CN113125754A - 23 antibody kit for monitoring human immune state and application - Google Patents

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

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CN113125754A
CN113125754A CN202110412899.8A CN202110412899A CN113125754A CN 113125754 A CN113125754 A CN 113125754A CN 202110412899 A CN202110412899 A CN 202110412899A CN 113125754 A CN113125754 A CN 113125754A
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季庆华
李鹏
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Zhejiang Plttech Health Technology Co ltd
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Abstract

The invention discloses a kit capable of detecting human immune states, which can be used for monitoring changes of human immune states and comprises 23 labeled antibodies, wherein the 23 antibodies are monoclonal antibodies CD3, CD56, gdTCR, CD14, IgD, CD85j, CD19, CD25(IL-2Ra), CD27, CD24, CD45RA, CD28, CD197(CCR7), CD33, CD161, CD94, CD127(IL-7Ra), CD38, CD20, CD16, HLA-DR, CD4 and CD 8. The kit disclosed by the invention can rapidly and accurately carry out deep analysis on immune cells in peripheral blood by collecting the peripheral blood through veins on a multi-color flow cytometer or mass spectrometer platform by utilizing 23 antibodies, and has strong specificity and high sensitivity.

Description

23 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 23 antibodies labeled, said 23 antibodies being monoclonal antibodies CD3, CD56, gdTCR, CD14, IgD, CD85j, CD19, CD25(IL-2Ra), CD27, CD24, CD45RA, CD28, CD197(CCR7), CD33, CD161, CD94, CD127(IL-7Ra), CD38, CD20, CD16, HLA-DR, CD4, CD 8.
The kit disclosed by the invention can be used for deeply detecting T and B lymphocytes, NK cells, monocytes, dendritic cells and subgroups in peripheral blood. 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.
In a preferred embodiment of the kit of the present invention, the 23 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.
As a preferred embodiment of the kit according to the invention, the fluorescein is selected from the group consisting of Biotin, FITC, PE, TRITC, APC, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor488, 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/zzzle, PE-594 CF, Pacific 594, Brillit Villiant 600, Brilliant Blue 421, Brilliant Violet 702, Brilliant 650, Bright Violet750, Bright yellow 7, Bright yellow 750, Bright yellow, eFluor 450, 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 Metallic element Antibodies Serial number Metallic element Antibodies
1 89Y CD3 13 157Gd CD197(CCR7)
2 139La CD56 14 159Tb CD33
3 141Pr gdTCR 15 162Dy CD161
4 143Nd CD14 16 166Er CD94
5 144Nd IgD 17 169Tm CD127(IL-7Ra)
6 146Nd CD85j 18 171Yb CD38
7 147Sm CD19 19 173Yb CD20
8 148Nd CD25(IL-2Ra) 20 174Yb CD16
9 152Sm CD27 21 175Lu HLA-DR
10 153Eu CD24 22 176Yb CD4
11 154Sm CD45RA 23 197Au CD8
12 156Gd CD28
In a preferred embodiment of the kit of the present invention, each of the 23 labeled antibodies is contained in the kit in an amount of 0.5 to 89% by volume.
The antibody in the kit of the invention is not limited to the antibody clone number, the fluorescent label color of the antibody, the mass number of the metal label, 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.
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 use of the kit of the present invention for detecting the immune status of a human, 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.
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 23 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 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 each of 23 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-1.5ml 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 kit provided by the invention provides a group of 23-color antibody combination schemes by utilizing 23 antibodies, realizes single-tube detection of a single sample by utilizing the kit provided by the invention on a multi-color flow cytometer or mass spectrum flow cytometer platform, can carry out deep analysis on immune cells through bioinformatics analysis, and can effectively monitor the immunocompetence of a physical examiner. 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 23 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 monitoring scheme based on CD3, CD56, gdTCR, CD14, IgD, CD85j, CD19, CD25(IL-2Ra), CD27, CD24, CD45RA, CD28, CD197(CCR7), CD33, CD161, CD94, CD127(IL-7Ra), CD38, CD20, CD16, HLA-DR, CD4 and CD8 by analyzing the expression abundance of peripheral blood immunocyte proteins, and each antibody is respectively marked by Biotin, FITC, PE, TRITC, APC, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor CyexAPC 750, Alexa Fluor APC 750, Alexa Fluor 5/APC 46750, Alexa Fluor CyexCyexA Fluor 5, Alexa Fluor750, Alexa Fluor 5/APC 46710, Alexa CyexCyexCyexCyexCy Cy CyexaCyexA/APC 750, Alexa Fluor CyexCyexCyexA PE/Dazzle 594, PE-CF594, Pacific Blue, Brilliant Violet 421, Brilliant Violet 510, Brilliant Violet570, Brilliant Violet 605, Brilliant Violet 650, Brilliant Violet 711, Brilliant Violet750, Brilliant Violet 785, Super Bright 436, Super Bright 600, Super Bright 645, Super Bright 702, eFluor 450, eFluor 506, eFluor 660, eFluor 710, eFluor 780, BD Horizon BB515, BD Horizon PE-CF594, Horizon BD 421, BD 480, BD 510, Horizon BV605, BD 49, BD 6326, BD 49, BD III BV III, BD 49, 34, III V, III, V, III, IV, III, V, III, IV, III, V, III, V, III, 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, 209Bi mass-spectrometrically detectable metal collocation. 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 23 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.
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 inventor of the present application monitors the immune state on the basis of a large number of experimental studies, establishes a corresponding flow cytometry or mass spectrometry flow cytometry scheme, and determines the immune state of a human body through 23 antibody combinations of labeled CD3, CD56, gdTCR, CD14, IgD, CD85j, CD19, CD25(IL-2Ra), CD27, CD24, CD45RA, CD28, CD197(CCR7), CD33, CD161, CD94, CD127(IL-7Ra), CD38, CD20, CD16, HLA-DR, CD4 and CD 8.
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 comprises labeled antibodies, wherein the antibodies comprise CD3, CD56, gdTCR, CD14, IgD, CD85j, CD19, CD25(IL-2Ra), CD27, CD24, CD45RA, CD28, CD197(CCR7), CD33, CD161, CD94, CD127(IL-7Ra), CD38, CD20, CD16, HLA-DR, CD4 and CD8 monoclonal antibodies, the antibodies are not limited to antibody clone numbers, and the antibodies are respectively labeled with fluorescein suitable for flow cytometry detection or metal elements suitable for mass cytometry detection.
Preferably, the fluorescein is selected from the group consisting of Biotin, FITC, PE, TRITC, APC, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor488, 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-CF, Pacific 594, Brilliant Violli 421, Brillit 78605, Brilliant Violet 510, Brilliant Fluolet 650, Brilliant Fluolet 750, Bright Fluoret 750, Bright Fluor Bright 450, Bright Fluor Bright 410, Bright fluoride 750, Bright Fluor Bright, Any one of 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.
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 content of each labeled antibody in the kit is 0.5-89%.
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 23 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 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 each of 23 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-1.5ml 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. 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 23 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 neutrophils and eosinophils in the test sample by flow cytometry or mass spectrometry flow cytometry, and containing a series of cell activation markers;
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
In one embodiment of the kit of the present invention, the 23 fluorescent or metal-labeled monoclonal antibodies are prepared, the usage ratio is determined according to the titration result of the antibodies, each antibody is added into a 1.5ml centrifuge tube according to the corresponding concentration, the total volume is made up to 50ul with a confining liquid, and the fluorescent antibodies need to be stored away from light, so as to obtain 1 person of the product of the present invention.
Example 2
Preparation of the kit:
preparing the 23 marked monoclonal antibodies, determining the use proportion according to the titration result of the antibodies, respectively mixing 1-100 parts of fluorescent antibodies according to corresponding concentrations, 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 same kit is used for detecting the same sample to be detected for multiple times, and the generated result has the characteristics of repeatability and reproducibility.
The mass spectrometry validity detection results of 23 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 representation of the positive rate of 23 markers for a sample flow-tested using the same kit is shown in table 1. B086-B095 represent blood samples with different subject numbers, and Table 1 shows that the abundance of various protein markers in immune cells is different among different individuals.
TABLE 1 detection results of 23marker for different individuals
B086 B087 B088 B089 B090 B091 B092 B093 B094 B095
CD127 0.377 0.382 0.507 0.227 0.236 0.308 0.405 0.435 0.413 0.383
CD14 0.141 0.114 0.105 0.234 0.211 0.118 0.179 0.176 0.151 0.129
CD16 0.103 0.074 0.116 0.141 0.308 0.139 0.074 0.143 0.155 0.204
CD161 0.192 0.124 0.242 0.209 0.276 0.180 0.141 0.271 0.253 0.268
CD19 0.075 0.029 0.107 0.082 0.038 0.037 0.076 0.047 0.092 0.075
CD197 0.298 0.350 0.402 0.121 0.111 0.167 0.277 0.294 0.306 0.264
CD20 0.067 0.027 0.106 0.077 0.038 0.036 0.077 0.055 0.097 0.075
CD24 0.077 0.030 0.108 0.083 0.039 0.038 0.077 0.048 0.093 0.076
CD25 0.034 0.041 0.099 0.030 0.029 0.017 0.081 0.074 0.088 0.105
CD27 0.486 0.641 0.586 0.218 0.273 0.313 0.417 0.375 0.443 0.417
CD28 0.381 0.509 0.556 0.213 0.225 0.258 0.431 0.413 0.463 0.444
CD3 0.600 0.751 0.637 0.488 0.428 0.611 0.640 0.606 0.571 0.519
CD33 0.181 0.145 0.133 0.280 0.242 0.212 0.206 0.208 0.203 0.174
CD38 0.118 0.393 0.181 0.153 0.208 0.100 0.214 0.249 0.187 0.273
CD4 0.373 0.321 0.341 0.183 0.230 0.255 0.363 0.314 0.371 0.324
CD45RA 0.311 0.314 0.339 0.247 0.215 0.222 0.413 0.409 0.308 0.311
CD56 0.138 0.072 0.122 0.180 0.282 0.202 0.095 0.176 0.178 0.220
CD8 0.213 0.362 0.283 0.299 0.222 0.349 0.268 0.282 0.214 0.207
CD85j 0.217 0.183 0.251 0.339 0.467 0.320 0.287 0.356 0.332 0.324
gdTCR 0.044 0.057 0.050 0.030 0.003 0.044 0.027 0.032 0.025 0.008
HLA_DR 0.216 0.386 0.243 0.336 0.295 0.319 0.285 0.313 0.376 0.216
IGD 0.044 0.012 0.072 0.065 0.020 0.010 0.043 0.037 0.054 0.031
CD94 0.149 0.065 0.065 0.169 0.226 0.108 0.135 0.171 0.146 0.127
The kit for detecting the human immune state provided by the invention utilizes the expression abundance of corresponding proteins detected by 23 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;
detecting the number of samples Immune status score Physical condition of the subject
107 example (c) <0.5 Has no bad living habits and good physical condition
93 examples of >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 comprising 23 antibodies labeled, said 23 antibodies being monoclonal antibodies CD3, CD56, gdTCR, CD14, IgD, CD85j, CD19, CD25(IL-2Ra), CD27, CD24, CD45RA, CD28, CD197(CCR7), CD33, CD161, CD94, CD127(IL-7Ra), CD38, CD20, CD16, HLA-DR, CD4, CD 8.
2. The kit of claim 1, wherein the 23 antibodies are labeled with fluorescein or a metal element, respectively.
3. The kit of claim 2, wherein said fluorescein is selected from the group consisting of Biotin, FITC, PE, TRITC, APC, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor488, 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/efour 750, APC/FireTM750, PerCP/Cy5.5, PerCP-eFlourr 710, PE/7, PE/Cy5, PE/Dazzlile 594, PE-CF, Pacific Blue, Brilliant Blue 421, Brilliant Super 600, Brilliant Super 702, Brilliant Super 750, Bright Super 702, Bright Violet 702, Bright Flutter 750, Bright/Cy 7, Bright Super, eFluor 450, 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 23 labeled antibodies is present in the kit in an amount of 0.5 to 89% 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 23 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 the dark for 10-20 min, preferably 15min, or 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 each of 23 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 muL-1.5 ml of resuspended 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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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113899893A (en) * 2021-09-30 2022-01-07 杭州联科生物技术股份有限公司 Development method of IVD (in-vitro visual inspection) streaming product
CN114720358A (en) * 2022-04-11 2022-07-08 浙江普罗亭健康科技有限公司 Antibody combination for replacing side scattered light signals in mass spectrum flow type blood tumor immunophenotyping and application

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108226016A (en) * 2018-01-12 2018-06-29 浙江普罗亭健康科技有限公司 The mass spectrum flow cytometer detection kit of the accurate parting of tumor vaccine cells subgroup
CN109254147A (en) * 2018-10-12 2019-01-22 东莞暨南大学研究院 Human peripheral blood immune cell function fully assesses kit and appraisal procedure
CN109490544A (en) * 2018-09-13 2019-03-19 浙江博真生物科技有限公司 A kind of kit and its application for detecting the human immunity age
CN110191948A (en) * 2016-11-02 2019-08-30 巴塞尔大学 Identifiable cell surface variant is immunized for cell therapy
CN110412287A (en) * 2019-07-11 2019-11-05 上海宸安生物科技有限公司 One kind being based on single celled immunocyte parting quantitative analysis method

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110191948A (en) * 2016-11-02 2019-08-30 巴塞尔大学 Identifiable cell surface variant is immunized for cell therapy
CN108226016A (en) * 2018-01-12 2018-06-29 浙江普罗亭健康科技有限公司 The mass spectrum flow cytometer detection kit of the accurate parting of tumor vaccine cells subgroup
CN109490544A (en) * 2018-09-13 2019-03-19 浙江博真生物科技有限公司 A kind of kit and its application for detecting the human immunity age
CN109254147A (en) * 2018-10-12 2019-01-22 东莞暨南大学研究院 Human peripheral blood immune cell function fully assesses kit and appraisal procedure
CN110412287A (en) * 2019-07-11 2019-11-05 上海宸安生物科技有限公司 One kind being based on single celled immunocyte parting quantitative analysis method

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113899893A (en) * 2021-09-30 2022-01-07 杭州联科生物技术股份有限公司 Development method of IVD (in-vitro visual inspection) streaming product
CN113899893B (en) * 2021-09-30 2024-01-30 杭州联科生物技术股份有限公司 Development method of IVD (in-line delivery) streaming product
CN114720358A (en) * 2022-04-11 2022-07-08 浙江普罗亭健康科技有限公司 Antibody combination for replacing side scattered light signals in mass spectrum flow type blood tumor immunophenotyping and application
CN114720358B (en) * 2022-04-11 2022-09-27 浙江普罗亭健康科技有限公司 Antibody combination for substituting side scattered light signals in mass spectrum flow type blood tumor immunology typing and application

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