A novel binding assay for detection of 14-3-3 protein in cerebrospinal fluid from Creutzfeldt-Jakob patients

Here we describe a simple and fast microplate assay for 14-3-3 protein, whi ch is based on the binding of 14-3-3 protein to a chemically synthesized pe ptide containing the phospholylated recognition sequence of this protein. T he peptide is covalently bound to maleimide-activated microplates through r eaction of the sulfhydryl group of a N-terminal cysteine residue linked to the peptide. The peptide-14-3-3 complexes formed are detected using as a se cond recognition principle - an anti-14-3-3 protein antibody. This assay wa s used for measurement of 14-3-3 protein in brain (bovine) and in cerebrosp inal fluid from patients with Creutzfeldt-Jakob disease (CJD) and control c ases with diseases other than CJD. This novel assay allowed also the detect ion of low 14-3-3 protein levels present in cerebrospinal fluid, without fu rther concentration, in contrast to conventional immunoblotting or immunoas say procedures.