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Updated: Aug 8, 2026
Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells
Published on: June 5, 2015
Homotypic cell aggregation induced by anti-CD44(Pgp-1) monoclonal antibodies and related to CD44(Pgp-1) expression
P C Belitsos1, J E Hildreth, J T August
1Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Insights
Monoclonal antibody H4C4 induces homotypic aggregation of hemopoietic cells by targeting the CD44 (Pgp-1) glycoprotein. This cell adhesion process involves the cytoskeleton and is temperature-sensitive.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Monoclonal antibodies (mAbs) are crucial tools in cell biology research.
- Homotypic aggregation is a key process in cellular interactions and tissue formation.
- The CD44 (Pgp-1) molecule's role in cell adhesion is under investigation.
Purpose of the Study:
- To investigate the function of a novel monoclonal antibody (mAb H4C4) in inducing cell aggregation.
- To identify the antigen recognized by mAb H4C4 and elucidate its role in hemopoietic cell interactions.
- To explore the molecular mechanisms underlying CD44-mediated cell aggregation.
Main Methods:
- Development and characterization of a monoclonal antibody (mAb H4C4).
- In vitro induction of homotypic aggregation in hemopoietic cells and cell lines.
- Protein purification, immunologic cross-reactivity, and tryptic fragment sequencing to identify the target antigen.
- Functional assays using inhibitors (cytochalasin B, sodium azide, etc.) and varying temperatures.
Main Results:
- mAb H4C4 induced in vitro homotypic aggregation of various hemopoietic cells.
- The antigen recognized by mAb H4C4 is an 85-kDa glycoprotein identified as CD44 (equivalent to murine Pgp-1).
- Cell aggregation was temperature-sensitive, inhibited by cytochalasin B, suggesting cytoskeletal involvement, and unaffected by other tested agents.
- An inverse relationship between spontaneous aggregation and cell-surface CD44 expression was observed.
Conclusions:
- CD44 (Pgp-1) plays a fundamental role in hemopoietic cell aggregation.
- The aggregation process mediated by CD44 involves the cellular cytoskeleton.
- mAb H4C4 is a valuable reagent for studying CD44-mediated cell adhesion.
Abstract:
The present study shows that a mAb (H4C4) developed against human peripheral blood adherent cells has the unusual property of inducing in vitro homotypic aggregation of several types of hemopoietic cells and cell lines. The Ag recognized by mAb H4C4 is a 85-kDa glycoprotein that corresponds to the human Ag CD44 (equivalent to murine Pgp-1), as determined by protein purification, immunologic cross-reactivity studies, and tryptic fragment sequencing. In addition to H4C4, other mAb directed against some, but not all, epitopes of CD44(Pgp-1) were capable of inducing cell aggregation. This process was temperature sensitive and was almost totally abrogated by cytochalasin B but was unaffected by sodium azide, colchicine, EGTA, trifluoperazine, or staurosporin. A role for CD44 (Pgp-1) in cell-to-cell adhesion was further indicated by an inverse relationship observed between spontaneous aggregation of some hemopoietic cell lines and cell-surface expression of CD44(Pgp-1). These observations provide evidence for a fundamental role of CD44(Pgp-1) in cellular aggregation phenomena with an involvement of the cytoskeleton.
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