Background
Key words: protein quantification / cell line development / IgG assay kit / high producer screening / high throughput
The patent pending PAIA technology provides a novel platform for the quantification of proteins, e.g. monoclonal antibodies. PAIA assays consist of functionalized beads to capture the protein of interest (the analyte) and a fluorescent marker to detect the analyte. In contrast to other bead-based assays, which measure the fluorescence intensity of marker that is bound to the beads [1], PAIA assays measure the fluorescence of unbound marker remaining in solution. We use protrusions on the bottom of the microplate that separate captured analyte-marker complexes from unbound fluorescent marker. Therefore, no washing steps are needed and the assay can be performed in an automation-friendly 384-well plate format (Figure 1A).The time to result, the amount of sample required, and the hands-on time are substantially reduced compared to other immunoassay formats. Hence, this novel approach is particularly suitable for high throughput analysis of protein containing samples like supernatants of antibody producing cells.
PAIA Human IgG Fc/Fab assay.(a) Description of the PAIA assay workflow: Each of the wells of the 384 well PAIA plate contains pre-dispensed dried capture beads. The depicted procedure is run in parallel in each of the wells. The unique structure of the plate allows detection of the unbound fluorescence marker only through the transparent protrusion which keeps the beads outside the detection zone and light path. (b) IgG1 and IgG2 quantification in buffer. (c) IgG1 quantification in cell culture media. It is recommended to prepare standards and samples in the same matrix. Medium 1: Gibco Freestyle CHO + 8 mM Glutamax, Medium 2: BD Select CHO + 8 mM L-Ala-L-Gln, Medium3: Biochrome ISF-1 + 10% FBS. (d) IgG1 calibration curves for several sample volumes. The dynamic range is adapted to higher (2 μL) or lower (10 μL) IgG concentrations by the variation of sample volume.