Human 14-3-3 protein: radioimmunoassay, tissue distribution, and cerebrospinal fluid levels in patients with neurological disorders

Abstract

An antiserum to human 14-3-3 protein has been produced in rabbits. The protein was a poor antigen and attempts to improve immunogenicity were unsuccessful. A radioimmunoassay was developed using the antiserum, 125I-14-3-3-2, and unlabelled 14-3-3-2 as standards. The assay had a sensitivity limit of 2.5 ng.ml-1. The minor component of human 14-3-3 protein (14-3-3-1 protein) cross-reacted to approximately 10% in the assay. Human tissues were surveyed for 14-3- protein by two-dimensional electrophoresis and by radioimmunoassay. Two-dimensional electrophoresis showed a 14-3-3 protein complex in brain, intestine, and testis, but not in other tissues. Radioimmunoassay showed that although brain had the highest concentration of 14-3-3 (13.3 microgram.mg-1 soluble protein), immunoreactivity was present in all tissues, with the concentration in intestine and testis approaching 50% of the brain level. Lower levels (less than 1.0 microgram.mg-1 soluble protein) were seen in liver, kidney, skeletal muscle, and erythrocytes. The immunoreactivity present in tissues other than brain showed the same molecular weight and charge characteristics and authentic 14-3-3 protein. The radioimmunoassay also detected 14-3-3 protein in serum (50 ng.ml-1) and in CSF (5-130 ng.ml). The immunoreactivity present in CSF appeared to be intact 14-3-3 protein. CSF 14-3-3 levels were measured in 82 patients with various neurological disorders. Measurements of this protein did not appear sufficiently discriminating to be of diagnostic value.