Additional benefits of the ECL format include the speed of the ECL analysis, which is approximately 2 min per 96-well plate for eight serotypes, and the lack of integrated fluids that may result in clogging issues typically experienced by bead-based systems

Additional benefits of the ECL format include the speed of the ECL analysis, which is approximately 2 min per 96-well plate for eight serotypes, and the lack of integrated fluids that may result in clogging issues typically experienced by bead-based systems. reference material (21). Giebink sera. Serum obtained from G. S. Giebink, University of Minnesota, is usually human immune serum from an adult following vaccination with Pneumovax 23. The Giebink serum is the Pn ECL assay control tested at three (10-fold serial) dilutions on each plate within an assay run (1:1,000, 1:10,000, and 1:100,000 dilutions). MSD assay method overview. MSD technology is based on ECL detection which utilizes a Sulfo-Tag label that emits light upon electrochemical stimulation. The mechanism for generation of ECL from ruthenium tris(bipyridine) complexes at an oxidizing electrode in the presence of tripropylamine read buffer has been previously documented (4). With a dedicated ECL plate reader, an electrical current is placed across the plate-associated electrodes, resulting in a series of electrically induced reactions leading to a luminescent signal. The multispot configuration used in development and validation was 10 spots/well in a 96-well plate format. Each well was coated with 5 ng PnPs per spot (unless specified otherwise for optimization studies) of the following serotypes: 3, 4, 6B, 9V, 14, 18C, 19F, and 23F. Each well also contained two bovine serum albumin (BSA) spots, which were used to assess the background reactivity of the assay (i.e., the response associated with serum and labeled secondary antibody in the absence of PnPs). Assay standard (89SF-2), controls, and test sera were diluted at appropriate dilutions in phosphate-buffered saline made up of 0.05% Tween 20 (PBS-T), 1% BSA, 5 g/ml CPs, 10 g/ml Pn25, and 10 g/ml Pn72 and incubated overnight at 4C (2 to 8C) or at ambient temperature for 45 min. Each antigen-coated plate was incubated at ambient temperature for 1 h on a shaker platform with blocking agent. PF-06305591 Plates were washed with 0.05% PBS-T, and 25 l per well of the preadsorbed and diluted test sera was added and incubated for SHC1 45 min at ambient temperature PF-06305591 on a shaker platform. Plates were washed with 0.05% PBS-T, and MSD Sulfo-Tag-labeled goat anti-human IgG secondary antibody was added to each well and incubated 1 h at ambient temperature on a shaker platform. Plates were washed with 0.05% PBS-T, and 150 l of MSD Read Buffer-T 4X (with surfactant) diluted 1:4 in water was added to each well. The plates were read using a MSD sector imager model no. 2400 or 6000. Assay optimization experiments. A DOE statistical approach was used to determine the optimal settings and conditions for the major controllable factors in the assay prior to validation. The following five major assay conditions were jointly assessed for optimization of the Pn MSD assay: blocking reagent (5% BSA, 1% human serum albumin [HSA], and SuperBlock buffer [Pierce]), antigen concentration (2.5 ng/spot and 7.5 ng/spot), incubation time for secondary antibody (30 min and 2 h), incubation time for samples (30 min and 2 h), and secondary antibody concentration (0.5 g/ml and 2.0 g/ml). Twelve samples consisting of matching pairs of pre- and postimmunization sera from adults in a previously completed Pneumovax 23 clinical trial were tested at 1:50, 1:250, and 1:1,250 dilutions on each of 24 plates, with each plate representing a unique experimental condition (e.g., 1% HSA, 2.5 ng/spot, 2-h incubation of secondary antibody, 30-min sample incubation, and 2.0-g/ml secondary antibody concentration). A second optimization experiment using DOE methodology was performed to evaluate alternative formulations of the serum diluent in an attempt to further reduce the background reactivity in the assay without diminishing the detection of antigen-specific response. The following four components of the serum diluent were evaluated: Tween 20, PBS, Triton. PF-06305591