A custom development customer approached Radix BioSolutions interested in a 6-plex panel examining 3 analytes for which xMAP assays were already available and 3 which required novel development. Multiple vendors were available for the already-developed xMAP assays and for reagents for the remaining assays.
Selecting the best mix of vendor kits and integrating them with one another and with novel assays presented some significant challenges in development. Buffer systems, pretreatment of sample, assay conditions and reagent concentrations had to be reconciled to accommodate the needs of all assays in a multiplex.
Leveraging our experience developing xMAP immunoassays and knowledge of vendor kits, we developed three novel assays for MIG, MIP-1d, and Eotaxin-2. Reagents for these developments were selected from the same vendor that manufactures 2 of the 3 already-developed assays used to multiplex. Maintaining consistency of reagents as much as possible contributed significantly to ease of multiplexing.
A 6-plex assay containing reagents from 2 different existing kits and 3 novel assays was built and its performance assessed. Standard curves were extended beyond those available as kit components and the assay format changed to overnight capture incubation. Intra- and inter-assay precision was under 10% for all analytes in samples using the final kit format.