Monoclonal anti-endothelial cell antibodies (AECA), characterization and binding properties

Mouse anti-endotheial cell mAb ware raised by fusion of splenocytes separated from mice immuized with human AECA IgG fraction and mouse non-secreting myeloma cells (NSO) using polyethylene glycol (PEG). Supematants from growing hybridomas were screened for specific antibody production by cyto-ELISA and FACS analysis using human or mouse endothelial cells. Following the two fusions twelve clones were identified as positive by initial screening. Antibody formation was stabilized following limiting dultion cloning In three of these clones (BGM, 3C8 and 732). The immunoreactivity of mAbs against panel of differant antigens is shown in Table. Table 1. Analysis of reactivity of mouse monoclonal antibodies against different antigens by ELISA (Mean absorbance at 405 nm ±SD) Clones Antigens BGM 3CB 7G2 S2.9 1.720±0.101 0.952±0.070 0.843±0.112 0.060±0.003 HUVEC Hep 2 0.094±0.009 0.072±0.013 0.101±0.020 0.082±0.030 H5V 0.652±0.050 1.350±0.150 1.02010.095 0.070±0.013 Matrix 0.073±0.008 0.095±0.014 0.084±0.012 0.051±0.004 CL 0.098±0.023 0.113±0.02S 0.12410.035 0.0940.012 PS 0.107±0.031 0.115±0.030 0.130±0.038 0.103±0.024 PR-3 0.074±0.011 0.082±0.009 0.07910.010 0.052±0.001 Gelatin 0.06210.013 0.085±0.010 0.079±0.010 0.080±0.013 BSA 0.073±0.011 0.06310.020 0.071±0.01S 0.067±0.008 The mAbs showed restricted reactivity with components associated with endothelial cells (HUVEC, H5V) and no reactivity with either Hep 2 cells, extracellular matrix or other antignes tested. The FACS analysis confirmed membrane specific binding of mAbs to endothellal cells.