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, which is based on the binding of 14-3-3 protein to a chemically synthesized peptide containing the phosphorylated recognition sequence of this protein. The peptide is covalently bound to maleimide-activated microplates through reaction 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 second recognition principle--an anti-14-3-3 protein antibody. This assay was used for measurement of 14-3-3 protein in brain (bovine) and in cerebrospinal fluid from patients with Creutzfeldt-Jakob disease (CJD) and control cases with diseases other than CJD. This novel assay allowed also the detection of low 14-3-3 protein levels present in cerebrospinal fluid, without further concentration, in contrast to conventional immunoblotting or immunoassay procedures.
Leuck
J
J
Institut für Physiologische Chemie, Universität Mainz, Germany.
Peoch
K
K
Laplanche
J L
JL
Grabar
E
E
Müller
W E
WE
Schröder
H C
HC
D048948:14-3-3 Proteins; D000368:Aged; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001665:Binding Sites; D001923:Brain Chemistry; D002417:Cattle; D007562:Creutzfeldt-Jakob Syndrome / Q000134:cerebrospinal fluid*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000134:cerebrospinal fluid / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D012680:Sensitivity and Specificity; D014446:Tyrosine 3-Monooxygenase / Q000134:cerebrospinal fluid* / Q000276:immunology / Q000378:metabolism
D048948:14-3-3 Proteins; D000906:Antibodies; D010455:Peptides; D010750:Phosphoproteins; D014446:Tyrosine 3-Monooxygenase