An Aggregation-Specific Enzyme-Linked Immunosorbent Assay: Detection of Conformational Differences between Recombinant PrP Protein Dimers and PrP ^Sc Aggregates

Abstract

Scaggregate is the cause of prion diseases. We developed a novel enzyme-linked immunosorbent assay (ELISA) that is specific for PrP aggregate by screening 30 anti-PrP monoclonal antibodies (MAbs) for their ability to react with recombinant mouse, ovine, bovine, or human PrP dimers. One MAb that reacts with all four recombinant PrP dimers also reacts with PrP Sc aggregates in ME7-, 139A-, or 22L-infected mouse brains. The PrP Sc aggregate is proteinase K resistant, has a mass of 2,000 kDa or more, and is present at a time when no protease-resistant PrP is detectable. This simple and sensitive assay provides the basis for the development of a diagnostic test for prion diseases in other species. Finally, the principle of the aggregate-specific ELISA we have developed may be applicable to other diseases caused by abnormal protein aggregation, such as Alzheimer's disease or Parkinson's disease.

Test for Detection of Disease-Associated Prion Aggregate in the Blood of Infected but Asymptomatic Animals

“…To improve the sensitivity of AS-ELISA, we combined AS-ELISA with FACTT and developed a new assay, AS-FACTT. Approximately 5% of recombinant PrP (rPrP) occurs as dimers (26). We first demonstrated that AS-FACTT is more sensitive than AS-ELISA for detecting rPrP dimers.…”

“…into 7-week-old CD-1 mice as described previously (37). The ME7 inoculant has a titer of about 10 8 50% infectious doses/ml (26,37). Sham-infected, age-and sex-matched CD1 mice, normal CD1 mice, and PrP CϪ/Ϫ mice were used as controls.…”

“…To detect PrP species in brain homogenates, brain samples containing 60 g of total protein were dissolved in 2ϫ sample buffer and heated at 95°C for 5 min before being separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described previously (26). The 12% polyacrylamide gel was then transferred onto a nitrocellulose membrane and probed by MAb 8H4.…”

“…Each brain homogenate was treated with 50 g/ml of proteinase K (Sigma, MO) at 37°C for 1 h as described previously (26). The protease was inactivated by the addition of PMSF to a final concentration of 3 M.…”

“…Recently, we developed a novel aggregation-specific (AS) enzyme-linked immunosorbent assay (ELISA), termed "AS-ELISA," for PrP Sc aggregates present in the brains of mice infected with the ME7, 139A, or 22L strain of PrP Sc (26). The assay can detect disease-associated PrP Sc aggregates in the brains of mice 70 days postinfection, a time when proteaseresistant PrP Sc is undetectable.…”