Monoclonal Antibody

Abstract (Set) A rat hybridoma producing a high-affinity IgG2a monoclonal antibody (B3B4) directed against the murine lymphocyte IgE receptor (FcR) was established by using purified FcR from FcR+ murine hybridoma B cells as immunogen. The monoclonal and polyclonal anti-FcR inhibited the binging of IgE to the murine lymphocyte FcR and were also used to isolate the FcR. B3B4 specifically recognized only the 49-Kd FcR or murine B lymphocyte as determined by immunoprecipitation and SDS-PAGE analysis. In addition to it's reaction with intact FcR, B3B4 also recognized FcR fragments that were present in the culture media of FcR+ hybridoma cells. The predominant fragments isolated were 38 Kd anf 28 Kd by SDS-PAGE analysis. When tested for reactivity with other cell types, B3B4 was highly specific for murine B lineage cells in that it did not significantly react with FcR on macrophages and T cells and, in addition, did not react with the high affinity mast cell FcR. B3B4 completely blocked IgE resetting, and a reciprocal inhibition of binding was seen in a dose-dependent fashion between IgE and B3B4, indicating a close proximity of the IgE and B3B4 binding sites. Saturation binding analysis indicated that the Fab' fragment of B3B4 bound to twice as many sites/cell as IgE, suggesting that there are two identical B3B4 determinants per 49-Kd FcR or that the IgE binding site is formed by the association of at least two 49-Kd FcR. However, unlike IgE, neither B3B4 nor F(ab')2-B3B4 were very effective in causing FcR upregulation or murine hybridoma B cells; in fact, B3B4 prevented this upregulation when added in combination with IgE. These results suggest that a site-specific interaction provided only IgE may be essential for the ligand-specific upregulation. Both polyclonal and monoclonal antibodies will be useful in further studies concerning the functional relationship between the membrane FcR and the soluble FcR fragments.

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