ABSTRACT
Plaque assays are used to measure the infectious titer of viral samples. These assays are multi-day and low-throughput and may be subject to analyst variability from biased or subjective manual plaque counting. Typically, on day 1, cells are adhered to plates overnight. On day 2, cells are infected with virus. After 3 additional days, plaques are fixed, stained with a horseradish peroxidase (HRP)-conjugated antibody and a HRP substrate, and counted by eye. Manual-based visual counting of plaques is time-consuming and laborious and may be subject to variability between analysts. Also, the assay must proceed for several days to allow the plaques to increase to sufficiently large sizes for manual identification. Here, we integrate fluorescent detection and automated plaque counting to increase the sensitivity and speed of the assay. First, we stain plaques with a fluorescent-labeled antibody. Second, we implement a plate-based cell imager to perform non-biased, non-subjective plaque counting. The integration of these two technologies decreases the assay length by 40%, from 5 days to 3 days, because plaque size, plaque signal to noise, and manual visualization are no longer limiting. This optimized plaque assay is sensitive, fast, and robust and expands the throughput and usage of this method for measuring plaque formation.
ABSTRACT
The long circulating half-life of serum albumin, the most abundant protein in mammalian plasma, derives from pH-dependent endosomal salvage from degradation, mediated by the neonatal Fc receptor (FcRn). Using yeast display, we identified human serum albumin (HSA) variants with increased affinity for human FcRn at endosomal pH, enabling us to solve the crystal structure of a variant HSA/FcRn complex. We find an extensive, primarily hydrophobic interface stabilized by hydrogen-bonding networks involving protonated histidines internal to each protein. The interface features two key FcRn tryptophan side chains inserting into deep hydrophobic pockets on HSA that overlap albumin ligand binding sites. We find that fatty acids (FAs) compete with FcRn, revealing a clash between ligand binding and recycling, and that our high-affinity HSA variants have significantly increased circulating half-lives in mice and monkeys. These observations open the way for the creation of biotherapeutics with significantly improved pharmacokinetics.