1078. Optimization of a 96-well Plate Format Assay to Evaluate Concentration-Dependent Activity of a Monoclonal Antibody Against the O Antigen O25b from ST131-H30 Escherichia coli

Abstract Background Escherichia coli ST131 is a major challenge for global human health. Among ST131 isolates, the H30 type is prevalent and displays multiple drug resistance to classes. A monoclonal antibody (mAb) specific for the O antigen, O25b, which is conserved in ST131-H30 clonal lineage, was developed for diagnostic and prophylactic therapy. We optimized a 96-well plate assay to test different concentrations of the mAb against E. coli clinical isolates. Methods Initial growth curves were performed against 69 ST131-O25b, 9 ST131-O25a, 32 ST131 non-O25, and 29 non-ST131 E. coli isolates in 40% active human serum (HS). Additional conditions evaluated for growth inhibition by the mAb included 3 media types, 3 HS sources, pH variations, addition of polysorbate 80 (P80), inoculum concentration, length of incubation, and varying calcium and magnesium concentrations. Once optimal parameters were established, the assay was miniaturized into a 96-well format and 12 ST131-O25b and 5 non-ST131 were tested. Fluorescence and visual reading using alamarBlue (AB), a cell metabolism indicator, were performed at 8 and 10h in addition to plating for viable colonies. Results After 10h incubation, only 36/69 (52.2%) ST131-O25b isolates exhibited visible growth in BHI plus 40% HS. A 2-part growth inhibition assay using BHI plus 50% HS and BHI with 25% HS without the addition of P80, calcium, or magnesium was considered the optimal growth condition for EC strains to test the effect of the mAb. By 10 hours, all ST131-O25b strains displayed a visual inhibition of growth between 0.3-5 mg/L of α-ST131 mAb and zero non-ST131 strains were inhibited (Figure). While reading AB on a fluorometer was more sensitive and less subjective than visual interpretation, both reading methods allow for endpoint determinations. Additionally, panels were able to go through one freeze-thaw cycle and still retain α-ST131:O25b mAb activity. Conclusion Concentration-dependent activity of a α-ST131:O25b mAb can be measured in vitro in a microtiter format; however, the growth inhibition by HS, while still providing enough complement for complement-mediated mAb killing, might be an obstacle. Additionally, assay reproducibility may be difficult without a consistent source of active HS. Figure Disclosures Jill Lindley, Bravos Biosciences (Research Grant or Support)ContraFect Corporation (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support) Timothy B. Doyle, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Brian D. VanScoy, B.S., 3-V Biosciences (Grant/Research Support)Achogen (Grant/Research Support)Amplyx Pharmaceuticals, Inc. (Grant/Research Support)Arixa Pharmaceuticals (Grant/Research Support)Arsanis Inc. (Grant/Research Support)B. Braun Medical Inc. (Grant/Research Support)Basilea Pharmaceutica (Grant/Research Support)BLC USA (Grant/Research Support)Boston Pharmaceuticals (Grant/Research Support)Bravos Biosciences, LLC (Grant/Research Support)Cidara Therapeutics Inc. 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