CN113933511B - Antibody composition and method for detecting acute B lymphocyte leukemia tiny residue - Google Patents
Antibody composition and method for detecting acute B lymphocyte leukemia tiny residue Download PDFInfo
- Publication number
- CN113933511B CN113933511B CN202111113241.3A CN202111113241A CN113933511B CN 113933511 B CN113933511 B CN 113933511B CN 202111113241 A CN202111113241 A CN 202111113241A CN 113933511 B CN113933511 B CN 113933511B
- Authority
- CN
- China
- Prior art keywords
- antibody
- cells
- acute
- detecting
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000003719 b-lymphocyte Anatomy 0.000 title claims abstract description 47
- 230000001154 acute effect Effects 0.000 title claims abstract description 41
- 208000032839 leukemia Diseases 0.000 title claims abstract description 40
- 239000000203 mixture Substances 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title abstract description 36
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 claims description 19
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 18
- 208000003747 lymphoid leukemia Diseases 0.000 claims description 14
- 108700012813 7-aminoactinomycin D Proteins 0.000 claims description 6
- 239000000980 acid dye Substances 0.000 claims description 6
- 108020004707 nucleic acids Proteins 0.000 claims description 6
- 150000007523 nucleic acids Chemical class 0.000 claims description 6
- 102000039446 nucleic acids Human genes 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 5
- 239000012091 fetal bovine serum Substances 0.000 claims description 5
- 239000003228 hemolysin Substances 0.000 claims description 5
- 229920002866 paraformaldehyde Polymers 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- YXHLJMWYDTXDHS-IRFLANFNSA-N 7-aminoactinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=C(N)C=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 YXHLJMWYDTXDHS-IRFLANFNSA-N 0.000 claims description 4
- 108010006464 Hemolysin Proteins Proteins 0.000 claims description 4
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 3
- 101100485276 Arabidopsis thaliana XPO1 gene Proteins 0.000 claims description 2
- 101100407739 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) PET18 gene Proteins 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 101
- 230000014509 gene expression Effects 0.000 abstract description 57
- 238000001514 detection method Methods 0.000 abstract description 44
- 230000002159 abnormal effect Effects 0.000 abstract description 12
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- 238000012216 screening Methods 0.000 abstract description 3
- 206010028980 Neoplasm Diseases 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 49
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 23
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 23
- 210000000130 stem cell Anatomy 0.000 description 20
- 230000035945 sensitivity Effects 0.000 description 18
- 238000001228 spectrum Methods 0.000 description 15
- 210000004881 tumor cell Anatomy 0.000 description 12
- 239000000427 antigen Substances 0.000 description 11
- 102000036639 antigens Human genes 0.000 description 11
- 108091007433 antigens Proteins 0.000 description 11
- 210000001185 bone marrow Anatomy 0.000 description 11
- 239000006285 cell suspension Substances 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 11
- 238000000684 flow cytometry Methods 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 210000004698 lymphocyte Anatomy 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 9
- 239000010931 gold Substances 0.000 description 9
- 229910052737 gold Inorganic materials 0.000 description 9
- 210000003714 granulocyte Anatomy 0.000 description 9
- 210000001616 monocyte Anatomy 0.000 description 9
- 238000003745 diagnosis Methods 0.000 description 8
- 208000025321 B-lymphoblastic leukemia/lymphoma Diseases 0.000 description 7
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 7
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 7
- 208000017426 precursor B-cell acute lymphoblastic leukemia Diseases 0.000 description 7
- 208000007660 Residual Neoplasm Diseases 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 5
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 238000012795 verification Methods 0.000 description 5
- 102100031585 ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Human genes 0.000 description 4
- 101000777636 Homo sapiens ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Proteins 0.000 description 4
- 230000001464 adherent effect Effects 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 102000003729 Neprilysin Human genes 0.000 description 3
- 108090000028 Neprilysin Proteins 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000013068 control sample Substances 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 230000009977 dual effect Effects 0.000 description 3
- 239000000834 fixative Substances 0.000 description 3
- 238000003260 vortexing Methods 0.000 description 3
- 108010005465 AC133 Antigen Proteins 0.000 description 2
- 102000005908 AC133 Antigen Human genes 0.000 description 2
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 2
- 102100027221 CD81 antigen Human genes 0.000 description 2
- 102100025473 Carcinoembryonic antigen-related cell adhesion molecule 6 Human genes 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 2
- 101000914326 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 6 Proteins 0.000 description 2
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 2
- 108010029180 Sialic Acid Binding Ig-like Lectin 3 Proteins 0.000 description 2
- 102000001555 Sialic Acid Binding Ig-like Lectin 3 Human genes 0.000 description 2
- 230000010100 anticoagulation Effects 0.000 description 2
- 238000000149 argon plasma sintering Methods 0.000 description 2
- 210000002798 bone marrow cell Anatomy 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- 229960002897 heparin Drugs 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000010206 sensitivity analysis Methods 0.000 description 2
- 102000004008 5'-Nucleotidase Human genes 0.000 description 1
- 108700004024 5'-Nucleotidase Proteins 0.000 description 1
- 102100022464 5'-nucleotidase Human genes 0.000 description 1
- 108010027122 ADP-ribosyl Cyclase 1 Proteins 0.000 description 1
- 102000018667 ADP-ribosyl Cyclase 1 Human genes 0.000 description 1
- 102100022749 Aminopeptidase N Human genes 0.000 description 1
- 102100038080 B-cell receptor CD22 Human genes 0.000 description 1
- 108010021800 B7-2 Antigen Proteins 0.000 description 1
- 102000007697 B7-2 Antigen Human genes 0.000 description 1
- 108010071965 CD24 Antigen Proteins 0.000 description 1
- 102000007645 CD24 Antigen Human genes 0.000 description 1
- 102000013925 CD34 antigen Human genes 0.000 description 1
- 108050003733 CD34 antigen Proteins 0.000 description 1
- 108010084313 CD58 Antigens Proteins 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 description 1
- 101000757160 Homo sapiens Aminopeptidase N Proteins 0.000 description 1
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 description 1
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 description 1
- 101000998120 Homo sapiens Interleukin-3 receptor subunit alpha Proteins 0.000 description 1
- 101001063392 Homo sapiens Lymphocyte function-associated antigen 3 Proteins 0.000 description 1
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 1
- 101000884271 Homo sapiens Signal transducer CD24 Proteins 0.000 description 1
- 108010052781 Interleukin-3 Receptor alpha Subunit Proteins 0.000 description 1
- 102000018883 Interleukin-3 Receptor alpha Subunit Human genes 0.000 description 1
- 102100033493 Interleukin-3 receptor subunit alpha Human genes 0.000 description 1
- 102100030984 Lymphocyte function-associated antigen 3 Human genes 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 101710160107 Outer membrane protein A Proteins 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 102100038081 Signal transducer CD24 Human genes 0.000 description 1
- 108010077690 Tetraspanin 28 Proteins 0.000 description 1
- 101000980463 Treponema pallidum (strain Nichols) Chaperonin GroEL Proteins 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000013394 immunophenotyping Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000006371 metabolic abnormality Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 208000025113 myeloid leukemia Diseases 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57426—Specifically defined cancers leukemia
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Oncology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Hospice & Palliative Care (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an antibody composition and a method for detecting small residues of acute B lymphocyte leukemia, wherein the antibody composition comprises an anti-CD 19 antibody, an anti-CD 10 antibody, an anti-CD 34 antibody and an anti-CD 22 antibody. The invention can realize that only one tube is needed to complete the detection of 17 indexes, the sample size requirement is low, the framework antibody does not need to be reused, the reagent cost is low, the designed 17 detection indexes cover the expression modes of various abnormal cells, the detection rate of leukemia tiny residual cells is improved, and the invention can be used for detecting tiny residual focus after treatment and also can be used for screening tumor markers of primary patients.
Description
Technical Field
The invention belongs to the technical field of biomedical detection, and particularly relates to an antibody composition, a kit and a detection method for detecting acute B lymphocyte leukemia tiny residues.
Background
Acute lymphoblastic leukemia (acute lymphoblastic leukemia, ALL) is a malignant clonal proliferative disease of lymphocytes in an early differentiation stage, and is a group of highly heterogeneous diseases, wherein the incidence rate of acute B-lymphoblastic leukemia (B-ALL) accounts for about 85% of acute lymphoblastic leukemia, is mainly characterized by abnormal proliferation of primary and naive myelogenous cells in bone marrow and peripheral blood, and clinically presents as anemia, hemorrhage, infection and fever, infiltration of viscera, metabolic abnormality and the like, and most cases are urgent and serious, and prognosis is dangerous, if not treated in time, and life-threatening.
At present, the complete remission rate of leukemia is greatly improved by combined chemotherapy, but leukemia recurrence is a main problem of the current leukemia treatment, and the root of the recurrence mainly comes from residual leukemia cells in the body. In general, the state in which a small number of leukemia cells remain in the body after therapeutic remission of this leukemia is defined as minimal residual disease (minimal residual disease, MRD).
Clinically, the treatment scheme is regulated according to the level of MRD, and the dosage is regulated so as to achieve the purpose of curing. Therefore, whether MRD can be accurately determined is of great clinical value. The method can be used for detecting acute lymphoblastic leukemia tiny residues at present by cell morphology, chromosome karyotype analysis, molecular biology related gene detection, multiparameter flow cytometry analysis and other methods.
Cell morphology analysis has lower sensitivity, and residual tumor cells after treatment are possibly less, so that morphology has certain limitation; the chromosome karyotype analysis experiment period is longer; although the molecular biology method has high sensitivity, it cannot be used in cases where the karyotype is normal. The method for detecting the tiny residue by the multiparameter Flow Cytometry (FCM) based on the leukemia-related immunophenotype has the characteristics of rapidness, simplicity and convenience, and is an effective method for detecting the tiny residue currently accepted.
The analysis method adopted by the multiparameter flow cytometry to detect the residual leukemia cells is mainly used for monitoring leukemia-related immunophenotype (LAIP) or 'different from normal expression mode' (DFN), the LAIP method needs index screening during initial diagnosis immunophenotyping, otherwise, diagnosis is easy to miss, and the DFN method has higher requirements on the experience and expertise level of analysts. The multi-parameter flow cytometry can be used for detection by using a traditional flow cytometer, but is influenced by a detection channel, currently, a detection scheme with 6-10 colors is used more, each sample needs to be detected differently, not only the framework antibody needs to be repeatedly detected, but also the required sample quantity is more, and the sensitivity is not high.
The key point of detecting residual leukemia cells by using multiparameter flow cytometry is to identify normal differentiation precursor cells and abnormal leukemia cells, and it is difficult to use only a single leukocyte differentiation antigen as a specific immune marker for detecting minimal residual disease, so that at present, the cell classification is carried out by using a combination of antigens differentially expressed in acute lymphoblastic leukemia cells and normal bone marrow cells.
In the prior study, the MRD detection generally uses a cell membrane pan B cell marker CD19 assisted by SSC or CD45 (CD 19/SSC or CD45/CD19 double parameters) to gate for finding target B cells, but for patients after CD19-CAR-T cell infusion treatment, after treatment, part of normal or abnormal B cell surface CD19 antigen has shown to lose expression or weaken, immune escape occurs, and therefore missed diagnosis caused by the weakening or losing of CD19 expression can occur. At present, high-efficiency and accurate detection of tiny residual levels by using immune markers with high sensitivity and specificity is still lacking.
According to the above, a detection technology with high sensitivity, high accuracy, high detection rate, simplicity and rapidness is needed clinically to monitor the tiny residual focus of acute B-lymphocyte leukemia in time.
Disclosure of Invention
Based on the above, an object of the present invention is to provide an antibody composition for detecting minute residues of acute B-lymphocyte leukemia, wherein the detection index designed by the antibody composition covers the expression patterns of various abnormal cells of acute B-lymphocyte leukemia, and can overcome the defect of easy missed diagnosis.
The specific technical scheme for realizing the aim of the invention is as follows:
an antibody composition for detecting acute B-lymphoblastic leukemia minimal residual, said antibody composition comprising: anti-CD 19 antibodies, anti-CD 10 antibodies, anti-CD 34 antibodies, and anti-CD 22 antibodies.
In some of these embodiments, the antibody composition further comprises an anti-CD 45 antibody, an anti-CD 20 antibody, an anti-CD 24 antibody, an anti-CD 38 antibody, an anti-CD 81 antibody, an anti-CD 58 antibody, an anti-CD 13 antibody, an anti-CD 33 antibody, an anti-CD 66c antibody, an anti-CD 73 antibody, an anti-CD 123 antibody, and an anti-CD 86 antibody.
In some embodiments, the anti-CD 19 antibody, anti-CD 10 antibody, anti-CD 34 antibody, anti-CD 20 antibody, anti-CD 45 antibody, anti-CD 22 antibody, anti-CD 24 antibody, anti-CD 38 antibody, anti-CD 81 antibody, anti-CD 58 antibody, anti-CD 13 antibody, anti-CD 33 antibody, anti-CD 66c antibody, anti-CD 73 antibody, anti-CD 123 antibody, and anti-CD 86 antibody are numbered sequentially: j3-119, H10a, 581, 2H7, H130, S-HCL-1, ML5, HIT2, JS-81, AICD58, L138, D3HL60.251, B6.2/CD66, AD2, 9F5 and 2331.
In some embodiments, the antibodies are all fluorescein-labeled antibodies, and the fluorescein labeled with anti-CD 19 antibody, anti-CD 10 antibody, anti-CD 34 antibody, anti-CD 20 antibody, anti-CD 45 antibody, anti-CD 22 antibody, anti-CD 24 antibody, anti-CD 38 antibody, anti-CD 81 antibody, anti-CD 58 antibody, anti-CD 13 antibody, anti-CD 33 antibody, anti-CD 66c antibody, anti-CD 73 antibody, anti-CD 123 antibody, and anti-CD 86 antibody is in order: PC7, BV786, ECD, BV421, BV510, APC-CY7, PC5.5, BV480, FITC, PE, PE, BV650, BV605, BV750, and V450.
In some embodiments, the anti-CD 19 antibody, anti-CD 10 antibody, anti-CD 34 antibody, anti-CD 20 antibody, anti-CD 45 antibody, anti-CD 22 antibody, anti-CD 24 antibody, anti-CD 38 antibody, anti-CD 81 antibody, anti-CD 58 antibody, anti-CD 13 antibody, anti-CD 33 antibody, anti-CD 66c antibody, anti-CD 73 antibody, anti-CD 123 antibody, and anti-CD 86 antibody have titers of 2.5 μl, 0.625 μl, 5 μl, 1.25 μl, 0.625 μl, 4 μl, 2.5 μl, 5 μl, 2.5 μl, 1.25 μl, 2.5 μl, 4 μl, respectively.
In some of these embodiments, the antibody is a monoclonal antibody.
The invention also provides application of the antibody composition in preparation of a kit for detecting acute B lymphocyte leukemia tiny residues.
The invention also provides a kit for detecting the tiny residue of the acute B lymphocyte leukemia, which can be used for detecting the tiny residue of the acute B lymphocyte leukemia, and is quick, sensitive and accurate.
The specific technical scheme for realizing the aim of the invention is as follows:
a kit for detecting acute B-lymphoblastic leukemia minimal residual, comprising the above antibody composition.
In some of these embodiments, the kit further comprises a nucleic acid dye 7-AAD.
In some embodiments, the kit further comprises a hemolysin, a wash solution and a fixative solution, wherein the wash solution is a PBS solution containing 0.8% -1.2% fetal bovine serum, and the fixative solution is a PBS solution containing 0.8% -1.2% paraformaldehyde.
The invention also provides a method for detecting the small residues of the acute B lymphocyte leukemia, which can realize that the detection of 17 indexes can be completed by only one tube, and has the advantages of less sample size requirement, sensitivity and rapidness.
The specific technical scheme for realizing the aim of the invention is as follows:
a method for detecting acute B-lymphoblastic leukemia minimal residual, said method obtaining expression patterns and intensities of individual fluorescein-labeled antibodies of non-diagnostic interest, comprising the steps of:
(1) Adding nucleic acid dye 7-ADD and fluorescein labeled antibody composition into flow tube, and adding 1×10 antibody 8 ～5×10 8 Single cell suspension of each sample cell to be detected is uniformly mixed and incubated for 15-20 minutes at room temperature and in a dark place;
the fluorescein-labeled antibody composition is as follows: the fluorescein PC7, BV786, ECD, BV421, BV510, APC-CY7, PC5.5, BV480, FITC, PE, PE, BV650, BV605, BV750, and V450 labeled anti-CD 19 antibody, anti-CD 10 antibody, anti-CD 34 antibody, anti-CD 20 antibody, anti-CD 45 antibody, anti-CD 22 antibody, anti-CD 24 antibody, anti-CD 38 antibody, anti-CD 81 antibody, anti-CD 58 antibody, anti-CD 13 antibody, anti-CD 33 antibody, anti-CD 66c antibody, anti-CD 73 antibody, anti-CD 123 antibody, and anti-CD 86 antibody were used sequentially;
the titer of the antibodies was 2.5. Mu.L, 0.625. Mu.L, 5. Mu.L, 1.25. Mu.L, 0.625. Mu.L, 4. Mu.L, 2.5. Mu.L, 5. Mu.L, 2.5. Mu.L, 1.25. Mu.L, 2.5. Mu.L, 4. Mu.L in this order;
(2) Adding 380-420 mul of hemolysin into the flow tube, uniformly mixing, standing in a dark place until hemolysis is clear, centrifuging, discarding the supernatant, washing with PBS solution containing 0.8-1.2% of fetal bovine serum, centrifuging, discarding the supernatant, and re-suspending cells with PBS solution containing 0.8-1.2% of paraformaldehyde to obtain cell suspension of a sample to be detected;
(3) Detecting the cell suspension of the sample to be detected in the step (2) by using a full spectrum flow cytometer, and circling a target cell group in a cell scatter diagram of the sample to be detected; the gate setting mode of the full spectrum flow cytometer is as follows: sequentially removing dead cells, fragments and adherent cells, and gating by using an anti-CD 45 antibody-SSC, an anti-CD 19 antibody-anti-CD 10 antibody, an anti-CD 19 antibody-anti-CD 34 antibody, an anti-CD 22 antibody-anti-CD 34 antibody and an anti-CD 22 antibody-anti-CD 10 antibody, wherein target cell groups, namely original B cells and naive B cells, are precisely circled by a double-parameter scatter diagram;
(4) Analyzing the expression mode and intensity of each fluorescein-labeled antibody in the target cell population of the sample to be detected, comparing the expression mode with the expression mode template of the normal control B progenitor cell antibody, and detecting whether each fluorescein-labeled antibody of the sample to be detected falls on the expression mode template of the normal control B progenitor cell antibody;
the method for establishing the expression pattern template of the normal control B progenitor cell antibody comprises the following steps: detecting cell suspension of 20-40 normal control samples by using a full spectrum flow cytometer, circling the B progenitor cells in a normal control sample cell scatter diagram, analyzing the expression mode and the intensity of each fluorescein-labeled antibody in the B progenitor cells of the normal control sample to obtain the expression mode of the normal cells, and establishing an expression mode template of the normal control B progenitor cell antibody; the gating mode of the full spectrum flow cytometer is as described in the step (3).
In some embodiments, the fluorescein-labeled antibody of step (4) is one or more of an anti-CD 19 antibody-anti-CD 34 antibody, an anti-CD 19-antibody CD38, an anti-CD 19 antibody-anti-CD 13 antibody, an anti-CD 19 antibody-anti-CD 33 antibody, an anti-CD 19 antibody-anti-CD 58 antibody, an anti-CD 19 antibody-anti-CD 66c antibody, an anti-CD 19 antibody-anti-CD 123 antibody, an anti-CD 19 antibody-anti-CD 81 antibody, an anti-CD 19 antibody-anti-CD 86 antibody, an anti-CD 19 antibody-anti-CD 73 antibody, an anti-CD 19 antibody-anti-CD 20 antibody, an anti-CD 19 antibody-anti-CD 22 antibody, an anti-CD 19 antibody-anti-CD 24 antibody pair.
In some of these embodiments, the centrifugation in step (2) is a 500g centrifugal force, centrifugation for 5min.
Compared with the prior art, the invention has the following beneficial effects:
(1) The antibody composition for detecting the micro residue of the acute B lymphocyte leukemia at least comprises an antibody composition consisting of an anti-CD 19 antibody, an anti-CD 10 antibody, an anti-CD 34 antibody and an anti-CD 22 antibody, and four antibodies of the anti-CD 19 antibody, the anti-CD 10 antibody, the anti-CD 34 antibody and the anti-CD 22 antibody are used as marker skeleton antibodies for gating, so that target cells can be accurately locked, the influence of immune escape of target cells of a CAR-T targeted therapeutic drug on a detection result can be effectively avoided, and the missed diagnosis phenomenon caused after CAR-T targeted therapy can be prevented.
(2) The antibody composition for detecting the tiny residues of the acute B lymphocyte leukemia comprises 12 antibodies and nucleic acid dyes 7-AAD, and the 17 detection indexes cover the expression modes of various abnormal cells of the acute B lymphocyte leukemia, such as over-expression (for observing the expression of CD58 antigen, CD66c antigen, CD73 antigen, CD123 antigen and CD86 antigen), deletion expression (for observing the expression of CD38 antigen, CD81 antigen and CD24 antigen), cross-line expression (for observing the expression of CD13/CD33 antigen, because the CD13/CD33 antigen belongs to a myeloid common antigen and should not be expressed in a B cell line), and space-time staggered expression (for observing the expression of CD20 antigen and CD34 antigen); the antibodies are respectively matched with the antibodies of the specific fluorescein, and the different antibodies are matched with the specific fluorescein, so that when the method is applied to flow cytometry to detect the tiny residues of the acute B lymphocyte leukemia, all the fluorescein of each channel can achieve excellent dyeing effect.
(3) The method for detecting the acute B lymphocyte leukemia micro-residue by using the full spectrum flow cytometer can realize that only one tube is needed to simultaneously complete the detection of 17 clinical indexes which occur at high frequency, has little sample size requirement, does not need to be reused for skeleton antibody, has low reagent cost, improves the detection rate of leukemia micro-residue cells, and effectively overcomes the defects of no unified standard and easy missed diagnosis in the prior art; the standardized detection method and analysis flow can effectively improve the accuracy of MRD detection, and the detection sensitivity is further improved to 10 by improving the number of cells obtained from a single tube sample from 50 ten thousand to 500 ten thousand -5 (sensitivity is mainly influenced by the number of cells, the traditional flow cytometer is limited in detection channels, if the same number of indexes are detected, a plurality of tubes are needed to be separated, so that the number of cells in each tube is small, the full spectrum flow cytometer can complete detection by only making one tube, so that the collected samples are all used in one tube, the number of cells which can be obtained is large), and the flow cytometer can be used for detecting tiny residual focus after treatment, can also be used for screening tumor markers of primary patients, and provides a basis for clinical diagnosis and treatment.
Drawings
FIG. 1 is a two-parameter scattergram of 7AAD-SSC in example 3 of the present invention.
FIG. 2 is a scatter plot of FSC-SSC dual parameters in example 3 of the present invention.
FIG. 3 is se:Sub>A scatter plot of FSC-A-FSC-H parameters in example 3 of the present invention.
FIG. 4 shows the distribution of lymphocytes, monocytes, granulocytes, CD45 weak positive and CD45 negative after gating with anti-CD 45 antibody-SSC in example 3 of the present invention.
FIG. 5 shows the target cell population found in example 3 of the present invention.
FIGS. 6 to 17 are graphs showing the expression intensities of fluorescent antibodies in the target cell population of the sample to be tested in example 3 of the present invention.
FIG. 18 is a two-parameter scattergram of 7AAD-SSC in example 4 of the present invention.
FIG. 19 is a scatter plot of FSC-SSC dual parameters in example 4 of the present invention.
FIG. 20 is se:Sub>A scatter plot of FSC-A-FSC-H parameters in example 4 of the present invention.
FIG. 21 shows the distribution of lymphocytes, monocytes, granulocytes, CD45 weak positive and CD45 negative after gating with anti-CD 45 antibody-SSC in example 4 of the present invention.
FIG. 22 shows the target cell population found in example 4 of the present invention.
FIGS. 23 to 34 are graphs showing the expression intensities of fluorescent antibodies in the target cell population of the sample to be tested in example 4 of the present invention.
FIG. 35 is a graph showing the sensitivity analysis of a conventional flow assay.
FIG. 36 is a graph showing the operation of sensitivity analysis in example 5 of the present invention.
FIG. 37 is a two-parameter scatter plot of FSC-SSC in comparative example 1.
FIG. 38 is se:Sub>A two-parameter scatter plot of FSC-A-FSC-H of comparative example 1.
FIG. 39 shows the distribution of lymphocytes, monocytes, granulocytes, CD45 weak positive and CD45 negative after gating with anti-CD 45 antibody-SSC in comparative example 1.
FIG. 40 is a cell population of interest found in comparative example 1.
FIGS. 41 to 45 are graphs showing the expression intensities of fluorescent antibodies in the target cell population of the sample to be tested in the comparative example.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Unless otherwise indicated, it may be carried out according to methods set forth in "procedure for temporary examination" and references cited herein, as would be familiar to those skilled in the art. Wherein, the raw materials of the used reagents are all commercial products and can be purchased through public channels.
In the present invention, antibodies per se are well known to the person skilled in the art, which specifically bind (anti) the corresponding antigen. The antibody may be a monoclonal antibody or a polyclonal antibody, and in the embodiment of the present invention, a monoclonal antibody is preferable. The antibody may be of murine origin or rabbit origin. In the present invention, the antigens of each antibody, i.e., each cell surface antigen (CD 19, CD10, CD34, CD20, CD45, CD22, CD24, CD38, CD81, CD58, CD13, CD33, CD66c, CD73, CD123 and CD 86), are human cell surface antigens.
The invention is described in further detail below with reference to specific embodiments and figures.
EXAMPLE 1 antibody composition for detecting acute B-lymphoblastic leukemia minimal residual
An antibody composition for detecting minute residues of acute B-lymphoblastic leukemia of this example, comprising (each antibody is named antigen and fluorescent label): CD19-PC7, CD10-BV786, CD34-ECD, CD20-BV421, CD45-BV510, CD22-APC, CD24-APC-CY7, CD38-PC5.5, CD81-BV480, CD58-FITC, CD13-PE, CD33-PE, CD66c-BV650, CD73-BV605, CD123-BV750, and CD86-V450. These fluorescein-linked antibodies can be obtained directly by purchasing through public channels, and the product numbers and the manufacturers of the antibodies in the embodiment of the invention are shown in Table 1.
The monoclonal antibodies obtained in the market were sampled separately and subjected to concentration gradient verification to determine the optimal amounts of each antibody, and the specific amounts are shown in Table 1.
TABLE 1 antibody information and amounts
Example 2 kit for detecting acute B lymphocyte leukemia micro residue
The kit for detecting acute B-lymphocyte leukemia tiny residues in this embodiment comprises:
(1) The antibody composition of example 1, in which the titer of each antibody is shown in table 1.
(2) Nucleic acid dye 7-AAD
(3) Haemolysin
(4) Washing solution (PBS+1% fetal bovine serum)
(5) Fixing solution (PBS+1% paraformaldehyde)
(6) And a flow tube for use with a full spectrum flow cytometer.
Example 3 method for detecting acute B lymphocyte leukemia micro residue
By using the kit of example 2, a full spectrum flow cytometer was used to detect microscopic residues of the bone marrow sample cells to be tested (from the gold domain medical flow cytometry laboratory, the treated B-ALL patient, the collected bone marrow aspirate, heparin anticoagulation) acute B-lymphoblastic leukemia were added during collection. The method specifically comprises the following steps:
1. establishing an expression Pattern template for a Normal control antibody
(1) The amount of nucleic acid dye 7-ADD, fluorescein-conjugated monoclonal antibody of Table 1 of example 1 was added to a flow tube.
(2) Mixing bone marrow cells of 40 normal control groups (bone marrow of non-B-ALL patient, sample containing B progenitor cells, and derived from gold domain medical flow cell laboratory) at 1×10 concentration 8 ～5×10 8 40 parts of normal control single cell suspension were prepared per ml.
(3) 100 μl of normal control single cell suspension was added to the flow tube, mixed well by vortexing, and incubated at room temperature in the dark for 15min.
(4) After incubation, 400 μl of hemolysin was added to the flow tube, vortexing was performed, standing in the dark, after hemolysis was performed and light transmission, the flow tube was centrifuged at 500g for 5min, the supernatant was discarded, 2ml of washing solution (PBS+1% fetal bovine serum) was added, vortexing was performed, 500g of centrifugal force was performed, centrifugation was performed for 5min, the supernatant was discarded, and 400 μl of fixative (PBS+1% paraformaldehyde) was added to resuspend the cells to obtain a normal control cell suspension.
(5) Full spectrum flow cytometry using a CYTEK-NL-CLC (instrument model:NL-CLCV 16B 14R8 (SN: NL-1044)) detects 40 normal control cell suspensions, acquires 500-1000 thousands of cell populations, circles 40 normal control B progenitor cells, analyzes the expression pattern and intensity of each fluorescein-labeled antibody in 40 normal control sample B progenitor cells to obtain the expression pattern of the normal cells, and establishes an expression pattern template of the normal control B progenitor cell antibodies;
the gate setting mode of the full spectrum flow cytometer is as follows:
(a) Dead cells were removed using a 7AAD-SSC two-parameter scatter plot;
(b) Debris was removed using an FSC (Forward Angle light Scattering) -SSC double parameter scatter plot;
(c) Removing adherent cells using an FSC-se:Sub>A (arese:Sub>A, se:Sub>A) -FSC-H (height, H) dual-parameter scattergram;
(d) The individual haemocytes phylum were set up using anti-CD 45 antibody-SSC, and lymphocytes, monocytes, granulocytes were roughly observed, and whether obvious tumor cells or abnormal cells were present. Depending on the expression of the anti-CD 45 antibody-SSC, the cell population can be divided into 5 regions, respectively granulocyte (Gran), monocyte (Mono), lymphocyte (Lym), CD45 weak positive (CD 45 DIM), CD45 negative (CD 45 neg);
(e) Gating with anti-CD 19 antibody-anti-CD 10 antibody, anti-CD 19 antibody-anti-CD 34 antibody, anti-CD 22 antibody-anti-CD 34 antibody and anti-CD 22 antibody-anti-CD 10 antibody, and precisely circling B progenitor cells using a two-parameter scatter plot.
2. Detection ofAnalysis of antibody expression patterns of test sample cells
The steps (1) to (4) are the same as those described above, wherein the concentration of the catalyst in the step (2) is 1X 10 8 ～5×10 8 Single cell suspension of individual/ml of bone marrow sample cells to be tested.
(5) Detecting a cell suspension of a sample to be detected by using a CYTEK-NL-CLC full spectrum flow cytometer, obtaining 500-1000 thousands of cell groups, and circling a target cell group in a scatter diagram of the sample to be detected;
the gating method and the gating result of the full spectrum flow cytometer are as follows:
(a) Dead cells were removed using a 7AAD-SSC two-parameter scatter plot (fig. 1);
(b) Debris was removed using an FSC (Forward Angle light Scattering) -SSC double parameter scatter plot (FIG. 2);
(c) Adherent cells were removed using an FSC-se:Sub>A (arese:Sub>A, se:Sub>A) -FSC-H (height, H) dual-parameter scattergram (fig. 3);
(d) The individual haemocytes phylum were set up using anti-CD 45 antibody-SSC, and lymphocytes, monocytes, granulocytes were roughly observed, and whether obvious tumor cells or abnormal cells were present. Depending on the expression of anti-CD 45 antibody-SSC, the cell population can be divided into 5 regions (fig. 4), 5 regions, respectively granulocytes (Gran) (middle upper region in fig. 4), monocytes (Mono) (right upper region in fig. 4), lymphocytes (Lym) (right lower region in fig. 4), CD45 weak positive (CD 45 DIM) (middle lower region in fig. 4), CD45 negative (CD 45 neg) (left region in fig. 4); typically, the B progenitor cells are located within the CD45 DIM region; whereas tumor cells may appear in the CD45 DIM or CD45 negative regions.
(e) Gating with anti-CD 19 antibody-anti-CD 10 antibody, anti-CD 19 antibody-anti-CD 34 antibody, anti-CD 22 antibody-anti-CD 34 antibody and anti-CD 22 antibody-anti-CD 10 antibody, and precisely circling the target cell population, i.e., primitive B cells and naive B cells, by a two-parameter scatter plot.
The target cell population of the test sample of this example was CD19+CD10+ cells, which were located in the CD45 DIM region at a ratio of 16.83% (FIG. 5).
(6) And analyzing the expression intensity of each fluorescent antibody in the target cell population of the sample to be tested (preferably, the following antibody pairs: anti-CD 19-anti-CD 34 antibody, anti-CD 19-antibody CD38, anti-CD 19-anti-CD 13 antibody, anti-CD 19-anti-CD 33 antibody, anti-CD 19-anti-CD 58 antibody, anti-CD 19-anti-CD 66c antibody, anti-CD 19-anti-CD 123 antibody, anti-CD 19-anti-CD 81 antibody, anti-CD 19-anti-CD 86 antibody, anti-CD 19-anti-CD 73 antibody, anti-CD 19-anti-CD 20 antibody, anti-CD 19-anti-CD 22 antibody, anti-CD 19-anti-CD 24 antibody), and comparing the fluorescent antibody with the expression pattern template of the normal control B progenitor cell antibody established in the step 1, and if each fluorescent antibody in the target cell population of the sample to be tested falls on the expression pattern template of the normal control B progenitor cell antibody, the cell population is suspected to be a normal cell population, otherwise tumor cell population.
The results are shown in FIGS. 6 to 17.
As can be seen from fig. 6 to 17, the cell population antibody expression patterns of the sample to be tested all fall into the normal control B progenitor cell antibody expression pattern template, each antibody has a normal expression pattern, and most of the primitive B cells and the naive B cells fall into the gates of the normal control expression pattern template, which indicates that the primitive B cells and the naive B cells are normal, and the immunophenotypes of the primitive B cells and the naive B cells are CD58-, CD33-, CD13-, CD20-, cd22+, cd34+ in a small amount, CD123-, cd24+, cd38+, cd81+, CD66c-, CD73-, CD86-, and the expression intensities and the expression patterns of the primitive B cells and the naive B cells are normal. Namely, the target cell group of the sample to be detected is judged to be the cell group with normal immunophenotype in the antibody expression pattern template of the normal control B progenitor cells.
By adopting morphological analysis of bone marrow smear, no obvious residual tumor cells are seen, which indicates that the bone marrow is completely relieved and is consistent with the results.
EXAMPLE 4 method for detecting acute B-lymphoblastic leukemia minimal residual disease
The kit for detecting acute B lymphocyte leukemia micro-residue of example 2 is used, a full spectrum flow cytometer is used for detecting the bone marrow sample cells to be detected to detect the micro-residue of acute B lymphocyte leukemia of the sample cells to be detected (abnormal cells, namely bone marrow puncture material of a patient with B-ALL, which is confirmed by diagnosis), heparin is added for anticoagulation during collection, and the micro-residue is derived from a gold domain medical flow cytometry laboratory), and the kit comprises the following steps:
steps (1) - (4) are the same as steps (1) - (4) in example 3 to obtain resuspended cells of the sample to be tested.
(5) Detecting a cell suspension of a sample to be detected by using a CYTEK-NL-CLC full spectrum flow cytometer, obtaining 500-1000 thousands of cell groups, and circling a target cell group in a scatter diagram of the sample to be detected;
the gating method and the gating result of the full spectrum flow cytometer are as follows:
(a) Dead cells were removed using a 7AAD-SSC two-parameter scatter plot (fig. 18);
(b) Debris was removed using FSC (Forward scatter) -SSC dual parameter scatter plot (fig. 19);
(c) Adherent cells were removed using an FSC-se:Sub>A (arese:Sub>A, se:Sub>A) -FSC-H (height, H) dual-parameter scattergram (fig. 20);
(d) The individual haemocytes phylum were set up using anti-CD 45 antibody-SSC, and lymphocytes, monocytes, granulocytes were roughly observed, and whether obvious tumor cells or abnormal cells were present. Depending on the expression of anti-CD 45 antibody-SSC, the cell population can be divided into 5 regions (fig. 21), 5 regions, respectively granulocytes (Gran) (middle upper region in fig. 21), monocytes (Mono) (right upper region in fig. 21), lymphocytes (Lym) (right lower region in fig. 21), CD45 weak positive (CD 45 DIM) (middle lower region in fig. 21), CD45 negative (CD 45 neg) (left region in fig. 21); typically, the B progenitor cells are located within the CD45 DIM region; whereas tumor cells may appear in the CD45 DIM or CD45 negative regions.
(e) Gating with anti-CD 19 antibody-anti-CD 10 antibody, anti-CD 19 antibody-anti-CD 34 antibody, anti-CD 22 antibody-anti-CD 34 antibody and anti-CD 22 antibody-anti-CD 10 antibody, and precisely circling the target cell population, i.e., primitive B cells and naive B cells, by a two-parameter scatter plot.
The target cell population of the test sample of this example was CD19+CD10+ cells, located in the CD45 DIM region, at a ratio of 77.55% (FIG. 22).
(6) The expression intensity of each fluorescent antibody in the target cell population of the sample to be tested (preferably, the following antibody pairs: anti-CD 19-anti-CD 34 antibody, anti-CD 19-antibody CD38, anti-CD 19-anti-CD 13 antibody, anti-CD 19-anti-CD 33 antibody, anti-CD 19-anti-CD 58 antibody, anti-CD 19-anti-CD 66c antibody, anti-CD 19-anti-CD 123 antibody, anti-CD 19-anti-CD 81 antibody, anti-CD 19-anti-CD 86 antibody, anti-CD 19-anti-CD 73 antibody, anti-CD 19-anti-CD 20 antibody, anti-CD 19-anti-CD 22 antibody, anti-CD 19-anti-CD 24 antibody) is analyzed, and the comparison is performed with the normal control B progenitor cell expression pattern template established in step 1 of example 3. If each fluorescent antibody in the target cell population of the sample to be tested falls on the normal control B progenitor cell expression pattern template, the target cell population is suspected to be a normal cell population, otherwise tumor cell population.
The results are shown in FIGS. 23 to 34.
As can be seen from fig. 23 to 34, the target cell population antibody expression pattern of the test sample is partially located inside the gate and partially located outside the gate, and the immunophenotype of the CD19 positive cells is cd58+, cd33-, cd13-, cd20-, cd22+, cd34+, cd123+, cd24+, cd38-, cd81+, cd66c+, cd73+, cd86-, which indicates that the cell population is B-ALL tumor cells, which is consistent with the clinical test results.
EXAMPLE 5 methodological verification of the detection method of the invention
1. Accuracy (Specificity)
To evaluate the accuracy of the detection method of the present invention, 5 cases of bone marrow sample cells (samples were derived from gold domain medical flow cell laboratories) were selected for clinical confirmation of positive and negative, and the detection was performed by the method of example 3 of the present invention and compared with the conventional flow detection method (the same antibody composition as example 3 of the present invention was used and the same gating method as example 3 of the present invention was used) to evaluate the accuracy of the method of the present invention. The test results are shown in Table 2.
Table 2 accuracy check results
/>
As can be seen from Table 2, the detection method of the present invention has good consistency with the conventional flow detection method in terms of the expression profile and expression pattern of the detected CD molecules by using the antibody composition and the gate setting method of the present invention.
2. Precision (Precision)
Because a large number of bone marrow samples are difficult to obtain, 2 clinical MRD positive samples (all the samples are from a gold domain medical flow cytometry laboratory) are selected for precision evaluation, 3 repeated determinations are respectively carried out according to a specified operation method in a relatively short time under stable conditions, and the MRD results and the variation of antigen expression patterns are evaluated to investigate the random errors of the method.
The measurement results are shown in tables 3 and 4.
TABLE 3 results of precision test of first sample
/>
The results in Table 3 show that the MRD measurement result CV value was 0.01% and the antigen expression pattern was consistent, and the precision result in the batch was "pass".
TABLE 4 results of precision test of second sample
The results in Table 4 show that the MRD measurement result CV value was 0.49% and the antigen expression pattern was consistent, and the precision result in the batch was "pass".
3. Sensitivity (Assay Sensitivity)
1 sample positive for clinical B-ALL MRD (sample is from gold domain medical flow cell laboratory) was selected according to positive sample and normal sample (sample is from gold domain medical flow cell laboratory) 1:1, 1:9, 1:99, 1:999, 1:9999, diluting the positive sample, detecting by adopting a traditional flow detection method, recording the proportion of the minimum MRD which can be detected, and determining the sensitivity of the traditional flow detection method.
The results of the sensitivity verification of the conventional flow assay are shown in table 5.
TABLE 5 sensitivity verification results for conventional flow detection methods
The actual detection value is taken as an abscissa, the expected value is taken as an ordinate, and a working curve is drawn, as shown in fig. 35, wherein the working curve is y=0.99964x+0.05288, and r 2 =0.9999, the sensitivity of the conventional flow detection method was determined to be 0.01%.
The same clinical B-ALL MRD positive specimen (the sample is from the gold domain medical flow cell laboratory) was selected according to the positive sample and the normal sample (the sample is from the gold domain medical flow cell laboratory) of 1:1, 1:79, 1:799, 1: 7999. 1: 79999. 1:799999, 0:1, positive samples were diluted, tested using the method of example 3, the proportion of the minimum MRD detectable was recorded, and the sensitivity of the method of the invention was determined.
The measurement results are shown in Table 6.
TABLE 6 sensitivity verification results of the inventive method
| Dilution ratio | MRD expected value (%) | MRD actual detection value (%) | Difference (%) |
| 1:1 | 93.4000 | 91.5126 | -2.02 |
| 1:79 | 1.0378 | 0.8194 | -21.04 |
| 1:799 | 0.1037 | 0.0950 | -8.39 |
| 1：7999 | 0.0104 | 0.0105 | 0.96 |
| 1：79999 | 0.0010 | 0.0011 | 10.00 |
| 1：799999 | 0.0001 | 0.0001 | 0.00 |
| 0:1 | 0 | 0.0000 | N/A |
The actual detection value is taken as an abscissa, the expected value is taken as an ordinate, and a working curve is drawn, as shown in fig. 36, wherein the working curve is y= 0.9831x-0.7454, and R 2 = 0.99974, the correlation is good, and the sensitivity of the method of the invention is determined to be 0.001%.
It follows that the sensitivity of the method of the present invention far exceeds that of conventional flow detection methods.
Comparative example 1
Referring to the method for detecting acute B-lymphocyte leukemia cells of example 4, the test sample cells of example 4 were subjected to acute B-lymphocyte leukemia tiny residue detection (gating anti-CD 45 antibody-SSC, differing from example 4 in that the antibody composition used in comparative example 1 was anti-CD 45 antibody, anti-CD 66c antibody, anti-CD 34 antibody, anti-CD 19 antibody, anti-CD 10 antibody and anti-CD 58 antibody, and the detection equipment used was a conventional flow cytometer). The expression intensity of the fluorescent antibody of the cell population of interest against the anti-CD 34 antibody-anti-CD 19 antibody, anti-CD 19 antibody-anti-CD 58 antibody, anti-CD 19 antibody-anti-CD 66c antibody, anti-CD 19 antibody-anti-CD 10 antibody was analyzed and compared with the expression pattern template of the normal control B progenitor antibody in example 3.
The results are shown in FIGS. 36 to 42. The results showed that with the antibody composition of this comparative example, the target cells were all in the normally expressed gate, and it could not be judged as abnormal tumor cells.
The results of the detection of the anti-CD 81 antibody by adding the anti-CD 24 antibody are shown in FIG. 43 and FIG. 44, and the results show that the expression pattern of the target cell is different from that of the normal control B progenitor cell antibody, and thus it can be judged as an abnormal tumor cell.
The results of this comparative example show that there may be missed diagnosis due to fewer antibody pairs detected.
Comparative example 2
Referring to the method for detecting acute B-lymphocyte leukemia cells of example 4, the test sample cells of example 4 were subjected to acute B-lymphocyte leukemia tiny residue detection (gating anti-CD 45 antibody-SSC, anti-CD 34 antibody, and the difference from example 4 is that the antibody composition used in comparative example 2 was anti-CD 117 antibody, anti-CD 45 antibody, anti-CD 34 antibody, anti-CD 13 antibody, anti-CD 333 antibody, anti-HLA-DR antibody, anti-CD 7 antibody, anti-CD 11B antibody, anti-CD 15 antibody and anti-CD 38 antibody). The expression intensities of the cell population fluorescent antibodies of interest against CD34 antibody-anti-CD 117 antibody, anti-CD 34 antibody-anti-CD 13 antibody, anti-CD 34 antibody-anti-CD 33 antibody, anti-CD 34 antibody-anti-CD 7 antibody, anti-CD 34 antibody-anti-CD 15 antibody, anti-CD 34 antibody-anti-CD 11B antibody, anti-CD 34 antibody-anti-CD 38 antibody were analyzed and compared with the expression pattern template of the normal control B progenitor antibody in example 3.
The result shows that: since the detected antibody pairs are mainly expressed in myeloid primary cells for monitoring myeloid leukemia, primary B cells and naive B cells cannot be effectively identified and cannot be used for the detection of microscopic residues of acute B lymphocyte leukemia.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (7)
1. An antibody composition for detecting acute B-lymphocyte leukemia micro-residues, wherein the antibody composition consists of an anti-CD 19 antibody, an anti-CD 10 antibody, an anti-CD 34 antibody, an anti-CD 22 antibody, an anti-CD 20 antibody, an anti-CD 45 antibody, an anti-CD 24 antibody, an anti-CD 38 antibody, an anti-CD 81 antibody, an anti-CD 58 antibody, an anti-CD 13 antibody, an anti-CD 33 antibody, an anti-CD 66c antibody, an anti-CD 73 antibody, an anti-CD 123 antibody, and an anti-CD 86 antibody.
2. The antibody composition for detecting micro-remnants of acute B-lymphoblastic leukemia according to claim 1, wherein said anti-CD 19 antibody, anti-CD 10 antibody, anti-CD 34 antibody, anti-CD 20 antibody, anti-CD 45 antibody, anti-CD 22 antibody, anti-CD 24 antibody, anti-CD 38 antibody, anti-CD 81 antibody, anti-CD 58 antibody, anti-CD 13 antibody, anti-CD 33 antibody, anti-CD 66c antibody, anti-CD 73 antibody, anti-CD 123 antibody and anti-CD 86 antibody have the following clone numbers in order: j3-119, H10a, 581, 2H7, H130, S-HCL-1, ML5, HIT2, JS-81, AICD58, L138, D3HL60.251, B6.2/CD66, AD2, 9F5 and 2331.
3. The antibody composition for detecting micro-remnants of acute B-lymphoblastic leukemia according to claim 1, wherein said anti-CD 19 antibody, anti-CD 10 antibody, anti-CD 34 antibody, anti-CD 20 antibody, anti-CD 45 antibody, anti-CD 22 antibody, anti-CD 24 antibody, anti-CD 38 antibody, anti-CD 81 antibody, anti-CD 58 antibody, anti-CD 13 antibody, anti-CD 33 antibody, anti-CD 66c antibody, anti-CD 73 antibody, anti-CD 123 antibody and anti-CD 86 antibody have a titer of 2.5 μl, 0.625 μl, 5 μl, 1.25 μl, 0.625 μl, 4 μl, 2.5 μl, 5 μl, 2.5 μl, 1.25 μl, 2.5 μl, 4 μl in order.
4. The antibody composition for detecting microscopic residues of acute B lymphoblastic leukemia according to claim 1, wherein said antibodies are all fluorescein-labeled antibodies, said labeled fluorescein is in the order of anti-CD 19, anti-CD 10, anti-CD 34, anti-CD 20, anti-CD 45, anti-CD 22, anti-CD 24, anti-CD 38, anti-CD 81, anti-CD 58, anti-CD 13, anti-CD 33, anti-CD 66c, anti-CD 73, anti-CD 123, and anti-CD 86: PC7, BV786, ECD, BV421, BV510, APC-CY7, PC5.5, BV480, FITC, PE, PE, BV650, BV605, BV750, and V450.
5. The antibody composition for detecting small residues of acute B-lymphoblastic leukemia according to any one of claims 1 to 4, wherein said antibody is a monoclonal antibody.
6. A kit for detecting acute B-lymphoblastic leukemia minimal residual, characterized in that said kit comprises the antibody composition according to any one of claims 1 to 5.
7. The kit for detecting microscopic residues of acute B-lymphoblastic leukemia according to claim 6, wherein said kit further comprises nucleic acid dye 7-AAD, hemolysin, a washing solution and a fixing solution, wherein said washing solution is a PBS solution containing 0.8% -1.2% fetal bovine serum, and said fixing solution is a PBS solution containing 0.8% -1.2% paraformaldehyde.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111113241.3A CN113933511B (en) | 2021-09-18 | 2021-09-18 | Antibody composition and method for detecting acute B lymphocyte leukemia tiny residue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111113241.3A CN113933511B (en) | 2021-09-18 | 2021-09-18 | Antibody composition and method for detecting acute B lymphocyte leukemia tiny residue |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113933511A CN113933511A (en) | 2022-01-14 |
| CN113933511B true CN113933511B (en) | 2024-03-22 |
Family
ID=79276555
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111113241.3A Active CN113933511B (en) | 2021-09-18 | 2021-09-18 | Antibody composition and method for detecting acute B lymphocyte leukemia tiny residue |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113933511B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104471393A (en) * | 2012-06-14 | 2015-03-25 | 鹿特丹伊拉斯姆斯大学医疗中心 | Methods, reagents and kits for detecting minimal residual disease |
| CN109655616A (en) * | 2018-12-19 | 2019-04-19 | 广州金域医学检验中心有限公司 | Detect the composite reagent and system of acute myeloid leukemia cell |
| CN109884313A (en) * | 2019-02-22 | 2019-06-14 | 武汉康圣达医学检验所有限公司 | The detection kit of B-lineage Acute Lymphocyte Leukemia minimal residual |
-
2021
- 2021-09-18 CN CN202111113241.3A patent/CN113933511B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104471393A (en) * | 2012-06-14 | 2015-03-25 | 鹿特丹伊拉斯姆斯大学医疗中心 | Methods, reagents and kits for detecting minimal residual disease |
| CN107422122A (en) * | 2012-06-14 | 2017-12-01 | 鹿特丹伊拉斯姆斯大学医疗中心 | For detecting method, reagent and the kit of minimal residual disease |
| CN109655616A (en) * | 2018-12-19 | 2019-04-19 | 广州金域医学检验中心有限公司 | Detect the composite reagent and system of acute myeloid leukemia cell |
| CN109884313A (en) * | 2019-02-22 | 2019-06-14 | 武汉康圣达医学检验所有限公司 | The detection kit of B-lineage Acute Lymphocyte Leukemia minimal residual |
Non-Patent Citations (5)
| Title |
|---|
| CD81联合CD10及CD34在流式细胞术监测儿童B-ALL微小残留病变中的应用价值;方宗宇 等;山东医药;第59卷;第75-78页 * |
| Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia;Theunissen P 等;Blood;第129卷(第3期);第347-357页 * |
| 免疫分型分化抗原与儿童B-ALL早期微小残留病的相关性研究;王月芳;张鸽;江咏梅;高举;;中国实验血液学杂志(第05期);第57-64页 * |
| 急性淋巴细胞白血病免疫表型与微小残留病检测的意义;张丽;罗文达;陈葆国;郭群依;;现代中西医结合杂志(第09期);第1147-1150页 * |
| 流式细胞术检测急性白血病微小残留病变68例临床分析;张静;李芹;陈辉;朴文花;;宁夏医科大学学报(第12期);第106-108页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113933511A (en) | 2022-01-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2516795C (en) | Circulating tumor cells (ctc's): early assessment of time to progression,_survival and response to therapy in metastatic cancer patients | |
| CN109655616B (en) | Combined reagent and system for detecting acute myeloid leukemia cells | |
| CN113777327B (en) | Antibody composition for leukemia/lymphoma immunophenotyping primary screening and application thereof | |
| KR101716555B1 (en) | Methods for detecting 5t4-positive circulating tumor cells and methods of diagnosis of 5t4-positive cancer in a mammalian subject | |
| de Tute | Flow cytometry and its use in the diagnosis and management of mature lymphoid malignancies | |
| Kraan et al. | Flow cytometric immunophenotyping of cerebrospinal fluid | |
| JP6485759B2 (en) | Method for detecting malignancy of peripheral circulating tumor cell unit and kit thereof | |
| CN107402296A (en) | The immunofluorescence dyeing and interpretation method of a kind of circulating tumor cell | |
| CN103826750A (en) | Assay to capture and detect circulating multiple myeloma cells from blood | |
| CN114460311B (en) | Reagent composition for B-ALL/LBL immunophenotyping and application thereof | |
| CN113484520A (en) | Molecular marker and application thereof in detection of angioimmunoblastic T-cell lymphoma | |
| CN115856302A (en) | Antibody composition for mature B cell tumor immunophenotyping, kit, application and system thereof | |
| Statuto et al. | Cytofluorimetric and immunohistochemical comparison for detecting bone marrow infiltration in non-Hodgkin lymphomas: a study of 354 patients | |
| CN116482370B (en) | Antibody combination for screening plasma cell tumor treatment target and/or abnormal phenotype and application thereof | |
| CN109752548B (en) | Combined reagent and system for evaluating prognosis of chronic lymphocytic leukemia | |
| CN117192121A (en) | Antibody composition for MDS and/or AML minimal residual disease detection and application thereof | |
| Chen et al. | Immunophenotyping by multiparameter flow cytometry | |
| CN113933511B (en) | Antibody composition and method for detecting acute B lymphocyte leukemia tiny residue | |
| JP4590109B2 (en) | Products and methods for single-parameter and multi-parameter phenotyping of cells | |
| Gupta et al. | Flowcytometric comparative analysis in acute leukemias between Indian and proposed minimal screening panel | |
| Rashed et al. | Flow Cytometric immunophenotyping of acute leukaemia in adult and its comparison with cytomorphology | |
| RU2639382C1 (en) | Method for estimation of bone marrow aspiration quality during monitoring of minimum residual disease in case of multiple myeloma | |
| WO2018016656A1 (en) | Method for diagnosing myelodysplastic syndrome | |
| Witkowski et al. | Comparison of various diagnostic methods in assessing platelet count in patients with immune thrombocytopenia | |
| CN116338191A (en) | Lung cancer exosome protein marker based on flow technology and application thereof in aspect of lung cancer screening |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Country or region after: China Address after: 510700 No. 10, helix 3 Road, International Biological Island, Huangpu District, Guangzhou City, Guangdong Province Applicant after: GUANGZHOU KINGMED CENTER FOR CLINICAL LABORATORY Address before: 510000, No. 2429, Xingang East Road, Guangzhou, Guangdong, Haizhuqu District Applicant before: GUANGZHOU KINGMED CENTER FOR CLINICAL LABORATORY Country or region before: China |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |