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RESEARCH PROFILES New assay for the prion protein
WILLIAM HADLOW, ROCKY MOUNTAIN LABS (RET.)
Other factors, such as pH and ionic to match the specificity of the antibody. Assays for the abnormal prion protein strength, also induce dissociation of “You had to have a new peptide for (PrP), which is believed to cause a the complex. To address these probeach antibody,” explains Schmerr. The group of fatal neurodegenerative dislems, the researchers used a short capilresearchers have already shown that the eases called transmissible spongiform lary, a high separation voltage, and a new noncompetitive format works with encephalopathies (TSEs), typically rely relatively low concentration of running a wide range of monoclonal antibodies. on the postmortem analysis of brain tisbuffer. A rapid separation, sue. Although PrP is present however, was key to prein low amounts in the blood venting dissociation. of infected animals, no rouAnother problem with tine diagnostic tests for PrP CE-based protein analysis is are being used at the preclinadsorption of proteins to ical stage of disease. Several the capillary wall. Capillaries researchers are hoping to are typically coated to overchange that. come protein adsorption, Among them are Mary Jo but most coatings reduce Schmerr and her colleagues at electroosmotic flow, and Iowa State University–Ames this increases the separation Laboratory, the Veterinary time. To address this probLaboratories Agency (U.K.), lem, the researchers added and the German Primate carboxymethyl--cyclodexCenter. As described in the trin to the running buffer. July 15 issue of Analytical “That actually has made the Chemistry (pp 4489– 4494), electrophoresis part of the the researchers have developed Researchers are developing new diagnostic blood tests for detecting testing very robust. In eara CE-based immunoassay for the early stages of TSEs, such as scrapie in sheep. lier studies, we used BSA detecting PrP in blood sam[bovine serum albumin] to coat the Another advantage of the noncomples from infected animals. They have petitive format is enhanced sensitivity. It capillary, but carboxymethyl--cyclosuccessfully used the assay to test scrapiedextrin is much, much better, and it’s infected sheep as well as deer and elk with is about 10 more sensitive than previmore reproducible,” says Schmerr. ously reported competitive assays, says chronic wasting disease, although they One important aspect of the new Schmerr. Detection limits for recombihave not yet tried it on bovine blood to assay is the time when blood samples nant PrP (rPrP), which is PrP that has detect “mad cow” disease because of the are collected. The test works well for been synthesized in E. coli, were estidifficulty in obtaining samples. samples that were taken early in the premated to be ~6 ng/mL for the nonThe new immunoassay uses a noncompetitive assay. Recombinant PrP was clinical stage of disease, says Schmerr. competitive format in which fluoresBut it does not work as well if the samused as a surrogate for PrP because of cently labeled protein A (FITC-PrA) ples are taken later, from the infected the limited availability of blood samples binds to an antibody (Ab) that forms a animals. “We think that might be a biothat contain PrP from infected animals. complex with PrP. The complex is sepalogical problem. If we had more blood The main difference between rPrP and rated and analyzed by capillary zone tests available, we could figure this out a PrP is that rPrP is not glycosylated. electrophoresis (CZE). Isolation of the little better,” she says. Nonetheless, a The researchers used CZE rather FITC-PrA-Ab-PrP complex by CZE can preclinical test that could diagnose anithan other techniques because it is simbe performed in
