CN113125755A - 9 antibody kit for monitoring human immune state and application thereof - Google Patents

9 antibody kit for monitoring human immune state and application thereof Download PDF

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CN113125755A
CN113125755A CN202110413987.XA CN202110413987A CN113125755A CN 113125755 A CN113125755 A CN 113125755A CN 202110413987 A CN202110413987 A CN 202110413987A CN 113125755 A CN113125755 A CN 113125755A
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林铖
王甜
孔全明
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Zhejiang Plttech Health Technology Co ltd
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Abstract

The invention discloses a detection kit for monitoring human immune state, which consists of CD3, CD4, CD8, TCRgd, CD16, CD19, CD56, CD14 and HLA-DR monoclonal antibody. The antibodies are respectively provided with fluorescein suitable for flow type or metal elements suitable for mass flow type, and the immune state of a human body is evaluated by detecting T lymphocytes, B lymphocytes, NK cells, monocytes (monocytes) and dendritic cells (Dendriticcell) in human peripheral blood, so that the immune state and change of the human body are detected and monitored, and further the tracking, prognosis and curative effect evaluation of clinical treatment are realized.

Description

9 antibody kit for monitoring human immune state and application thereof
Technical Field
The invention relates to the field of cytobiology and biomedical detection, in particular to a flow detection kit for detecting human immune state, which is used for evaluating human immune state and predicting disease risk.
Background
Peripheral blood lymphocytes are classified into T lymphocytes, B lymphocytes, NK lymphocytes according to biological function and cell surface antigen expression. T lymphocytes are mainly involved in cellular immunity and express CD3 antigen; b cells are primarily involved in humoral immunity, expressing the CD19 antigen; NK cells express CD16 and/or CD56 and exert cytotoxic effects in the body spontaneously independent of antigen stimulation. Among these, T cells include helper T cells (Th) and suppressor T cells (Ts), which express CD4 and CD8, respectively. Additionally gdT cells of CD3+ CD4-CD 8-gdTcCR + were present. Monocytes predominantly express CD14, whereas dendritic cells can be identified by HLA-DR and the markers in the present kit.
Lymphocyte subpopulation analysis is an important indicator for detecting immune function, and generally reflects the current immune function, state and balance level of the organism. Moreover, the occurrence and development of various diseases are closely related to the state change of an immune system, the evaluation of the immune state is important for the disease risk of certain diseases, and the auxiliary diagnosis of certain diseases, such as autoimmune diseases, immunodeficiency diseases, malignant tumors, hematopathy, allergic diseases and the like, and has important significance in analyzing the pathogenesis, observing the curative effect and detecting the prognosis.
CN201811191483.2 discloses an autoimmune disease patient immune function assessment kit and an assessment method, the kit comprises: antihuman CD3, CD4, CD8, CD19, CD21, CD24, CD25, CD27, CD28, CD38, CD57, CD127, CD45RA, CXCR3, CXCR5, CCR4, CCR6, CCR7, HLA-DR, PD-1, IgD and IgM antibodies, all of which carry a fluorescein label.
CN201811191491.7 discloses a kit and a method for comprehensively evaluating human peripheral blood immune cell functions, wherein the kit comprises anti-human CD3, CD4, CD8, CD19, CD21, CD24, CD25, CD27, CD28, CD38, CD56, CD57, CD94, CD127, CD45RA, CXCR3, CXCR5, CCR4, CCR6, CCR7, HLA-DR, PD-1, P30, P46, NKG2D, KIR (NKB1), gamma delta, V delta 2, IgD and IgM antibodies, and all the antibodies carry fluorescein labels.
Disclosure of Invention
The technical problem to be solved by the invention is a kit for detecting the immune state of a human body, and the kit is used for detecting the proportion of T lymphocytes, B lymphocytes, NK cells, monocytes and dendritic cells in peripheral blood and evaluating the immune state of the human body. Therefore, the invention provides a kit for detecting the composition of peripheral blood lymphocytes and evaluating the immune state of a human body.
The kit consists of CD3, CD4, CD8, TCRgd, CD16, CD19, CD56, CD14 and HLA-DR monoclonal antibodies, and is not limited to antibody clone numbers; the method is not limited to the color of the fluorescent label of the antibody, the mass number of the metal label, or other methods for labeling and detecting the antibody.
The detection instrument used by the detection kit is not limited to a fluorescence flow cytometer and a mass spectrometer.
The technical problem solved by the invention can be further realized by adopting the following measures.
A kit, wherein the reagents in the kit consist of CD3, CD4, CD8, TCRgd, CD16, CD19, CD56, CD14, HLA-DR monoclonal antibodies;
the CD3, CD4, CD8, TCRgd, CD16, CD19, CD56, CD14 and HLA-DR monoclonal antibodies respectively carry 9 fluorescein suitable for flow cytometry or 9 metal elements suitable for mass flow; the 9 fluorescein or metal elements are different from each other.
Preferably, the aforementioned kit comprises a fluorescein selected from the group consisting of Biotin, FITC, PE, TRITCAlexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor750, APC/Alexa Fluor750, APC/Cy7, APC/eflor 750, APC/FireTM750, PerCP/Cy5.5, PerCP-eFlourr 710, PE/Cy7, PE/Cy5, PE/Dazle, PE-CF594, Pacific, Brilliant Violli, Brilli Villi 421, Brilliant 600, Villiant 702, Villiant 650, Villiant 750, Villiant 710, Villiant 702, Villiant 750, Villiant 450, Villiant, Epluor 780, BD Horizon BB515, BD Horizon PE-CF594, BD Horizon BV421, BD Horizon BV480, BD Horizon BV510, BD Horizon BV605, BD Horizon BV650, BD Horizon BV711, BD Horizon BV786, BD Horizon BUV395, BD Horizon BUV496, BD Horizon BUV737, BD Horizon BUV805, BD Horizon APC R700, Cy3, Cy5, Cy7 vant Violet 570, Brilliant Violet 605, Brilliant Violet 650, Brilliant Violet 711, Brilliant Violet 750, Brilliant Violet 785, Super Bright 436, Fluor 450, BD 480, Horizon BV 515, BD 700.
Preferably, the metal element In the kit is any one selected from 89Y, 106Cd, 110Cd, 111Cd, 112Cd, 113Cd, 114Cd, 115In, 116Cd, 139La, 141Pr, 142Nd, 143Nd, 144Nd, 145Nd, 146Nd, 147Sm, 148Nd, 149Sm, 150Nd, 151Eu, 152Sm, 153Eu, 154Sm, 155Gd, 156Gd, 157Gd, 158Gd, 159Tb, 160Gd, 161Dy, 162Dy, 163Dy, 164Dy, 165Ho, Er, 167Er, 168Er, 169Tm, 170Er, 171Yb, 172Yb, 173Yb, 174Yb, 175Lu, 176Yb, 197Au, 198Pt and 209 Bi.
Preferably, the labeled antibody of the kit comprises the following components: the fluorescent or metal-labeled CD3, CD4, CD8, TCRgd, CD16, CD19, CD56, CD14 and HLA-DR account for 0.5-96 percent of the total volume percentage.
The technical problem to be solved by the invention is solved by the following technical scheme. The preparation method of the kit provided by the invention comprises the following steps: 1) carrying out fluorescence labeling or metal labeling on the antibody; 2) determining the amount of antibody used by titration; 3) and (3) putting the metered marked antibody into a reagent bottle, uniformly mixing, sealing, and keeping the fluorescent antibody away from light.
The technical problem solved by the invention is realized by adopting the following technical scheme. The invention provides application of a kit in detecting the immune state of a human body.
Preferably, the use of the aforementioned kit for detecting the immune status of a human, said kit being used for detecting the proportion of immune cells in peripheral blood;
the proportion of immune cells in the peripheral blood is distinguished by the unique immunophenotypic characteristics of the surface of each type of immune cell.
Preferably, the kit is used for detecting the immune state of a human body, wherein the detection sample is from the peripheral blood of the human body, and the human body comprises physical testers of all age groups.
Preferably, the kit is used for detecting the immune age of a human body, wherein the kit is used for detecting the ratio of T lymphocytes, B lymphocytes, NK cells, monocytes and dendritic cells in human peripheral blood mononuclear cells.
Preferably, the kit is used for detecting the immune age of a human body, wherein the monitoring comprises the following steps: 1) isolating peripheral blood mononuclear cells; 2) detecting the number and proportion of lymphocyte subpopulations in the peripheral blood of the sample by a flow cytometer or a mass spectrometer; 3) and determining the immune state of the detection sample according to the quantity and the proportion of the immune cell subsets in the peripheral blood.
Preferably, the kit is used for monitoring the immune state of a human body, and the flow detection preparation of the detection sample comprises the following steps:
1) collecting not less than 100 mu L of human peripheral blood as a detection sample; immediately detecting the sample after collection, or refrigerating at 2-8 ℃ and detecting within 48 h;
2) respectively sucking 0.5-5 mu L of 9 reagents from the kit into a flow tube, adding the detection sample into the flow tube, and uniformly mixing the reagents by oscillation to obtain a first pre-sample;
3) incubating the first pre-sample at normal temperature in a dark place for 10-20 min, preferably 15min, or at 2-8 ℃ for 20-40 min, preferably 30min to obtain a second pre-sample;
4) adding 300 mu L-2 mL of erythrocyte lysate into the second pre-sample, oscillating and uniformly mixing, and incubating for 2min at normal temperature in a dark place to obtain a third pre-sample;
5) centrifuging the third pre-sample to obtain a fourth pre-sample; the centrifugation condition of the centrifugation is 500 Xg, normal temperature and 5 min;
6) discarding the supernatant of the fourth pre-sample, and adding PBS buffer solution into the residual sample after discarding the supernatant to carry out resuspension to obtain a detection sample;
7) and (4) detecting the detection sample on a flow cytometer.
The mass spectrometric detection preparation of the detection sample comprises the following steps:
1) collecting not less than 5mL of peripheral blood of a human body as a detection sample; immediately detecting the sample after collection, or refrigerating at 2-8 ℃ and detecting within 48 h;
2) separating a detection sample by using lymphocyte separating medium to obtain mononuclear cells;
3) counting the cells, taking 1X 106～3×106A cell;
4) using PBS buffer solution to prepare 194Pt (1mM) dead and live staining solution with final concentration of 0.25 mu M, taking 50-1.5 mL of 194Pt dead and live staining solution to resuspend cells, preferably 100 mu L, and staining on ice for 5 min;
5) adding FACS Buffer of 100-1.5 mL, preferably 500 μ L, into each sample, resuspending cells, centrifuging at 2-8 ℃ for 400g/5min, and discarding the supernatant;
6) adding 20-1.5 mL of confining liquid, preferably 50 μ L, into each sample, resuspending the cells, and confining on ice for 20 min-1 h, preferably 20 min;
7) respectively sucking 0.5-4 mu L of 9 metal antibodies from the kit into a centrifugal tube, and adjusting the total volume to 50 mu L by using confining liquid;
8) adding the detection sample into the centrifuge tube, wherein the total volume is 100 mu L, and uniformly mixing the detection sample and the centrifuge tube by oscillation;
9) gently blowing and beating the uniformly mixed cells, and dyeing on ice for 20-60 min, preferably 30 min;
10) adding 1mL of FACS Buffer into each sample, resuspending cells, centrifuging at 2-8 ℃ for 400g/5min, and discarding the supernatant;
11) preparing Ir dye solution with final concentration of 100-500 nM, preferably 250nM, by using Fix and Perm Buffer, taking 200 mu L-1.5 mL of resuspended cells from each sample, and incubating for 1h at room temperature;
12) discarding the supernatant, using 500 mu L-1.5 mL FACS Buffer, preferably 1mL, resuspending the cells, centrifuging at 2-8 ℃ for 800g/5min, discarding the supernatant, resuspending the cells with deionized water, and detecting the detection sample on a mass cytometry.
Through the technical route, the kit for monitoring the immune state of the human body and the application thereof provided by the invention have at least the following advantages:
with the change of modern human life style, many people are in a sub-health state, the kit detects the composition and proportion of peripheral blood lymphocytes, establishes an organism immune state scoring system, evaluates the autoimmunity, assists in diagnosing certain diseases, analyzes the pathogenesis, observes the curative effect and detects the prognosis;
the detection can find out whether the physical examiner has the problem of abnormal immune state. For people with abnormal immune state, the immunity is mediated by adjusting the self state or adopting a medical means to intervene and enhance the immunity under the guidance of doctors.
The kit provided by the invention provides a group of 9-color antibody combination schemes by utilizing 9 antibodies, single-tube detection of a single sample is realized by utilizing the kit provided by the invention on a multi-color flow cytometer or mass spectrum flow cytometer platform, quantitative analysis can be carried out on T lymphocytes, B lymphocytes, NK cells, monocytes and dendritic cells through manual circle gating, and the immunocompetence of a physical examiner can be effectively monitored.
The beneficial effects produced by adopting the invention are as follows:
1) by adopting the kit, peripheral blood is collected through veins, and then T lymphocytes, B lymphocytes, NK cells, monocytes and dendritic cells of the peripheral blood can be rapidly and accurately quantitatively analyzed;
2) the kit is suitable for monitoring the immune state of physical testers in all age groups;
3) the kit has strong specificity and high sensitivity; each antibody can be accurately measured only by 0.5 mu L, and the repeatability reproducibility of the measured data is good;
4) the sample is easy to obtain, the report period is short, and the detection time and the cost are effectively reduced.
The above description only outlines the technical scheme of the present invention, and the specific implementation can be implemented according to the content of the kit specification, and the following detailed description is given with reference to the preferred embodiment of the present invention.
Drawings
FIG. 1 shows the mass spectrometric validity test results of 9 antibodies in the kit of the present invention.
Detailed Description
To further illustrate the technical means and practical effects of the present invention for achieving the predetermined objects, the following embodiments are provided to describe the structure, features, specific embodiments and effects of the kit for monitoring human immune status and its applications according to the present invention in detail. In the following description, different "one embodiment" or "an embodiment" refers to not necessarily the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
The kit for monitoring the immune state of the human body, provided by the invention, is suitable for physical examination persons of all ages.
And analyzing the proportion of T lymphocytes, B lymphocytes, NK cells, monocytes and dendritic cells in peripheral blood by using a flow cytometer or a mass flow cytometer to evaluate the immune state.
According to the invention, related personnel analyze unique markers of T lymphocytes, B lymphocytes, NK cells, monocytes and dendritic cells, a monitoring scheme based on CD3, CD4, CD8, TCRgd, CD16, CD19, CD56, CD14, HLA-DR and the like is established, mass spectrometry is utilized to detect basic samples of hundreds of healthy people in a flow mode, an immune state model suitable for Chinese people and reflecting the steady state of peripheral blood lymphocytes is established, the correlation between the proportion of the individual peripheral blood lymphocytes and the immune state can be accurately evaluated, and therefore, whether the immunity is abnormal or disordered or not is judged, and the accurate health management of individuals is facilitated.
CD3 is expressed on T lymphocytes, thymocytes, and NK cells.
CD4 is expressed in helper T lymphocyte, peripheral blood monocyte, macrophage and dendritic cell, and is mainly used for diagnosis of T lymphocyte leukemia and lymphoma and immune function detection.
CD8 is expressed on killer or suppressor T lymphocytes, thymocytes, and certain NK cells.
TCRgd is expressed in T lymphocytes, and is a main effector cell involved in early anti-infection and anti-tumor immunity.
CD16 was expressed on normal granulocytes, NK cell nuclei large granular T cells.
CD19 is expressed on B cells late nuclear mature B cells.
CD56 is expressed on NK cells, partial T cells and monocytes, and a few proliferating myeloid cells may also be weakly expressed.
CD14 is expressed on monocytes.
HLA-DR is expressed on activated T cells.
The fluorescein-labeled or metal element-labeled CD3, CD4, CD8, TCRgd, CD16, CD19, CD56, CD14, and HLA-DR monoclonal antibodies used in the examples of the present invention are commercially available under the brands of, for example, american Biolegend and R & D, but the composition of the kit of the present invention is not limited to the raw materials of the brands involved in the examples.
Fluorescein used in the examples of the present invention is commercially available under the brand names ThermoFisher, BD, etc.
The metal elements and their labeling kits used in the examples of the present invention are commercially available under the U.S. Fluidigm brand.
On the basis of the previous research, the immune state is monitored, a corresponding flow cytometry or mass spectrum flow cytometry scheme is established, and the human immune state is measured through fluorescence labeled CD3, CD4, CD8, TCRgd, CD16, CD19, CD56, CD14 and HLA-DR, 9 antibody combinations.
The monitoring method is suitable for physical examination persons of all ages.
The optimal dosage of the antibody is determined by methods such as antibody titration, the dosage of a single antibody is in the range of 0.1-4 mu L, ideal grouping can be achieved, and the detection effect can reach the expectation.
The sample obtained when the immune state monitoring model is established is taken from peripheral blood of a healthy person.
200 healthy human samples are selected to be detected by peripheral blood T lymphocytes, B lymphocytes, NK cells, monocytes and dendritic cells.
Firstly, the proportion of T lymphocytes, B lymphocytes, NK cells, monocytes and dendritic cells in peripheral blood of a healthy human sample is detected. The method comprises the following steps:
the kit is composed of CD3, CD4, CD8, TCRgd, CD16, CD19, CD56, CD14 and HLA-DR monoclonal antibodies, is not limited to antibody clone numbers, and is respectively marked with 9 fluorescein suitable for flow cytometry detection or 9 metals suitable for mass cytometry detection.
Preferably, the aforementioned kit comprises a fluorescein selected from the group consisting of Biotin, FITC, PE, TRITCAlexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor750, APC/Alexa Fluor750, APC/Cy7, APC/eflor 750, APC/FireTM750, PerCP/Cy5.5, PerCP-eFlourr 710, PE/Cy7, PE/Cy5, PE/Dazle, PE-CF594, Pacific, Brilliant Violli, Brilli Villi 421, Brilliant 600, Villiant 702, Villiant 650, Villiant 750, Villiant 710, Villiant 702, Villiant 750, Villiant 450, Villiant, Epluor 780, BD Horizon BB515, BD Horizon PE-CF594, BD Horizon BV421, BD Horizon BV480, BD Horizon BV510, BD Horizon BV605, BD Horizon BV650, BD Horizon BV711, BD Horizon BV786, BD Horizon BUV395, BD Horizon BUV496, BD Horizon BUV737, BD Horizon BUV805, BD Horizon APC R700, Cy3, Cy5, Cy7 vant Violet 570, Brilliant Violet 605, Brilliant Violet 650, Brilliant Violet 711, Brilliant Violet 750, Brilliant Violet 785, Super Bright 436, Fluor 450, BD 480, Horizon BV 515, BD 700.
Preferably, the metal element In the kit is any one selected from 89Y, 115In, 139La, 141Pr, 142Nd, 143Nd, 144Nd, 145Nd, 146Nd, 147Sm, 148Nd, 149Sm, 150Nd, 151Eu, 152Sm, 153Eu, 154Sm, 155Gd, 156Gd, 157Gd, 158Gd, 159Tb, 160Gd, 161Dy, 162Dy, 163Dy, 164Dy, 165Ho, 166Er, 167Er, 168Er, 169Tm, 170Er, 171Yb, 172Yb, 173Yb, 174Yb, 175Lu, 176Yb, 197Au, 198pt and 209 Bi.
Preferably, the labeled antibody of the kit comprises the following components: the fluorescent or metal-labeled CD3, CD4, CD8, TCRgd, CD16, CD19, CD56, CD14 and HLA-DR are 0.5-96% by volume.
The preparation method of the kit comprises the following steps:
1) carrying out fluorescence or metal labeling on the antibody;
2) performing antibody titration;
3) and (4) subpackaging the labeled antibody according to the titration result.
The storage conditions of the kit are as follows: and (3) storing the fluorescent antibody kit in a dark place at the temperature of 2-8 ℃.
After the preparation of the kit is completed, the human peripheral blood from the 200 samples is detected.
Preferably, the kit is used for monitoring the immune state of a human body, and the flow detection preparation of the detection sample comprises the following steps:
1) collecting not less than 100 mu L of human peripheral blood as a detection sample; immediately detecting the sample after collection, or refrigerating at 2-8 ℃ and detecting within 48 h;
2) respectively sucking 0.5-5 mu L of 9 reagents from the kit into a flow tube, adding the detection sample into the flow tube, and uniformly mixing the reagents by oscillation to obtain a first pre-sample;
3) incubating the first pre-sample at normal temperature in a dark place for 10-20 min, preferably 15min, or at 2-8 ℃ for 20-40 min, preferably 30min to obtain a second pre-sample;
4) adding 300 mu L-2 mL of erythrocyte lysate into the second pre-sample, oscillating and uniformly mixing, and incubating for 2min at normal temperature in a dark place to obtain a third pre-sample;
5) centrifuging the third pre-sample to obtain a fourth pre-sample; the centrifugation condition of the centrifugation is 500 Xg, normal temperature and 5 min;
6) discarding the supernatant of the fourth pre-sample, adding PBS buffer (pH7.4) into the residual sample after discarding the supernatant, and resuspending to obtain a detection sample;
7) and (4) detecting the detection sample on a flow cytometer.
The mass spectrometric detection preparation of the detection sample comprises the following steps:
1) collecting not less than 5mL of peripheral blood of a human body as a detection sample; immediately detecting the sample after collection, or refrigerating at 2-8 ℃ and detecting within 48 h;
2) separating a detection sample by using lymphocyte separating medium to obtain mononuclear cells;
3) counting the cells, taking 1X 106～3×106A cell;
4) using PBS buffer solution (pH7.4) to prepare 194Pt (1mM) dead and live staining solution with final concentration of 0.25. mu.M, taking 50. mu.L-1.5 mL of 194Pt dead and live staining solution to resuspend cells, preferably 100. mu.L, and staining on ice for 5 min;
5) adding FACS Buffer of 100-1.5 mL, preferably 500 μ L, into each sample, resuspending cells, centrifuging at 2-8 ℃ for 400g/5min, and discarding the supernatant;
6) adding 20-1.5 mL of confining liquid, preferably 50 μ L, into each sample, resuspending the cells, and confining on ice for 20 min-1 h, preferably 20 min;
7) respectively sucking 0.5-4 mu L of 9 metal antibodies from the kit into a centrifugal tube, and adjusting the total volume to 50 mu L by using confining liquid;
8) adding the detection sample into the centrifuge tube, wherein the total volume is 100 mu L, and uniformly mixing the detection sample and the centrifuge tube by oscillation;
9) gently blowing and beating the uniformly mixed cells, and dyeing on ice for 20-60 min, preferably 30 min;
10) adding 1mL of FACS Buffer into each sample, resuspending cells, centrifuging at 2-8 ℃ for 400g/5min, and discarding the supernatant;
11) preparing Ir dye solution with final concentration of 100-500 nM, preferably 250nM, by using Fix and Perm Buffer, taking 200 mu L-1.5 mL of resuspended cells from each sample, and incubating for 1h at room temperature;
12) discarding the supernatant, using 500 mu L-1.5 mL FACS Buffer, preferably 1mL, resuspending the cells, centrifuging at 2-8 ℃ for 800g/5min, discarding the supernatant, resuspending the cells with deionized water, and detecting the detection sample on a mass cytometry.
The application of the kit is based on a flow cytometer or a mass spectrometer, and the kit is used for detecting the proportion of T lymphocytes, B lymphocytes, NK cells, monocytes and dendritic cells in human peripheral blood.
From the results of the measurement of the ratios of T lymphocytes, B lymphocytes, NK cells, monocytes, and dendritic cells in the peripheral blood of the 200 samples, a reference range was established, and this range was considered as an immune homeostasis.
The invention also provides application of the kit in monitoring the immune state of a human body.
Preferably, the kit is used for monitoring the immune state of a human body, and when the kit is used for physical examination of the immune state, the detection comprises the following steps:
1) detecting the proportion of T lymphocytes, B lymphocytes, NK cells, monocytes and dendritic cells of the detection sample by a flow cytometer or a mass spectrometer;
2) the immune status of the monitored sample is assessed against the homeostatic model described above according to the ratio of T lymphocytes, B lymphocytes, NK cells, monocytes, dendritic cells.
Example 1
Preparation of a reagent:
CD3-FITC, CD4-PerCP/Cy5.5, TCRgd-Pacific Blue, CD8-APC/Cy7, CD16-PE, CD19-PE/Cy7, CD56-APC, CD14-PE-CF594 and HLA-DR-BV510 nine fluorescence labeled monoclonal antibodies, wherein 12.5 mu L of each antibody is respectively taken and added into a plastic tube or a glass reagent bottle to be uniformly mixed, and the fluorescence antibodies need to be kept away from light to be stored, so that 100 parts of the product is prepared, and the using amount of each part of the product is 1.125 mu L.
Example 2
Preparation of a reagent:
CD3-FITC, CD4-PerCP/Cy5.5, TCRgd-Pacific Blue, CD8-APC/Cy7, CD16-PE, CD19-PE/Cy7, CD56-APC, CD14-PE-CF594 and HLA-DR-BV510 nine fluorescence labeled monoclonal antibodies, wherein each antibody is 25 muL respectively, and the antibodies are added into a plastic tube or a glass reagent bottle and uniformly mixed, and the fluorescence antibodies need to be stored in a dark place, so that 100 parts of the product are prepared, and the using amount of each part of the product is 2.25 muL.
Example 3
Preparation of a reagent:
CD3-FITC, CD4-PerCP/Cy5.5, TCRgd-Pacific Blue, CD8-APC/Cy7, CD16-PE, CD19-PE/Cy7, CD56-APC, CD14-PE-CF594 and HLA-DR-BV510 nine fluorescence labeled monoclonal antibodies, wherein each antibody is respectively 50 mu L, added into a plastic tube or a glass reagent bottle, and uniformly mixed, and the fluorescence antibodies need to be stored in a dark place, so that 100 parts of the product is prepared, and the using amount of each part of the product is 4.5 mu L.
Example 4
Preparation of a reagent:
CD3-FITC, CD4-PerCP/Cy5.5, TCRgd-Pacific Blue, CD8-APC/Cy7, CD16-PE, CD19-PE/Cy7, CD56-APC, CD14-PE-CF594 and HLA-DR-BV510 nine fluorescence labeled monoclonal antibodies, wherein each antibody is respectively 100 mu L, added into a plastic tube or a glass reagent bottle, and uniformly mixed, and the fluorescence antibodies need to be stored in a dark place, so that 100 parts of the product is prepared, and the using amount of each part of the product is 9 mu L.
Example 5
Preparation of a reagent:
CD3-FITC, CD4-PerCP/Cy5.5, TCRgd-Pacific Blue, CD8-APC/Cy7, CD16-PE, CD19-PE/Cy7, CD56-APC, CD14-PE-CF594 and HLA-DR-BV510 nine fluorescence labeled monoclonal antibodies, wherein each antibody is respectively 200 mu L, added into a plastic tube or a glass reagent bottle, and uniformly mixed, and the fluorescence antibodies need to be stored in a dark place, so that 100 parts of the product is prepared, and the using amount of each part of the product is 18 mu L.
Example 6
Preparation of a reagent:
CD3-FITC, CD4-PerCP/Cy5.5, TCRgd-Pacific Blue, CD8-APC/Cy7, CD16-PE, CD19-PE/Cy7, CD56-APC, CD14-PE-CF594 and HLA-DR-BV510 nine fluorescence labeled monoclonal antibodies, wherein 400 mu L of each antibody is respectively taken and added into a plastic tube or a glass reagent bottle to be uniformly mixed, and the fluorescence antibodies need to be stored in a dark place, so that 100 parts of the product are prepared, and the using amount of each part of the product is 36 mu L.
Example 7
Preparation of a reagent:
CD3-FITC, CD4-PerCP/Cy5.5, TCRgd-Pacific Blue, CD8-APC/Cy7, CD16-PE, CD19-PE/Cy7, CD56-APC, CD14-PE-CF594 and HLA-DR-BV510 nine monoclonal antibodies, wherein one antibody (any one of the antibodies) is taken as 12.5 mu L, and the rest antibodies are respectively taken as 400 mu L, and are added into a plastic tube or a glass reagent bottle and uniformly mixed, and the fluorescent antibodies need to be kept away from light, so that 100 parts of the product are prepared, and the using amount of the product corresponding to each part is 32.125 mu L.
Example 8
Preparation of a reagent:
CD3-FITC, CD4-PerCP/Cy5.5, TCRgd-Pacific Blue, CD8-APC/Cy7, CD16-PE, CD19-PE/Cy7, CD56-APC, CD14-PE-CF594 and HLA-DR-BV510 nine monoclonal antibodies, wherein one antibody (any one of the antibodies) is 25 muL, the rest antibodies are 400 muL respectively, the antibodies are added into a plastic tube or a glass reagent bottle and mixed uniformly, and the fluorescent antibodies need to be kept away from light, so that 100 human parts of the product are prepared, and the using amount of each part of the product is 32.25 muL.
Example 9
Preparation of a reagent:
CD3-FITC, CD4-PerCP/Cy5.5, TCRgd-Pacific Blue, CD8-APC/Cy7, CD16-PE, CD19-PE/Cy7, CD56-APC, CD14-PE-CF594 and HLA-DR-BV510 nine monoclonal antibodies, wherein 50 mu L of one antibody (any one antibody) is taken, 400 mu L of the rest antibodies are respectively taken, the antibodies are added into a plastic tube or a glass reagent bottle and uniformly mixed, and the fluorescent antibodies need to be kept away from light, so that 100 parts of the product can be prepared, and the using amount of the product corresponding to each part is 32.50 mu L.
Example 10
Preparation of a reagent:
CD3-FITC, CD4-PerCP/Cy5.5, TCRgd-Pacific Blue, CD8-APC/Cy7, CD16-PE, CD19-PE/Cy7, CD56-APC, CD14-PE-CF594 and HLA-DR-BV510 nine monoclonal antibodies, wherein one antibody (any one of the antibodies) is taken as 100 mu L, and the rest antibodies are respectively taken as 400 mu L, and are added into a plastic tube or a glass reagent bottle to be uniformly mixed, and the fluorescent antibodies need to be kept away from light, so that 100 parts of the product can be prepared, and the using amount of each part of the product is 33 mu L.
Example 11
Preparation of a reagent:
CD3-FITC, CD4-PerCP/Cy5.5, TCRgd-Pacific Blue, CD8-APC/Cy7, CD16-PE, CD19-PE/Cy7, CD56-APC, CD14-PE-CF594 and HLA-DR-BV510 nine monoclonal antibodies, wherein 200 mu L of one antibody (any one antibody) is taken, 400 mu L of the rest antibodies are respectively taken, the antibodies are added into a plastic tube or a glass reagent bottle and uniformly mixed, and the fluorescent antibodies need to be kept away from light, so that 100 parts of the product can be prepared, and the using amount of the product corresponding to each part is 34 mu L.
Example 12
Preparation of a reagent:
CD3-FITC, CD4-PerCP/Cy5.5, TCRgd-Pacific Blue, CD8-APC/Cy7, CD16-PE, CD19-PE/Cy7, CD56-APC, CD14-PE-CF594 and HLA-DR-BV510 nine monoclonal antibodies, wherein 400 mu L of one antibody (any one antibody) is taken, 12.5 mu L of the rest antibodies are respectively taken, the antibodies are added into a plastic tube or a glass reagent bottle and are uniformly mixed, and the fluorescent antibodies need to be kept away from light, so that 100 parts of the product are prepared, and the using amount of each part of the product is 5 mu L.
Example 13
Preparation of a reagent:
CD3-FITC, CD4-PerCP/Cy5.5, TCRgd-Pacific Blue, CD8-APC/Cy7, CD16-PE, CD19-PE/Cy7, CD56-APC, CD14-PE-CF594 and HLA-DR-BV510 nine monoclonal antibodies, wherein 400 mu L of one antibody (any one antibody) is taken, 25 mu L of the other antibodies are respectively taken, the antibodies are added into a plastic tube or a glass reagent bottle and uniformly mixed, and the fluorescent antibodies need to be kept away from light, so that 100 parts of the product can be prepared, and the using amount of each part of the product is 6 mu L.
Example 14
Preparation of a reagent:
CD3-FITC, CD4-PerCP/Cy5.5, TCRgd-Pacific Blue, CD8-APC/Cy7, CD16-PE, CD19-PE/Cy7, CD56-APC, CD14-PE-CF594 and HLA-DR-BV510 nine monoclonal antibodies, wherein 400 mu L of one antibody (any one antibody) is taken, 50 mu L of the rest antibodies are respectively taken, the antibodies are added into a plastic tube or a glass reagent bottle and uniformly mixed, and the fluorescent antibodies need to be kept away from light, so that 100 parts of the product can be prepared, and the using amount of each part of the product is 8 mu L.
Example 15
Preparation of a reagent:
CD3-FITC, CD4-PerCP/Cy5.5, TCRgd-Pacific Blue, CD8-APC/Cy7, CD16-PE, CD19-PE/Cy7, CD56-APC, CD14-PE-CF594 and HLA-DR-BV510 nine monoclonal antibodies, wherein 400 mu L of one antibody (any one antibody) is taken, 100 mu L of the rest antibodies are respectively taken, the antibodies are added into a plastic tube or a glass reagent bottle and uniformly mixed, and the fluorescent antibodies need to be kept away from light, so that 100 parts of the product can be prepared, and the using amount of each part of the product is 12 mu L.
Example 16
Preparation of a reagent:
CD3-FITC, CD4-PerCP/Cy5.5, TCRgd-Pacific Blue, CD8-APC/Cy7, CD16-PE, CD19-PE/Cy7, CD56-APC, CD14-PE-CF594 and HLA-DR-BV510 nine monoclonal antibodies, wherein 400 mu L of one antibody (any one antibody) is taken, 200 mu L of the rest antibodies are respectively taken, the antibodies are added into a plastic tube or a glass reagent bottle and uniformly mixed, and the fluorescent antibodies need to be kept away from light, so that 100 parts of the product can be prepared, and the using amount of the product corresponding to each part is 20 mu L.
Example 17
Preparation of a reagent:
115In-CD3, 197Au-CD4, 142Nd-TCRgd, 198Pt-CD8, 175Lu-CD16, 148Nd-CD19, 141Pr-CD56, 144Nd-CD14 and 176Yb-HLA-DR nine metal-labeled monoclonal antibodies, according to the titration result, 0.5-4 mu L of each antibody is respectively taken for each detection, and the antibodies are respectively added into 9 plastic tubes according to 1-100 parts, so that the product of the invention is prepared for 1-100 parts. The 9 antibodies are mixed in proportion before dyeing and are used as they are.
After the kit is prepared, the usage amount of the product is marked on the kit.
Multiple monitoring of the same test sample using the kit of the same embodiment produces reproducible results.
The mass spectrometry validity detection results of 9 antibodies in the kit are shown in figure 1.
Through experiments, a sample of the subject is detected by using the mass spectrometry flow antibody kit of the above embodiment. B001-B010 represent different numbers of the subjects, and the abundance of various protein markers in immune cells is different in different individuals as shown in Table 1.
Table 1 example of positive ratios of 9 markers for a mass spectrometry sample using the kit of example 3
B001 B002 B003 B004 B005 B006 B007 B008 B009 B010
CD3 0.256 0.306 0.439 0.405 0.499 0.311 0.388 0.377 0.525 0.510
CD4 0.171 0.241 0.227 0.229 0.354 0.186 0.237 0.310 0.197 0.208
CD8 0.091 0.062 0.210 0.182 0.104 0.108 0.163 0.083 0.169 0.268
CD14 0.457 0.148 0.182 0.230 0.160 0.261 0.198 0.140 0.101 0.167
CD16 0.138 0.232 0.114 0.279 0.219 0.268 0.297 0.166 0.144 0.105
CD19 0.062 0.267 0.213 0.124 0.232 0.234 0.170 0.206 0.219 0.155
CD56 0.136 0.215 0.093 0.159 0.098 0.126 0.164 0.225 0.146 0.150
TCRgd 0.004 0.013 0.016 0.010 0.050 0.028 0.008 0.006 0.161 0.054
HLA_DR 0.567 0.488 0.440 0.452 0.391 0.519 0.424 0.323 0.338 0.334
The kit for detecting the human immune state provided by the invention utilizes the expression abundance of corresponding proteins detected by 9 antibodies of a subject to perform dimensionality reduction analysis and machine learning calculation, and establishes an immune state scoring system for detection and evaluation of various groups of people.
The kit for monitoring the immune state provided by the invention is used for monitoring a physical examination sample and comparing the physical conditions of a physical examinee. As shown in table 2, when the immune status score is less than 0.5, it indicates that the subjects have good autoimmune status, good aging degree of immune system, and low immune injury degree, and is matched with the subjects having no bad life habits and good physical conditions; when the immune state score is more than 0.5, the autoimmune state of the subject is lower than the normal level, the aging degree of the immune system is high, the immune injury degree is high, and the immune system corresponds to the physical conditions of 'disordered work and rest, irregular diet and easy fatigue'.
TABLE 2 examination table of immune status and general physical condition of physical examination person
Detecting the number of samples Immune status score Physical condition of the subject
95 examples of <0.5 Has no bad living habits and good physical condition
105 example (c) >0.5 Disordered daily work and rest, irregular diet and easy fatigue
The kit can be used for rapidly and accurately detecting and evaluating the autoimmunity.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are still within the scope of the technical solution of the present invention.

Claims (10)

1. A kit of parts, characterized in that,
the reagent in the kit consists of CD3, CD4, CD8, TCRgd, CD16, CD19, CD56, CD14 and HLA-DR monoclonal antibody;
the CD3, CD4, CD8, TCRgd, CD16, CD19, CD56, CD14 and HLA-DR monoclonal antibodies respectively carry 9 fluorescein suitable for flow cytometry or 9 metal elements suitable for mass flow; the 9 fluorescein or metal elements are different from each other.
2. The kit according to claim 1,
the fluorescein is selected from Biotin, FITC, PE, TRITCAlexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor750, APC/Alexa Fluor750, APC/Cy7, APC/eflor 750, APC/FireTM750, PerCP/Cy5.5, PerCP-eFlour 710, PE/Cy7, PE/5, PE/Dazle BD 594, PE-CF, Pacific Blue 421, Brilliant Violet, Brilliant villi Villiant 510, Brilliant Fluorit 600, Brilliant Fluorit 750, Bright fluoride 710, Bright Fluorit 750, Bright Fluorit 750, Bright TM750, Brilliant Blue 750, Brilliant 5, Brilliant, Any nine of BD Horizon PE-CF594, BD Horizon BV421, BD Horizon BV480, BD Horizon BV510, BD Horizon BV605, BD Horizon BV650, BD Horizon BV711, BD Horizon BV786, BD Horizon BUV395, BD Horizon BUV496, BD Horizon BUV737, BD Horizon BUV805, BD Horizon APC R700, Cy3, Cy5, Cy7 iatt Violet 570, Brilliant Violet 605, Brilliant Violet 650, Brilliant Violet 711, Brilliant Violet 750, Brilliant Violet 785, Super Bright 436, eFluor 450, BD Horizon 480 BV, BD Horizon BB515, BD Horizon R700;
the metal elements are selected from any nine of 89Y, 106Cd, 110Cd, 111Cd, 112Cd, 113Cd, 114Cd, 115In, 116Cd, 139La, 141Pr, 142Nd, 143Nd, 144Nd, 145Nd, 146Nd, 147Sm, 148Nd, 149Sm, 150Nd, 151Eu, 152Sm, 153Eu, 154Sm, 155Gd, 156Gd, 157Gd, 158Gd, 159Tb, 160Gd, 161Dy, 162Dy, 163Dy, 164Dy, 165Ho, 166Er, 167Er, 168Er, 169Tm, 170Er, 171Yb, 172Yb, 173Yb, 174Yb, 175Lu, 176Yb, 197Au, 198Pt and 209 Bi.
3. The kit according to claim 1,
the reagent in the kit comprises the following components:
the total content of fluorescent or metal-labeled CD3, CD4, CD8, TCRgd, CD16, CD19, CD56, CD14 and HLA-DR is 0.5-96% by volume percentage.
4. A method for preparing the kit according to any one of claims 1 to 3, characterized in that it comprises the following steps:
1) carrying out fluorescence or metal labeling on the antibody;
2) performing antibody titration;
3) and (4) subpackaging the labeled antibody according to the titration result.
5. Use of a kit according to any one of claims 1 to 3 for monitoring the immune status of a human.
6. Use of a kit according to claim 5 for monitoring the immune status of a human, wherein the kit is used for detecting the proportion of immune cells in peripheral blood;
the proportion of immune cells in the peripheral blood is distinguished by the unique immunophenotypic characteristics of the surface of each type of immune cell.
7. Use of a kit according to claim 5 for monitoring the immune status of a human, wherein the sample to be monitored is derived from the peripheral blood of the human.
8. Use of a kit according to any of claims 5 for monitoring the immune status of a human, characterized in that the monitoring comprises the following steps:
1) isolating peripheral blood mononuclear cells;
2) detecting the number and proportion of lymphocyte subpopulations in the peripheral blood of the sample by a flow cytometer or a mass spectrometer;
3) and determining the immune state of the detection sample according to the quantity and the proportion of the immune cell subsets in the peripheral blood.
9. Use of a kit according to claim 8 for monitoring the immune status of a human, wherein the flow assay preparation of the test sample comprises the steps of:
1) collecting not less than 100 mu L of human peripheral blood as a detection sample; immediately detecting the sample after collection, or refrigerating at 2-8 ℃ and detecting within 48 h;
2) respectively sucking 0.5-5 mu L of 9 reagents from the kit into a flow tube, adding the detection sample into the flow tube, and uniformly mixing the reagents by oscillation to obtain a first pre-sample;
3) incubating the first pre-sample at normal temperature in a dark place for 10-20 min, preferably 15min, or at 2-8 ℃ for 20-40 min, preferably 30min to obtain a second pre-sample;
4) adding 300 mu L-2 mL of erythrocyte lysate into the second pre-sample, oscillating and uniformly mixing, and incubating for 2min at normal temperature in a dark place to obtain a third pre-sample;
5) centrifuging the third pre-sample to obtain a fourth pre-sample; the centrifugation condition of the centrifugation is 500 Xg, normal temperature and 5 min;
6) discarding the supernatant of the fourth pre-sample, and adding PBS buffer solution into the residual sample after discarding the supernatant to carry out resuspension to obtain a detection sample;
7) and (4) detecting the detection sample on a flow cytometer.
10. The use of a kit according to claim 8 for monitoring the immune status of a human, wherein the mass spectrometric preparation of the test sample comprises the steps of:
1) collecting not less than 5mL of peripheral blood of a human body as a detection sample; immediately detecting the sample after collection, or refrigerating at 2-8 ℃ and detecting within 48 h;
2) separating a detection sample by using lymphocyte separating medium to obtain mononuclear cells;
3) counting the cells, taking 1X 106～3×106A cell;
4) using PBS buffer solution to prepare 194Pt (1mM) dead and live staining solution with final concentration of 0.25 mu M, taking 50-1.5 mL of 194Pt dead and live staining solution to resuspend cells, preferably 100 mu L, and staining on ice for 5 min;
5) adding FACS Buffer of 100-1.5 mL, preferably 500 μ L, into each sample, resuspending cells, centrifuging at 2-8 ℃ for 400g/5min, and discarding the supernatant;
6) adding 20-1.5 mL of confining liquid, preferably 50 μ L, into each sample, resuspending the cells, and confining on ice for 20 min-1 h, preferably 20 min;
7) respectively sucking 0.5-4 mu L of 9 metal antibodies from the kit into a centrifugal tube, and adjusting the total volume to 50 mu L by using confining liquid;
8) adding the detection sample into the centrifuge tube, wherein the total volume is 100 mu L, and uniformly mixing the detection sample and the centrifuge tube by oscillation;
9) gently blowing and beating the uniformly mixed cells, and dyeing on ice for 20-60 min, preferably 30 min;
10) adding 1mL of FACS Buffer into each sample, resuspending cells, centrifuging at 2-8 ℃ for 400g/5min, and discarding the supernatant;
11) preparing Ir dye solution with final concentration of 100-500 nM, preferably 250nM, by using Fix and Perm Buffer, taking 200 mu L-1.5 mL of resuspended cells from each sample, and incubating for 1h at room temperature;
12) discarding the supernatant, using 500 mu L-1.5 mL FACS Buffer, preferably 1mL, resuspending the cells, centrifuging at 2-8 ℃ for 800g/5min, discarding the supernatant, resuspending the cells with deionized water, and detecting the detection sample on a mass cytometry.
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