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Published on: May 15, 2019
Biochemical analysis of preliminary clusters in the non-lineage panel
J Naessens1, J M Nthale, P Muiya
1International Livestock Research Institute, Nairobi, Kenya.
Insights
This study refined antibody clusters using flow cytometry and competition assays, identifying new antibodies for myeloid and erythroid cell antigens. These findings expand the repertoire of reagents for cell surface antigen research.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Monoclonal antibodies (mAbs) are crucial tools for cell surface antigen research.
- Previous workshops established antibody panels, but further characterization and expansion are needed.
- Flow cytometry and immunoprecipitation are key techniques for antibody validation.
Purpose of the Study:
- To validate and cluster monoclonal antibodies (mAbs) based on their binding characteristics.
- To identify and characterize novel cell surface antigens using antibody panels.
- To expand the availability of well-defined mAbs for non-lineage and lineage-specific antigens.
Main Methods:
- Preliminary clustering of mAbs using flow cytometry data.
- Competition assays to confirm mAbs binding to the same antigen.
- Immunoprecipitation to estimate antigen molecular weights.
- Characterization of antibody binding to various cell types.
Main Results:
- Selected mAb clusters binding to a broad range of cells for further validation.
- Confirmed specificity of mAbs within clusters through competition assays and molecular weight estimation.
- Identified a new antigen (BoWC14) on myeloid cells and a T lymphocyte subpopulation.
- Characterized a novel erythroid lineage-specific antigen (BoWC15).
Conclusions:
- This study successfully validated and expanded antibody panels for cell surface antigens.
- New reagents (BoWC14 and BoWC15) were identified, enhancing the tools for myeloid, T cell, and erythroid research.
- The findings contribute to a more comprehensive understanding of cell surface markers and their expression patterns.
Abstract:
After the preliminary clustering of the whole workshop panel using flowcytometry data, we selected those clusters that were composed of mAbs that bound a broad range of cells. To obtain evidence that mAbs in each of these preliminary clusters detected the same antigen, they were tested for their capacity to compete with each other for binding to a target cell and the molecular weights of their antigens were estimated after immunoprecipitation. Most preliminary clusters in the non-lineage panel contained control mAbs that had been characterised in one of the previous workshops, and this study therefore increased the number of mAbs available to each of these non-lineage antigens. One new interesting cluster, BoWC14, was described which defined an antigen on myeloid cells and a subpopulation of CD4, CD8 and WC1 T lymphocytes (BT3/8.12 and IL-A155). An additional cluster, which did not fit into any other panel, contained mAbs specific for an antigen restricted to the erythroid lineage and received the nomenclature BoWC15 (ANA8, IL-A135, IL-A137, IL-A138 and IL-A160).
