alpha-Synuclein (alphasyn) mutations, overexpression, misfolding, and aggregation are associated with Parkinson's disease. This protein has been intensively studied in neuronal systems. However, alphasyn is also present in extracellular fluids, such as cerebrospinal fluid and blood plasma. Recent studies have attempted to quantify its levels and compare these in various extracellular fluids of control and Parkinson's disease subjects. Data from these studies have been difficult to interpret, suggesting that more sensitive, standardized, and well-characterized assays of larger cohorts are required. Here, we describe the development of a new ELISA specifically for quantifying alphasyn in human plasma. An initial assay, using a commercial anti-alphasyn monoclonal antibody (211; Santa Cruz Biotechnology, Santa Cruz, CA) and based on a published protocol, was adapted for use in human plasma. In addition, we have developed a novel alphasyn-specific antibody for the assay that has very high sensitivity and signal:noise characteristics. Assays with either antibody showed high specificity for alphasyn, and detected it in a variety of sample types, including plasma. These assays can now be employed on large cohorts of patients and control subjects to determine whether plasma levels are altered in disease. Although measuring extracellular alphasyn levels may prove to be a useful biomarker of Parkinson's disease, it should also be a powerful tool for basic research aimed at understanding the normal and pathological physiology of alphasyn secretion. .

Hereditary predisposition to thrombosis due to activated protein C resistance (APCR) has been attributed to a missense mutation in the factor V gene at nucleotide 1691 (G to A), causing replacement of arginine at codon 506 with glutamine. Using an RFLP‐PCR assay to detect this mutation, we measured a prevalence of 3.3% in healthy Caucasians and 1.25% in healthy African‐Americans. In addition, we evaluated a total of 90 consecutive specimens submitted to the coagulation laboratory at the Medical College of Virginia for the presence of this mutation. We compared our results for 78 of these specimens with the values measured by a modified partial thromboplastin assay, the COATEST. Twelve of the 90 samples could not be tested using the COATEST because the patients were undergoing anticoagulant therapy. One of the latter 12 specimens was positive by the RFLP‐PCR test. Using the genetic test as the definitive assay and the cutoff value established for distinguishing between normal and abnormal results by the COATEST, the COATEST had a sensitivity of 50% and specificity of 93% for the detection of factor V mutation. Analysis of the 90 samples stratified by ethnic groups revealed a frequency of mutation of 13.3% for Caucasians and 6.88% for African‐Americans, although with the present sample size, the difference was not statistically significant. Although the COATEST is technically simpler to perform than the genetic test for diagnosing the presence of the factor V mutation, its use for this purpose is limited due to low sensitivity. Thus where this disorder is clinically suspected, submission of the specimen directly for genetic testing by RFLP‐PCR or equivalent assay should be considered. J. Clin. Lab. Anal. 11:328–335, 1997. © 1997 Wiley‐Liss, Inc.