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CN113109575A - 40 antibody kit for monitoring human immune state and application - Google Patents

40 antibody kit for monitoring human immune state and application Download PDF

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CN113109575A
CN113109575A CN202110413977.6A CN202110413977A CN113109575A CN 113109575 A CN113109575 A CN 113109575A CN 202110413977 A CN202110413977 A CN 202110413977A CN 113109575 A CN113109575 A CN 113109575A
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王甜
孔全明
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Zhejiang Plttech Health Technology Co ltd
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    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
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    • G01—MEASURING; TESTING
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    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

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Abstract

The invention discloses a kit capable of detecting human immune states and application thereof, wherein the kit can be used for monitoring changes of human immune states, and comprises 40 labeled antibodies, wherein the 40 antibodies are monoclonal antibodies CD45, gdTCR, CD196/CCR 45, CD45, IgD, CD123/IL-3 45 85 45, CD 45/IL-2 36274/PD-L45, CD278/ICOS, CD45, CD45 45, CD197/CCR 45, CD11 45, CD152/CTLA-4, CD161, CD185/CXCR 45, CD66 45, CD 183/45, CD 3645, CD127/IL-7Ra, CD/PD-1, CD45, CD 36194, CD45, CD. The kit disclosed by the invention can be used for rapidly and accurately carrying out deep analysis on immune cells in peripheral blood by collecting the peripheral blood through veins on a multi-color flow cytometer or a mass spectrometer flow cytometer platform by utilizing 40 antibodies, and has strong specificity and high sensitivity.

Description

40 antibody kit for monitoring human immune state and application
Technical Field
The invention relates to a detection kit in the fields of cell biology and biomedical detection, in particular to a detection kit for detecting human immune state, which can be used for evaluating human immune state and disease risk prediction.
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. The T cells comprise helper T cells (Th) and suppressor T cells (Ts), which respectively express CD4 and CD8, and the Th cells can be divided into multiple types such as Th1, Th2, Th17, Th19, Th22, Tfh, Treg and the like according to the difference of transcription factors and secreted cytokines, and can be divided into initial T cells, effector T cells and memory T cells according to the different activation states of the T cells.
Immunomodulatory therapy has achieved clinical efficacy in a variety of cancers, autoimmune diseases and inflammation, allergy, widely used therapeutic strategies such as hematopoietic stem cell transplantation, immune checkpoint suppression, CART therapy, etc., many immunotherapeutic trials have been accompanied by immune monitoring, providing important cues of immune cell status from immune subpopulations and single cell levels. Monitoring peripheral blood immune cells is an important tool for immunodiagnosis and biomarker screening, and is expected to better understand immune pathogenesis and pathology and determine new therapeutic targets.
Disclosure of Invention
The invention aims to provide a kit for detecting the immune state of a human body, and also provides a preparation method of the kit and application of the kit in detecting the immune state of the human body.
In order to achieve the purpose, the invention adopts the technical scheme that: a kit comprising 40 antibodies labeled, said 40 antibodies being monoclonal antibodies CD45, gdTCR, CD196/CCR 45, CD45, IgD, CD123/IL-3 45, CD 45/IL-2 36274/PD-L45, CD278/ICOS, CD45, CD 3645 45, CD197/CCR 45, CD11 45, CD152/CTLA-4, CD161, CD185/CXCR 45, CD66 45, CD183/CXCR 45, CD45 45, CD127/IL-7Ra, CD279/PD-1, CD45, CD 194/45, CD279/PD 45, CD 3611.
The kit disclosed by the invention deeply detects T and B lymphocytes, NK cells, monocytes, dendritic cells and subgroups in peripheral blood, and comprises a series of cell activation markers and immune checkpoint proteins. The application to monitoring and comprehensive deep analysis of the patient's peripheral immune system status during clinical studies not only helps elucidating the immune cell mechanisms of new therapies, but it also helps predict the patient's risk of disease progression by identifying activation states, immune checkpoint features, and helps predict clinical response to treatment.
As a preferred embodiment of the kit of the present invention, the 40 antibodies are labeled with fluorescein or metal element, respectively. The antibody in the kit of the present invention is preferably labeled with fluorescein or metal element, but not limited to fluorescein and metal element. The 40 antibodies are labeled with different fluorescein or 40 different metal elements, respectively.
As a preferred embodiment of the kit according to the invention, the fluorescein is selected from the group consisting of Biotin, FITC, PE, TRITC, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor750, APC/Cy7, APC/efour 750, APC/FireTM750, PerCP/Cy5.5, PerCP-eFlourr 710, PE/Cy7, PE/Cy5, PE/zzzle, PE-CF594, Pacific, Brillit Villiant 600, Brilliant 788, Brilliant yellow 650, Brilliant 702, Brilliant 600, Brilliant yellow 750, Bright Violet 702, Bright Fluant 750, Bright Flutter 750, Bright yellow 7, Bright yellow 750, Bright, eFluor 506, eFluor 660, eFluor 710, eFluor 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, Cy 7.
As a preferred embodiment of the kit of the invention, the metal element is 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, 166Er, 167Er, 168Er, 169Tm, 170Er, 171Yb, 172Yb, 173Yb, 174Yb, 175Lu, Yb 176, 197Au, 198Pt, 209 Bi.
In a preferred embodiment of the kit of the present invention, the antibody in the kit is labeled with a metal element, and the labeling of the antibody with the metal element corresponds to the following:
serial number Channel Antibodies Serial number Channel Antibodies
1 89Y CD45 21 159Tb CD11c
2 115ln CD3 22 160Gd CD33
3 141Pr CD56 23 161Dy CD152/CTLA-4
4 142Nd gdTCR 24 163Dy CD161
5 143Nd CD196/CCR6 25 164Dy CD185/CXCR5
6 144Nd CD14 26 165Ho CD66b
7 145Nd IgD 27 166Er CD183/CXCR3
8 146Nd CD123/IL-3R 28 167Er CD94
9 147Sm CD85j 29 168Er CD57
10 148Nd CD19 30 169Tm CD45RO
11 149Sm CD25/IL-2R 31 170Er CD127/IL-7Ra
12 150Nd CD274/PD-L1 32 171Yb CD279/PD-1
13 151Eu CD278/ICOS 33 172Yb CD38
14 152Sm CD39 34 173Yb CD194/CCR4
15 153Eu CD27 35 174Yb CD20
16 154Sm CD24 36 175Lu CD16
17 155Gd CD45RA 37 176Yb HLA-DR
18 156Gd CD86 38 197Au CD4
19 157Gd CD28 39 198Pt CD8a
20 158Gd CD197/CCR7 40 209Bi CD11b
In a preferred embodiment of the kit of the present invention, the labeled 40 antibodies each have a volume percentage of 0.5-80.5% in the kit.
The 40 monoclonal antibodies in the kit are not limited to antibody clone numbers, fluorescent label colors of the antibodies, mass numbers of metal labels or other antibody labeling and detecting methods.
In addition, the invention also provides a preparation method of the kit, which comprises the following steps:
(1) labeling the antibody with fluorescein or metal element;
(2) performing antibody titration;
(3) and (4) subpackaging the labeled antibody according to the titration result.
In the preparation method of the kit, the antibody is firstly subjected to fluorescence or metal labeling, then the use amount of the antibody is determined by titration, and finally the metered labeled antibody is put into a reagent bottle, uniformly mixed and sealed, and the fluorescent antibody needs to be stored in a dark place.
Finally, the invention also provides the application of the kit in detecting the immune state of the human body.
The kit can be used for detecting the immune cells in the peripheral blood, and the proportion of the immune cells in the peripheral blood is distinguished by the unique protein expression characteristics on the surfaces of various immune cells.
As a preferred embodiment of the application of the kit of the present invention in the detection of the immune state of a human body, when the kit is used for detecting the immune state of the human body, a detection sample is from the peripheral blood of the human body, and the human body comprises people of all ages.
As a preferred embodiment of the application of the kit of the invention in the detection of the immune state of a human body, the kit is used for the in-depth detection of T and B lymphocytes and NK cells, monocytes, dendritic cells and subpopulations in human peripheral blood and comprises a series of cell activation markers and immune checkpoint proteins.
As a preferred embodiment of the application of the kit of the invention in detecting the immune state of a human body, the kit comprises the following steps in detecting the immune state of the human body:
(1) isolating peripheral blood mononuclear cells;
(2) detecting the expression abundance of the antigen corresponding to the lymphocyte antibody in the peripheral blood of the sample by using a flow cytometer or a mass spectrometer;
(3) and (3) performing dimensionality reduction analysis and operation by using an algorithm according to the abundance of each protein expression of immune cells in peripheral blood, and determining the immune state of the detection sample.
When the kit is applied to detection of the immune state of a human body, the used detection instrument is preferably but not limited to a fluorescence flow cytometer and a mass spectrum flow cytometer.
As a preferred embodiment of the application of the kit of the present invention in detecting the immune state of a human body, the flow cytometry detection in the step (2) 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 40 reagents from the kit into a flow tube, adding the detection sample into the flow tube, and uniformly mixing the detection sample with the flow tube 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 ul-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.
As a preferred embodiment of the application of the kit of the present invention in detecting the immune state of a human body, the mass spectrometry flow cytometry detection in the step (2) 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 to 1-3 × 106A cell;
4) using PBS buffer solution to prepare 194Pt (1mM) dead and live staining solution with the final concentration of 0.25 mu M, taking 50 ul-1.5 ml of 194Pt dead and live staining solution to resuspend cells, preferably 100 mu L, and staining for 5min on ice;
5) adding 100 ul-1.5 ml FACS Buffer (preferably 500 ul) into each sample, resuspending cells, centrifuging at 2-8 ℃ for 400g/5min, and discarding the supernatant;
6) adding 20 ul-1.5 ml of sealing liquid, preferably 50ul, into each sample, resuspending the cells, and sealing on ice for 20 min-1 h, preferably 20 min;
7) respectively sucking 0.5-4 mu L of 40 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 heavy suspension cells from each sample, and incubating for 1h at room temperature;
12)12) discarding the supernatant, using 500 ul-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 (MS) flow cytometer.
The kit provided by the invention provides a group of 40-color antibody combination schemes by utilizing 40 antibodies, single-tube detection of a single sample is realized on a multi-color flow cytometer or mass spectrum flow cytometer platform by utilizing the kit provided by the invention, deep analysis can be carried out on immune cells through bioinformatics analysis, and the immunocompetence of a physical examiner can be effectively monitored. By adopting the kit, the immune cells in peripheral blood can be rapidly and accurately deeply analyzed by collecting the peripheral blood through veins; the kit is suitable for monitoring the immune state of physical testers in all age groups. The kit has strong specificity and high sensitivity; each antibody can be accurately measured only by 0.5 mu L-4 ul, and the repeatability reproducibility of the measured data is good; the sample is easy to obtain, the report period is short, and the detection time and the cost are effectively reduced.
With the change of modern human life style, many people are in a sub-health state, the kit disclosed by the invention can be used for deeply analyzing peripheral blood lymphocytes, establishing an organism immune state scoring system, evaluating the autoimmunity, assisting in diagnosing certain diseases, analyzing a pathogenesis, observing a curative effect and detecting prognosis. The kit can find whether a physical examiner has the problem of abnormal immune state through detection, and for people with abnormal immune state, the kit can condition the people by adjusting the self state or adopting a medical means to intervene and enhance the immunity under the guidance of doctors.
Drawings
FIG. 1 is a diagram showing the results of mass spectrometry validity detection of 40 antibodies in the kit of the present invention.
Detailed Description
To better illustrate the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific 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. The kit analyzes the protein expression abundance of peripheral blood immune cells by using a flow cytometer or a mass spectrometer, and evaluates the immune state by being assisted with bioinformatics analysis.
The inventor builds a scheme of monitoring basic antibody such as AluTC, AluTC or Fluoron, AluTC or Fluoron, AldTc or Fluoron, or AldTc, CD45, CD45 45, CD197/CCR 45, CD11 45, CD152/CTLA-4, CD161, CD185/CXCR 45, CD66 45, CD183/CXCR or CXCR 45, CD183/CXCR 45, CD 279/IL-7 Ra, CD279/PD-1, CD45, CD194/CCR 45, CD 279/IL-7 Ra, AluTC or Fluoron or AluTC or Fluoron or, Alexa Fluor 647, Alexa Fluor660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor750, APC/Alexa Fluor750, APC/Cy7, APC/efour 750, APC/FireTM750, PerCP/Cy5.5, PerCP-eFlour 710, PE/Cy7, PE/Cy5, PE/Dazzle 594, PE-CF594, Pacific Blue, Brilliant Violet 421, Brilliant Violet 510, Brilliant Violet 570, Brilliant Violet 605, Brilliant Violet 650, Brilliant Violet 711, BD 594, BD 750, Brilliant Violet 785, Brilliant 436, Super Brilliant 600, Brilliant Super horir Fluor 650, Hongzon Fluor 650, Brilliant BV, Brilliant Fluor 410, Brilliant BV Fluor 650, Brilliant BV, Brilliant Fluor 450, Brilliant BV, Brilliant Fluor 410, Brilliant BV, BD horizons BUV395, BD horizons BUV496, BD horizons BUV737, BD horizons BUV805, BD horizons APC R700, Cy3, Cy5, Cy7 and the like flow cytometer detectable fluorescence matching, or 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 173, 173Yb 174, 175Lu, Yb 176, 197Au 198, Pt, 209Bi and the like flow cytometer detectable fluorescence matching. The mass spectrum flow detection method is used for detecting basic samples of hundreds of healthy people, an 'immune state' model which is suitable for Chinese and reflects the steady state of peripheral blood lymphocytes is established, and the correlation between the proportion of the individual peripheral blood lymphocytes and the immune state can be accurately evaluated, so that whether the immunity is abnormal or disordered or not is judged, and the individual is helped to carry out correct health management.
The 40 kinds of monoclonal antibodies labeled with fluorescein or metal elements used in the present invention are commercially available, for example, but not limited to, U.S. Biolegend, BD, R & D, and the like.
The fluorescein used in the present invention is commercially available, for example, but not limited to, the brand name ThermoFisher, BD, etc.
The metal elements used in the present invention are commercially available, such as, but not limited to, the U.S. Fluidigm brand.
The present inventors monitored immune status based on a number of experimental studies and established corresponding flow cytometry or mass cytometry protocols, and tested human immune status by combining labeled antibodies of CD127(IL-7Ra), CD14, CD16, CD19, CD197(CCR7), CD 7 (IL-2Ra), CD 7, CD40 7, CD 7, HLA-DR, TCRgd, CD11 7, CD123(IL-3R), CD161, CD183(CXCR 7), CD185(CXCR 7), CD196(CCR 7), CD 7, CD 36278 (ICOS), CD279(PD-1), CD 7, CD 3685 7, CD IgD, CD152 (CD-4), CD274 (PD-L7), CD 7, CD 36194, fonxp 194, graxt, CD 36194, CD 7, CD 36194.
The human immune state monitoring method is suitable for physical testers of all ages, the optimal dosage of the antibody is determined by antibody titration and other methods, the dosage of a single antibody is within the range of 0.1-4 ul, ideal grouping can be achieved, and the detection effect is expected; the sample obtained when the immune state monitoring model is established is taken from peripheral blood of a healthy person; and (3) taking 200 healthy human samples to carry out expression abundance detection of antigens corresponding to peripheral blood immune cells.
When the kit is used for detecting the human body immunity state, the peripheral blood immune cells of a healthy human sample are firstly detected. The specific method comprises the following steps:
the kit of the invention is adopted for detection, the kit of the invention contains 40 marked antibodies, the 40 antibodies are CD45, CD3, CD56, gdTCR, CD196/CCR6, CD14, IgD, CD123/IL-3R, CD85j, CD19, CD25/IL-2R, CD274/PD-L1, CD278/ICOS, CD39, CD27, CD24, CD45RA, CD86, CD28, CD197/CCR7, CD11c, CD33, CD152/CTLA-4, CD161, CD185/CXCR5, CD66b, CD183/CXCR3, CD94, CD57, CD45RO, CD127/IL-7Ra, CD279/PD-1, CD38, CD194/CCR4, CD20, CD16, HLA-DR, CD4, CD8a, CD11b monoclonal antibodies, the antibodies are not limited to antibody clone numbers, and the antibodies are respectively marked with different fluorescein suitable for flow cytometry detection or 40 metal elements suitable for mass cytometry detection.
Preferably, the fluorescein is selected from Biotin, FITC, PE, TRITC, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor660, 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/Dazzle 594, PE-CF Blue 780, Pacific 780, Brilliant Villiant Ville 421, Brilliant Ville 605, Brilliant Ville 510, Brilliant 600, Brilliant vitr Fluoret 750, Bright et 750, Bright Fluoret 750, Bright ad 750, Bright Fluoret 750, Bright et 650, Bright ad 750, Bright ad 450, Bright Fluoret 750, Bright ad 450, Bright et 450, Bright Fluor Bright Fluoret 750, Bright Fluor, Any one of 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, and Cy 7.
Preferably, the metal element is selected from any one 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.
Preferably, the volume percentage of each labeled antibody in the 40 labeled antibodies in the kit is 0.5-80.5%.
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.
After the kit is prepared, the storage condition is 2-8 ℃, and the fluorescent antibody kit needs to be stored in a dark place. After the preparation of the kit is completed, the human peripheral blood from the 200 samples is detected.
Preferably, the sample to be tested is tested by a flow cytometer, and the preparation for flow test of the sample to be tested 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 40 reagents from the kit into a flow tube, adding the detection sample into the flow tube, and uniformly mixing the detection sample with the flow tube 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 ul-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.
Preferably, the mass cytometry is adopted to detect a sample to be detected, and the mass flow detection preparation of the sample to be detected 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 to 1-3 × 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 ul-1.5 ml of 194Pt dead and live staining solution to re-suspend cells, preferably 100. mu.L, and staining on ice for 5 min;
5) adding 100 ul-1.5 ml FACS Buffer (preferably 500 ul) into each sample, resuspending cells, centrifuging at 2-8 ℃ for 400g/5min, and discarding the supernatant;
6) adding 20 ul-1.5 ml of sealing liquid, preferably 50ul, into each sample, resuspending the cells, and sealing on ice for 20 min-1 h, preferably 20 min;
7) respectively sucking 0.5-4 mu L of 40 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 heavy suspension cells from each sample, and incubating for 1h at room temperature;
12) discarding the supernatant, using 500 ul-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, the kit is used for detecting T and B lymphocytes and NK cells, monocytes, dendritic cells and subgroups in human peripheral blood, and the kit comprises a series of cell activation markers and immune checkpoint proteins. Through the detection results of the immune cells in the peripheral blood of the 200 samples, namely the expression abundance of the corresponding protein detected by 40 antibodies of the testee, dimension reduction analysis and machine learning calculation are carried out, and an immune state scoring system is established for detecting and evaluating the autoimmune states of various crowds.
The invention also provides application of the kit in monitoring the immune state of a human body.
Preferably, when the kit is used for monitoring the immune state of a human body, the kit can detect the immune state of the human body, and the specific detection comprises the following steps:
1) detecting T and B lymphocytes and NK cells, monocytes, dendritic cells and subpopulations of the detection sample by flow cytometry or mass cytometry, and comprising a series of cell activation markers and immune checkpoint proteins;
2) and (3) according to the expression condition of each protein, evaluating the immune state of the detection sample by contrasting the steady state model.
Example 1
According to one embodiment of the kit, the 40 fluorescent or metal-labeled monoclonal antibodies are prepared, according to the titration result of the antibodies, the antibody concentration which can be clearly grouped or accords with the performance declared by a manufacturer is selected, each antibody is added into 1.5ml of centrifuge tube according to the corresponding concentration, the total volume is made up to 50 mu L by using a sealing solution, and the fluorescent antibodies need to be stored in a dark place, so that 1 person of the product is obtained.
Example 2
Preparation of the kit:
preparing the 40 marked monoclonal antibodies, selecting the antibody concentration which can be clearly grouped or accords with the performance declared by a manufacturer according to the titration result of the antibodies, respectively mixing 1-100 parts of fluorescent antibodies according to the corresponding concentration, and storing in a dark place to obtain 1-100 parts of the product.
After the kit is prepared, the usage amount of the product is marked on the kit.
The mass spectrometry validity detection results of 40 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.
A mass spectrometric positive ratio of 40 markers for a sample using the same kit is shown in table 1. B019-B028 represent different numbers of subjects, and the abundance of various protein markers in immune cells is shown to be different in different individuals through Table 1.
TABLE 1 detection results of 40 markers for different individuals
Figure BDA0003025135210000121
Figure BDA0003025135210000131
The kit for detecting the human immune state provided by the invention utilizes the expression abundance of corresponding proteins detected by 40 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.
200 samples of volunteers were monitored and compared with the physical condition of the volunteers using the kit for monitoring immune status provided by the present invention. 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 survey table of immune status test results and general physical condition of physical examination patients;
Figure BDA0003025135210000132
Figure BDA0003025135210000141
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 comprising 40 antibodies labeled, said 40 antibodies being monoclonal antibodies CD45, gdTCR, CD196/CCR 45, CD45, IgD, CD123/IL-3 45, CD 45/IL-2 36274/PD-L45, CD278/ICOS, CD45, CD45 45, CD197/CCR 45, CD11 45, CD152/CTLA-4, CD161, CD185/CXCR 45, CD66 45, CD183/CXCR 45, CD45 45, CD127/IL-7Ra, CD279/PD-1, CD45, CD194/CCR 45, CD 45-CD 45, DR 3611, HLA 3611.
2. The kit of claim 1, wherein the 40 antibodies are labeled with fluorescein or metal element, respectively.
3. The kit of claim 2, wherein said fluorescein is selected from the group consisting of Biotin, FITC, PE, TRITC, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor660, 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/zzzle, PE-594 CF594, Pacific 594, Brillit Villi 421, Brillit 600, Brilliant Violet 702, Bright Flutter 750, Bright yellow 7, Bright yellow 750, Bright yellow 8926, Bright yellow 750, eFluor 506, eFluor 660, eFluor 710, eFluor 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, Cy 7.
4. The kit of claim 2, wherein the metallic element is selected from the group consisting 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, 209 Bi.
5. The kit of claim 1, wherein each of the 40 labeled antibodies is present in the kit in an amount of 0.5 to 80.5% by volume.
6. The method for preparing the kit according to any one of claims 1 to 5, wherein the method comprises the following steps:
(1) labeling the antibody with fluorescein or metal element;
(2) performing antibody titration;
(3) and (4) subpackaging the labeled antibody according to the titration result.
7. Use of a kit according to any one of claims 1 to 5 for detecting the immune status of a human.
8. Use of a kit according to claim 7 for detecting the immune status of a human, comprising the steps of:
(1) isolating peripheral blood mononuclear cells;
(2) detecting the expression abundance of the antigen corresponding to the lymphocyte antibody in the peripheral blood of the sample by using a flow cytometer or a mass spectrometer;
(3) and (3) performing dimensionality reduction analysis and operation by using an algorithm according to the abundance of each protein expression of immune cells in peripheral blood, and determining the immune state of the detection sample.
9. The use of the kit of claim 8 for detecting the immune status of a human, wherein the flow cytometry detection in step (2) 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 40 reagents from the kit into a flow tube, adding the detection sample into the flow tube, and uniformly mixing the detection sample with the flow tube 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 ul-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 the kit of claim 8 for detecting the immune status of a human, wherein the mass spectrometric flow cytometry detection in step (2) 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 to 1-3 × 106A cell;
4) using PBS buffer solution to prepare 194Pt (1mM) dead and live staining solution with the final concentration of 0.25 mu M, taking 50 ul-1.5 ml of 194Pt dead and live staining solution to resuspend cells, preferably 100 mu L, and staining for 5min on ice;
5) adding 100 ul-1.5 ml FACS Buffer (preferably 500 ul) into each sample, resuspending cells, centrifuging at 2-8 ℃ for 400g/5min, and discarding the supernatant;
6) adding 20 ul-1.5 ml of sealing liquid, preferably 50ul, into each sample, resuspending the cells, and sealing on ice for 20 min-1 h, preferably 20 min;
7) respectively sucking 0.5-4 mu L of 40 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 heavy suspension cells from each sample, and incubating for 1h at room temperature;
12) discarding the supernatant, using 500 ul-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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