Optimization of experimental conditions for functional in vitro characterization of humanized antibodies specific for...

Staphylococcus aureus is a common nosocomial infection and its resistance to penicillin and methicillin antibiotics is a growing clinical problem. We previously described the development of a humanized anti-Staphylococcus enterotoxin B (SEB) antibody derived from the mouse antibody made by the 20B1 hybridoma. This antibody was humanized and characterized kinetically by surface plasmon resonance demonstrating that the humanized clones retained binding to SEB. Clones were then functionally characterized in an in vitro assay demonstrating that the murine 20B1, chimeric and humanized antibodies potently inhibited SEB-induced murine splenocyte proliferation assay. Here, we describe a human cell-based screening assay, optimized by varying multiple experimental parameters that resulted in an assay that was used to demonstrate full and potent neutralization by the parental, chimeric and humanized antibodies. The replacement of fetal bovine serum (FBS) with normal human serum (NHS) was found to be a crucial factor in the performance of the human cell based screening assay that enabled the calculation of mAb efficacy and potency. In addition, we found that anti-SEB antibodies showed similar efficacy and potency with a triple mutant Fc region (designed to be effector function null) or a wild-type Fc region, which is in contrast to previously described studies.