Rat and mouse monoclonal antibodies to human myelin basic protein.

BALB/c mice and Lewis rats were immunized with human myelin basic protein and its N- and C-terminal fragments. Mouse X mouse fusions produced seven monoclonal antibodies, all of the IgG class and directed against the N-terminal fragment. Five of the antibodies seemed to be against the same epitope, between amino acid residues 92 and 118. One antibody bound between residues 45 and 91, and the remaining antibody reacted with both peptides 1-44 and 45-91. Three monoclonal antibodies, all of the IgM class, were obtained by rat X rat hybridization. Two monoclonal antibodies, raised against whole myelin basic protein and the C-terminal fragment, respectively, each bound to peptide 118-178. The remaining antibody, raised against the N-terminal fragment, bound to peptide 45-91. These monoclonal antibodies are of interest for use in clinical radioimmunoassays and for immunohistochemical investigation of the structural relationships of the myelin sheath.

Isolation of PGP 9.5, a new human neurone-specific protein detected by high-resolution two-dimensional electrophoresis.

Protein gene product (PGP) 9.5 is a new brain-specific protein originally detected by high-resolution two-dimensional electrophoresis of the soluble proteins of human brain and other organs. We have purified this protein from human brain and raised a rabbit antihuman PGP 9.5 antiserum. The protein has a monomer molecular weight of approximately 27,000 and is present in brain at concentrations at least 50 times greater than in other organs. Immunoperoxidase labelling has localised PGP 9.5 to neurones in the human cerebral cortex with no evidence of staining of glial elements. PGP 9.5 is estimated to be present in brain at concentrations of 200-500 micrograms/g wet weight and represents a major protein component of neuronal cytoplasm. This new neurone-specific cytoplasmic marker may prove useful in studies of neuronal development and in the detection of neuronal damage in disease of the nervous system.

Purification, properties, and immunohistochemical localisation of human brain 14-3-3 protein.

A protein has been purified from human brain that appears to be the human equivalent of bovine 14-3-3 protein. On polyacrylamide gel electrophoresis the protein migrates as a faster major component, termed 14-3-3-2 protein, and a slower minor component, termed 14-3-3-1 protein, which consists of approximately 12% of the total protein. Both 14-3-3-1 and 14-3-3-2 have a native molecular weight of approximately 67,000. 14-3-3-2 appears to have the subunit composition alpha beta; 14-3-3-1 has the composition beta'beta'. Peptide mapping with Staphylococcus aureus V8 proteinase shows that alpha and beta subunits are unrelated but the beta and beta' subunits show some common peptides. Immunoperoxidase labelling shows that 14-3-3 is localised in neurones in the human cerebral cortex. 14-3-3 shows no enolase, creatine kinase, triose phosphate isomerase, ATPase, cyclic nucleotide-dependent protein kinase, or purine nucleoside phosphorylase activity. 14-3-3 does not bind calcium and does not appear to be related to calmodulin, calcineurin, tubulin, neurofilament proteins, clathrin-associated proteins, or tropomyosin. The functional significance of this neuronal protein remains obscure.

Cellular localization of aldolase C subunits in human brain.

An antiserum specific for the brain-type aldolase C subunit has been used to investigate the cellular localization of this protein in human brain. Immunoperoxidase labeling at the light microscope level showed heavy staining of Purkinje cells in the cerebellum and of astrocytes in the cerebrum. Faint staining of occasional large neurons in the gray matter of the cerebral cortex was also seen.

Immunohistochemical localization of creatine kinase-BB isoenzyme to astrocytes in human brain.

Indirect immunoperoxidase labelling at the light microscope level using a specific antisera to brain type creatine kinase-BB isoenzyme has localized the protein to astrocytes in the white matter of human cerebrum. No specific staining of neurones or of other glial elements was detected.

Radioimmunoassay of brain-type creatine kinase-BB isoenzyme in human tissues and in serum of patients with neurological disorders.

A sensitive radioimmunoassay for human brain type creatine kinase-BB isoenzyme has been used to measure the protein in serum as a potential index of central nervous system damage in patients with neurological disorders. The MB form of the enzyme (but not the MM form) partially cross-reacts in the radioimmunoassay but shows non-parallel dilution characteristics which distinguish it from the BB form of the enzyme. A survey of human tissues shows immunoreactive creatine kinase-BB in all human organs, with intestine and prostate approaching 35% of the brain concentration in contrast to the particularly low levels found in skeletal muscle, liver, spleen, and erythrocytes. No serum factors interfering in the assay were detected, and the normal serum level of creatine kinase-BB was found to be approximately 1-3 ng/ml. Serum containing high levels of immunoreactivity showed dilution characteristics paralleling the standard curve in the radioimmunoassay. Measurements of the protein in 47 patients with a variety of neurological disorders showed significantly raised mean levels in patients with dementia and with head injuries, isolated raised levels were also found in other disorders.

Purification, radioimmunoassay, and distribution of human brain 14-3-2 protein (nervous-system specific enolase) in human tissues.

Human 14-3-2 protein, a nervous-system specific enolase (EC 4.2.1.11) isoenzyme, has been purified from human brain and a sensitive radioimmunoassay has been developed for its detection. A systematic survey of human organs has shown that immunoreactive nervous-system specific enolase is present in all human organs but at levels less than 3% of those found in human brain, with especially low levels in liver, kidney and skeletal muscle, and with the highest levels in adrenal and large intestine. In all organs immunoreactive nervous-system specific enolase occurs in two forms representing the heterodimer and homodimer forms of the enzyme, and in all tissues except brain the heterodimer predominates. The presence of nervous-system specific enolase in other organs is unlikely to be explicable by innervation alone since significant quantities are found in red blood cell haemolysates. Tissues which contain amine precursor uptake and decarboxylation cells, for which the protein has been claimed to be a specific molecular marker, do not contain significantly higher levels of immunoreactive nervous-system specific enolase than other tissues. Both the heterodimer and homodimer forms of the enolase appear to be expressed at low levels in all tissues.

Immunoreactive nervous system of specific enolase (14-3-2 protein) in human serum and cerebrospinal fluid.

Nervous system-specific 14-3-2 protein is an acidic enolase (EC 4.2.1.11) isoenzyme which is found in high concentrations in nervous tissue and which is also considered to be a molecular marker for amine precursor uptake and decarboxylation (APUD) cells. A radioimmunoassay for this protein shows levels of immunoreactivity of 10 ng/ml and 5 ng/ml in human cerebrospinal fluid and serum respectively. The immunoreactivity in concentrated cerebrospinal fluid dilutes out in parallel with the standard curve in the assay, has a similar molecular weight to the immunoreactivity in soluble protein extracts of whole human brain, and on polyacrylamide gel electrophoresis migrates as two forms representing the homodimer and probably the heterodimer species of the enzyme. The protein also occurs at relatively high levels in erythrocytes and an approximately 1% haemolysis would be sufficient to account for the normal serum level of immunoreactivity. Clinical measurements of 14-3-2 protein in neurological disorders or APUD-system disease could be potentially useful in cerebrospinal fluid but are unlikely to be of value in serum.