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Characterization of EN4 monoclonal antibody: a reagent with CD31 specificity
V L Burgio1, S Zupo, S Roncella
1Dipartimento di Medicina Sperimentale, Università La Sapienza, Rome, Italy.
Abstract:
EN4 MoAb was originally described as a MoAb that reacts specifically with human endothelial cells, and the reagent was not assigned to any of the presently known CD. Here, we provide evidence indicating that EN4 reacts with the CD31 antigen. Thus, EN4 stains strongly murine fibroblasts transfected with the human CD31 gene. Furthermore, SDS-PAGE analysis of immunoprecipitates of cell lysates from surface-iodinated Jurkart T cells demonstrated that EN4 and reference CD31 MoAb recognized the same antigen, of 130 kD mol. wt. Finally, both EN4 and CD31 gave the same pattern of reactivity when tested on tonsillar or peripheral blood lymphoid cells by FACS analysis or by immunohistochemistry on sections of a variety of human tissues. EN4, however, proved consistently more efficient than the reference anti-CD31 MoAb as judged by both the intensity of fluorescence or of tissue staining. This property has thus allowed a better characterization of the tissue and cellular distribution of CD31.
Insights
The EN4 monoclonal antibody (MoAb) identifies the CD31 antigen, crucial for endothelial cells. This finding enhances understanding of CD31 distribution in tissues and cells.
Area of Science:
- Immunology
- Cell Biology
- Hematology
Background:
- The EN4 monoclonal antibody (MoAb) was initially characterized for its specific reactivity with human endothelial cells.
- Its classification within the known Cluster of Differentiation (CD) system was previously undetermined.
Purpose of the Study:
- To investigate and confirm the antigen recognized by the EN4 MoAb.
- To establish the relationship between EN4 and the CD31 antigen.
- To evaluate the utility of EN4 for characterizing CD31 expression.
Main Methods:
- Transfection of murine fibroblasts with the human CD31 gene to assess EN4 reactivity.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation of surface-iodinated Jurkat T cells.
- Flow cytometry (FACS) analysis and immunohistochemistry on human tissues using EN4 and reference anti-CD31 MoAb.
Main Results:
- EN4 demonstrated strong staining of murine fibroblasts expressing human CD31.
- SDS-PAGE analysis confirmed that EN4 and a reference CD31 MoAb recognize the same 130 kDa antigen.
- Both antibodies exhibited similar reactivity patterns on lymphoid cells and human tissues, but EN4 provided more intense staining.
Conclusions:
- EN4 MoAb is confirmed to react with the human CD31 antigen.
- EN4 exhibits superior staining efficiency compared to existing anti-CD31 MoAb.
- The enhanced efficiency of EN4 facilitates improved characterization of CD31's tissue and cellular distribution.