scholarly journals A High-Throughput Hybridoma Selection Method Using Fluorometric Microvolume Assay Technology

2008 ◽  
Vol 13 (3) ◽  
pp. 210-217 ◽  
Author(s):  
Rozanne Lee ◽  
Mylinh Tran ◽  
Mark Nocerini ◽  
Meina Liang
Keyword(s):  
Cell Surface ◽  
High Throughput ◽  
Antigen Expression ◽  
Surface Antigens ◽  
Antibody Binding ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cell Surface Antigens ◽  
Homogeneous Assay ◽  
Exquisite Specificity ◽  
Promising Type

Monoclonal antibodies (mAb) are not only useful reagents but also represent a promising type of therapeutics due to their high affinity and exquisite specificity for their antigens. A critical step in mAb generation is to identify antigen-specific antibodies. Although enzyme-linked immunosorbent assay (ELISA) has been broadly applied for antibody selection against secreted antigens, an inherent disadvantage for ELISA is the difficulty in identifying antibodies that recognize the native conformation of cell surface antigens. To overcome this drawback, the authors have developed a high-throughput cell-based antibody binding assay using fluorometric microvolume assay technology (FMAT). This method offers a homogeneous assay for detection of antibody binding to its antigen on the cell surface. To distinguish antibodies that bind to antigen on the cell surface from those that bind nonspecifically to cells, the binding is assessed using both antigen-expressing cells and related cells devoid of the antigen expression. This assay can detect antibodies at a concentration as low as 5 ng/mL and cell surface antigen as low as 9000 copies per cell. Results demonstrate that the FMAT method provides a sensitive and homogeneous assay to detect antibody binding to cell surface antigens and is amenable for high-throughput hybridoma selection. ( Journal of Biomolecular Screening 2008:210-217)

Screening Hybridomas for Cell Surface Antigens by High-Throughput Homogeneous Assay and Flow Cytometry

2014 ◽  
pp. 81-103
Author(s):  
Alejandro Uribe-Benninghoff ◽  
Teresa Cabral ◽  
Efthalia Chronopoulou ◽  
Jody D. Berry ◽  
Cindi R. Corbett
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
High Throughput ◽  
Surface Antigens ◽  
Cell Surface Antigens ◽  
Homogeneous Assay

scholarly journals Immunologic classification of lymphocytic leukemias based on monoclonal antibody-defined cell surface antigens

Blood ◽  
1982 ◽  
Vol 59 (2) ◽  
pp. 207-215
Author(s):  
RW Schroff ◽  
KA Foon ◽  
RJ Billing ◽  
JL Fahey
Keyword(s):  
Cell Surface ◽  
B Cell ◽  
Cell Lineage ◽  
Antigen Expression ◽  
Surface Antigens ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Cell Surface Antigens ◽  
Prolymphocytic Leukemia ◽  
Hla Dr

A panel of monoclonal antibodies reactive with normal lymphocyte subsets was used to classify cases of lymphocytic leukemia on the basis of cell surface antigen expression. The antibodies employed were commercially available and included a common framework HLA-DR antibody, two pan-T antibodies (Leu-1 and OKT-3), and antibodies defining cytotoxic/suppressor (Leu-2 and OKT-8) and helper/inducer (Leu-3 and OKT-4) subpopulations of normal T lymphocytes. Cases of ALL could be subgrouped into non-T non-B, pre-T and T-ALL on the basis of reactivity with HLA-DR, Leu-1, and OKT-3 antibodies. Leukemic cells from patients with T-cell CLL could be divided into Leu-2/OKT-8 reactive and Leu- 3/OKT-4 reactive subpopulations, as well as a subgroup in which the majority of cells were unreactive with either of these antibodies. With the exception of one individual, all Sezary cell leukemias expressed a phenotypic pattern similar to that of the Leu-3 subgroup of T-CLL. Malignancies of B-cell lineage (B-CLL, prolymphocytic leukemia, and lymphosarcoma) that were examined were reactive with both the HLA-DR and Leu-1 antibodies. On the contrary, normal B lymphocytes and lymphoid cell lines of B-cell origin did not express surface antigens recognized by the Leu-1 antibody.

scholarly journals Immunologic classification of lymphocytic leukemias based on monoclonal antibody-defined cell surface antigens

Blood ◽  
1982 ◽  
Vol 59 (2) ◽  
pp. 207-215 ◽  
Author(s):  
RW Schroff ◽  
KA Foon ◽  
RJ Billing ◽  
JL Fahey
Keyword(s):  
Cell Surface ◽  
B Cell ◽  
Cell Lineage ◽  
Antigen Expression ◽  
Surface Antigens ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Cell Surface Antigens ◽  
Prolymphocytic Leukemia ◽  
Hla Dr

Abstract A panel of monoclonal antibodies reactive with normal lymphocyte subsets was used to classify cases of lymphocytic leukemia on the basis of cell surface antigen expression. The antibodies employed were commercially available and included a common framework HLA-DR antibody, two pan-T antibodies (Leu-1 and OKT-3), and antibodies defining cytotoxic/suppressor (Leu-2 and OKT-8) and helper/inducer (Leu-3 and OKT-4) subpopulations of normal T lymphocytes. Cases of ALL could be subgrouped into non-T non-B, pre-T and T-ALL on the basis of reactivity with HLA-DR, Leu-1, and OKT-3 antibodies. Leukemic cells from patients with T-cell CLL could be divided into Leu-2/OKT-8 reactive and Leu- 3/OKT-4 reactive subpopulations, as well as a subgroup in which the majority of cells were unreactive with either of these antibodies. With the exception of one individual, all Sezary cell leukemias expressed a phenotypic pattern similar to that of the Leu-3 subgroup of T-CLL. Malignancies of B-cell lineage (B-CLL, prolymphocytic leukemia, and lymphosarcoma) that were examined were reactive with both the HLA-DR and Leu-1 antibodies. On the contrary, normal B lymphocytes and lymphoid cell lines of B-cell origin did not express surface antigens recognized by the Leu-1 antibody.

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