anti-human CD25 antibody, anti-human CD27 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human
CD57 antibody, anti-human CD127 antibody, anti-human CD 45 RA antibody, anti-human CXCR3 antibody, anti-human CXCR5 antibody, anti-human CCR4 are anti-
Body, anti-human CCR6 antibody, anti-human CCR7 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human IgD antibody, anti-human IgM
Antibody is all made of the encapsulation of antibody reagent pipe.
Preferably, the antibody reagent pipe is completely opaque brown plastic tube, in other words, the brown plastic tube
Light transmittance be zero.
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 CD4 antibody, anti-human CD8 antibody, anti human CD 19 antibody, anti-human CD21
It is antibody, anti-human CD24 antibody, anti-human CD25 antibody, anti-human CD27 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human
CD57 antibody, anti-human CD127 antibody, anti-human CD 45 RA antibody, anti-human CXCR3 antibody, anti-human CXCR5 antibody, anti-human CCR4 are anti-
Body, anti-human CCR6 antibody, anti-human CCR7 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human IgD antibody, anti-human IgM
Antibody is liquid preparation, and liquid preparation uses more convenient dissolving step before capable of saving pulvis use, and energy
Caused detection error problem when avoiding pulvis dissolution 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 CD4 antibody, anti-human CD8 antibody, anti human CD 19 antibody, anti-human CD21 antibody,
Anti-human CD24 antibody, anti-human CD25 antibody, anti-human CD27 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human CD57 are anti-
It is body, anti-human CD127 antibody, anti-human CD 45 RA antibody, anti-human CXCR3 antibody, anti-human CXCR5 antibody, anti-human CCR4 antibody, anti-human
CCR6 antibody, anti-human CCR7 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human IgD antibody, in anti-human IgM antibodies
When at least one is pulvis, autoimmune disease patient's immune function assessment kit further includes phosphate buffer
(PBS), the phosphate buffer can be placed in reagent bottle.By providing phosphate buffer, preparation has been saved for user
The time of phosphate buffer keeps the use of antibody more convenient.
Optionally, the anti-CD3antibody, anti-human CD4 antibody, anti-human CD8 antibody, anti human CD 19 antibody, anti-human CD21
It is antibody, anti-human CD24 antibody, anti-human CD25 antibody, anti-human CD27 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human
CD57 antibody, anti-human CD127 antibody, anti-human CD 45 RA antibody, anti-human CXCR3 antibody, anti-human CXCR5 antibody, anti-human CCR4 are anti-
Body, anti-human CCR6 antibody, anti-human CCR7 antibody, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human IgD antibody, anti-human IgM
The fluorescein label of antibody is selected from PerCP-Cy5.5, BV510, FITC, AF647, PE-Cy7, BV421, PE, AF647, APC-
H7、BB515。
The present invention also provides a kind of autoimmune disease patient immune function appraisal procedures, include the following steps:
Step 1 provides autoimmune disease patient's immune function assessment kit described above；
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 autoimmune disease patient's immune function assessment kit has carried out in detail above
Thin 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 autoimmune disease patient's immune function assessment kit in all antibody be liquid
When 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 list
A nucleus is mixed, and is protected from light incubation 10 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)
Minute to 30 minutes (such as 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes), obtains cell detection sample；
It is first when at least a kind of antibody is pulvis in autoimmune disease patient's immune function assessment kit
Pulvis is first dissolved as by antibody-solutions using appropriate phosphate buffer (PBS), is divided from the antibody of all liq form later
1 μ l-5 μ l (such as 1 μ l, 2 μ l, 3 μ l, 4 μ l, 5 μ l) is not taken, 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.
In conclusion autoimmune disease patient's immune function assessment kit of the invention can combine fluidic cell
Instrument is used to carry out comprehensive assessment to the immune cell function of autoimmune disease patient, easy to use and safe to the human body.This
The autoimmune disease patient immune function appraisal procedure of invention can be to the immunocyte of autoimmune disease patient
Function carries out comprehensive assessment, 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 autoimmune disease patient's immune function assesses kit characterized by comprising with fluorescein-labeled
Anti-CD3antibody is marked with fluorescein-labeled anti-human CD4 antibody, with fluorescein-labeled anti-human CD8 antibody, band fluorescein
Anti human CD 19 antibody, with fluorescein-labeled anti-human CD21 antibody, with fluorescein-labeled anti-human CD24 antibody, band fluorescein
Label anti-human CD25 antibody, with fluorescein-labeled anti-human CD27 antibody, with fluorescein-labeled anti-human CD28 antibody, with glimmering
The anti-human CD38 antibody of light element label resists with fluorescein-labeled anti-human CD57 antibody, with fluorescein-labeled anti-human CD127
Body resists with fluorescein-labeled anti-human CD 45 RA antibody, with fluorescein-labeled anti-human CXCR3 antibody, with fluorescein-labeled
People CXCR5 antibody, with fluorescein-labeled anti-human CCR4 antibody, with fluorescein-labeled anti-human CCR6 antibody, band fluorescein mark
Note anti-human CCR7 antibody, with fluorescein-labeled anti-human HLA-DR antibodies, with fluorescein-labeled anti-human PD-1 antibody, with glimmering
Light element label anti-human IgD antibody, with fluorescein-labeled anti-human IgM antibodies.
2. autoimmune disease patient's immune function as described in claim 1 assesses kit, which is characterized in that described anti-
It is people CD3 antibody, anti-human CD4 antibody, anti-human CD8 antibody, anti human CD 19 antibody, anti-human CD21 antibody, anti-human CD24 antibody, anti-human
CD25 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human CD57 antibody, anti-human CD127 antibody, resists anti-human CD27 antibody
Human CD 45 RA antibody, anti-human CXCR3 antibody, anti-human CXCR5 antibody, anti-human CCR4 antibody, anti-human CCR6 antibody, anti-human CCR7 are anti-
Body, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human IgD antibody, anti-human IgM antibodies are monoclonal antibody.
3. autoimmune disease patient's immune function as described in claim 1 assesses kit, which is characterized in that described anti-
It is people CD3 antibody, anti-human CD4 antibody, anti-human CD8 antibody, anti human CD 19 antibody, anti-human CD21 antibody, anti-human CD24 antibody, anti-human
CD25 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human CD57 antibody, anti-human CD127 antibody, resists anti-human CD27 antibody
Human CD 45 RA antibody, anti-human CXCR3 antibody, anti-human CXCR5 antibody, anti-human CCR4 antibody, anti-human CCR6 antibody, anti-human CCR7 are anti-
Body, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human IgD antibody, any one in anti-human IgM antibodies for pulvis or
Liquid preparation.
4. autoimmune disease patient's immune function as claimed in claim 3 assesses kit, which is characterized in that described anti-
It is people CD3 antibody, anti-human CD4 antibody, anti-human CD8 antibody, anti human CD 19 antibody, anti-human CD21 antibody, anti-human CD24 antibody, anti-human
CD25 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human CD57 antibody, anti-human CD127 antibody, resists anti-human CD27 antibody
Human CD 45 RA antibody, anti-human CXCR3 antibody, anti-human CXCR5 antibody, anti-human CCR4 antibody, anti-human CCR6 antibody, anti-human CCR7 are anti-
Body, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human IgD antibody, anti-human IgM antibodies are all made of the encapsulation of antibody reagent pipe, institute
Stating antibody reagent pipe is light transmittance in 10% brown plastic tube below.
5. autoimmune disease patient's immune function as claimed in claim 4 assesses kit, which is characterized in that described anti-
The light transmittance of body Reagent Tube is zero, i.e., completely opaque.
6. autoimmune disease patient's immune function as claimed in claim 4 assesses kit, which is characterized in that described anti-
When the antibody contained in body Reagent Tube is liquid preparation, the volume of antibody is 0.5ml-5ml in the antibody reagent pipe.
7. autoimmune disease patient's immune function as claimed in claim 3 assesses kit, which is characterized in that described anti-
It is people CD3 antibody, anti-human CD4 antibody, anti-human CD8 antibody, anti human CD 19 antibody, anti-human CD21 antibody, anti-human CD24 antibody, anti-human
CD25 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human CD57 antibody, anti-human CD127 antibody, resists anti-human CD27 antibody
Human CD 45 RA antibody, anti-human CXCR3 antibody, anti-human CXCR5 antibody, anti-human CCR4 antibody, anti-human CCR6 antibody, anti-human CCR7 are anti-
Body, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human IgD antibody, anti-human IgM antibodies are liquid preparation.
8. autoimmune disease patient's immune function as claimed in claim 3 assesses kit, which is characterized in that described anti-
It is people CD3 antibody, anti-human CD4 antibody, anti-human CD8 antibody, anti human CD 19 antibody, anti-human CD21 antibody, anti-human CD24 antibody, anti-human
CD25 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human CD57 antibody, anti-human CD127 antibody, resists anti-human CD27 antibody
Human CD 45 RA antibody, anti-human CXCR3 antibody, anti-human CXCR5 antibody, anti-human CCR4 antibody, anti-human CCR6 antibody, anti-human CCR7 are anti-
At least one of body, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human IgD antibody, anti-human IgM antibodies are pulvis, described
It further includes phosphate buffer that autoimmune disease patient's immune function, which assesses kit,.
9. autoimmune disease patient's immune function as claimed in claim 3 assesses kit, which is characterized in that described anti-
It is people CD3 antibody, anti-human CD4 antibody, anti-human CD8 antibody, anti human CD 19 antibody, anti-human CD21 antibody, anti-human CD24 antibody, anti-human
CD25 antibody, anti-human CD28 antibody, anti-human CD38 antibody, anti-human CD57 antibody, anti-human CD127 antibody, resists anti-human CD27 antibody
Human CD 45 RA antibody, anti-human CXCR3 antibody, anti-human CXCR5 antibody, anti-human CCR4 antibody, anti-human CCR6 antibody, anti-human CCR7 are anti-
Body, anti-human HLA-DR antibodies, anti-human PD-1 antibody, anti-human IgD antibody, anti-human IgM antibodies fluorescein label be selected from PerCP-
Cy5.5、BV510、FITC、AF647、PE-Cy7、BV421、PE、AF647、APC-H7、BB515。
10. a kind of autoimmune disease patient immune function appraisal procedure, which comprises the steps of:
The autoimmune disease patient's immune function of step 1, offer as described in any one of claim 1 to claim 9
Assess kit；
Peripheral blood mononuclear cells is extracted from 2ml-10ml human peripheral blood；
Step 2, when autoimmune disease patient's immune function assessment kit in all antibody be liquid preparation
When, it takes 1 μ l-5 μ l respectively from all antibody, is all mixed with the peripheral blood mononuclear cells, in 2 DEG C of -6 DEG C of conditions
Under be protected from light incubation 10 minutes to 30 minutes, obtain cell detection sample；
When at least a kind of antibody is pulvis in autoimmune disease patient's immune function assessment kit, adopt first
Pulvis is dissolved as antibody-solutions with phosphate buffer, takes 1 μ l-5 μ l respectively from the antibody of all liq form later, entirely
Portion is 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, is obtained thin
Born of the same parents detect 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811191483.2A CN109406775A (en) | 2018-10-12 | 2018-10-12 | Autoimmune disease patient's immune function assesses kit and appraisal procedure |
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| CN110687292A (en) * | 2019-11-11 | 2020-01-14 | 北京大学人民医院(北京大学第二临床医学院) | Application of double negative B cell and regulatory B10 cell in preparation of autoimmune disease diagnostic reagent |
| CN111579779A (en) * | 2020-05-09 | 2020-08-25 | 中山大学附属第一医院 | Marker for evaluating immune cell function of hepatitis B patient and application thereof |
| CN111848779A (en) * | 2019-08-07 | 2020-10-30 | 北京市农林科学院 | Chicken complement receptor 2(ChCR2) monoclonal antibody and application thereof |
| CN112379099A (en) * | 2020-09-21 | 2021-02-19 | 华中科技大学同济医学院附属协和医院 | Detection method for predicting immunological index of HBsAg clearance in CHB patient |
| CN115166252A (en) * | 2022-07-07 | 2022-10-11 | 泰州宸安生物科技有限公司 | Lymphocyte subset grouping and quantitative detection kit, detection method and application thereof |
| TWI812274B (en) * | 2022-06-10 | 2023-08-11 | 何鈞軒 | Method of detecting immune efficacy and system thereof |
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| CN112379099A (en) * | 2020-09-21 | 2021-02-19 | 华中科技大学同济医学院附属协和医院 | Detection method for predicting immunological index of HBsAg clearance in CHB patient |
| TWI812274B (en) * | 2022-06-10 | 2023-08-11 | 何鈞軒 | Method of detecting immune efficacy and system thereof |
| CN115166252A (en) * | 2022-07-07 | 2022-10-11 | 泰州宸安生物科技有限公司 | Lymphocyte subset grouping and quantitative detection kit, detection method and application thereof |
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