A universal method for detection of amyloidogenic misfolded proteins.

Diseases associated with the misfolding of endogenous proteins, such as Alzheimer's disease and type II diabetes, are becoming increasingly prevalent. The pathophysiology of these diseases is not totally understood, but mounting evidence suggests that the misfolded protein aggregates themselves may be toxic to cells and serve as key mediators of cell death. As such, an assay that can detect aggregates in a sensitive and selective fashion could provide the basis for early detection of disease, before cellular damage occurs. Here we report the evolution of a reagent that can selectively capture diverse misfolded proteins by interacting with a common supramolecular feature of protein aggregates. By coupling this enrichment tool with protein specific immunoassays, diverse misfolded proteins and sub-femtomole amounts of oligomeric aggregates can be detected in complex biological matrices. We anticipate that this near-universal approach for quantitative misfolded protein detection will become a useful research tool for better understanding amyloidogenic protein pathology as well as serve as the basis for early detection of misfolded protein diseases.

Infectious titer assay for adeno-associated virus vectors with sensitivity sufficient to detect single infectious events.

A highly sensitive assay for determination of infectious titers of recombinant adeno-associated virus (AAV) by limiting dilution analysis is described. This assay is capable of detecting single infectious events and can therefore provide an absolute rather than relative measure of infectivity. The assay utilizes a HeLa-derived AAV2 Rep/Cap-expressing cell line, D7-4, grown in 96-well plates and infected with replicate 10-fold serial dilutions of AAV2 vectors in the presence of adenovirus type 5. Forty-eight hours after infection, vector genome replication is determined by quantitative PCR (Q-PCR). A linear relationship between vector genome input and replicated copy number (slope = 2670 copies per vector genome) was determined, enabling detection of one infectious event per well by Q-PCR. The observed binomial distribution of the end-point data confirmed that single infectious events could be detected, and allowed calculation of infectious titers by the Kärber method. Analysis of an AAV2 reference vector, AAV-hFIX16, in 21 independent determinations gave an average ratio of AAV vector genomes (VG) to infectious units (IU) of 8.3 +/- 4.2 VG/IU, a value close to the theoretical limit. No significant differences in vector particle-to-infectious unit ratios were observed between vectors purified by column chromatography (9.3 +/- 5.0 VG/IU, n = 7) and cesium chloride gradient ultracentrifugation (6.4 +/- 3.2 VG/IU, n = 7).