Micro-volume wall-less immunoassays using patterned planar plates.

Miniaturization of immunoassays has numerous potential advantages over traditional ELISAs. Here we present a novel approach using patterned planar plates (PPPs). These 'wall-less' plates consist of a 16 × 24 array of 2 mm diameter hydrophilic regions surrounded by a hydrophobic polytetrafluoroethylene (PTFE) coating. Assays are performed by adding 2 µL droplets to the hydrophilic areas. These droplets are overlaid with an immiscible mixture of perfluorocarbon liquid (PFCL) that essentially eliminates evaporation. During wash steps, a thin film of PFCL covers the hydrophobic coating and prevents its wetting by wash buffer; as a result, the hydrophilic wells remain intact and inter-well cross-contamination is prevented. We compared the performance of three immunoassays using PPPs versus traditional 384-well ELISA plates. These included assays for soluble FcRH5 in human serum, SDF-1 in mouse serum, and human IgG in mouse plasma. The results show that the PPP assays were closely comparable to the ELISAs in terms of sensitivity, linearity of dilution, and sample quantitation. Moreover, the PPP assays were rapid to perform, easily adapted from ELISA protocols, and used 10- to 50-fold less sample and reagent volume as compared to 384- or 96-well plate ELISAs. As an additional advantage, PPPs conform to established microplate dimensional standards making them compatible with pre-existing equipment and workflows. PPPs therefore represent an attractive and broadly applicable approach to flexible miniaturization of plate-based immunochemical assays.

Development and preclinical characterization of a humanized antibody targeting CXCL12.

PURPOSE: Our goal was to develop a potent humanized antibody against mouse/human CXCL12. This report summarized its in vitro and in vivo activities. EXPERIMENTAL DESIGN: Cell surface binding and cell migration assays were used to select neutralizing hamster antibodies, followed by testing in several animal models. Monoclonal antibody (mAb) 30D8 was selected for humanization based on its in vitro and in vivo activities. RESULTS: 30D8, a hamster antibody against mouse and human CXCL12α, CXCL12ß, and CXCL12γ, was shown to dose-dependently block CXCL12α binding to CXCR4 and CXCR7, and CXCL12α-induced Jurkat cell migration in vitro. Inhibition of primary tumor growth and/or metastasis was observed in several models. 30D8 alone significantly ameliorated arthritis in a mouse collagen-induced arthritis model (CIA). Combination with a TNF-α antagonist was additive. In addition, 30D8 inhibited 50% of laser-induced choroidal neovascularization (CNV) in mice. Humanized 30D8 (hu30D8) showed similar in vitro and in vivo activities as the parental hamster antibody. A crystal structure of the hu30D8 Fab/CXCL12α complex in combination with mutational analysis revealed a "hot spot" around residues Asn(44)/Asn(45) of CXCL12α and part of the RFFESH region required for CXCL12α binding to CXCR4 and CXCR7. Finally, hu30D8 exhibited fast clearance in cynomolgus monkeys but not in rats. CONCLUSION: CXCL12 is an attractive target for treatment of cancer and inflammation-related diseases; hu30D8 is suitable for testing this hypothesis in humans.