A positive 14-3-3 assay is a criterion for probable Creutzfeldt-Jakob disease (CJD). Cerebrospinal fluid (CSF) 14-3-3 is usually detected by immunoblot using an antibody that recognizes all of the 14-3-3 isoforms. In a few cases, the antibody recognizes an inferior band and this pattern is associated with false positive results. We analyzed 43 CSF (26 CJD, 17 controls) samples using antibodies against specific isoforms (beta, epsilon, gamma, tau, xi) and compared the results with those obtained with the standard antibody. The anti-gamma and anti-beta antibody achieved similar results but the presence of atypical patterns made the standard antibody more accurate for the CJD diagnosis. To study the nature of the inferior band, CSF samples were probed with antibodies against light chain immunoglobulins, and immunoblots of human IgG with the standard antibody. The experiments suggested a cross-reaction of the anti-14-3-3 antibody with light chain immunoglobulins.