Highly sensitive enzyme immunoassay for bovine chromogranin A: application for studies of regional distribution in bovine central nervous system.
Bovine chromogranin A (CGA) was purified by three steps of column chromatography to a single-band purity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antibody against this preparation was purified by a CGA-coupled Sepharose column, and F(ab')2 and Fab' of the antibody IgG were prepared by enzymatic digestion. An enzyme immunoassay (EIA) system was developed with the F(ab')2 immobilized on polystyrene balls and the Fab' labeled with beta-D-galactosidase. The EIA was able to detect 1 pg of CGA and was three to four orders more sensitive compared with any radioimmunoassay systems hitherto reported. Several neural acidic proteins (dopamine beta-hydroxylase, neuron specific enolase, S-100a protein, and brain-type creatine kinase) showed no cross-reaction. Using this EIA, CGA was detected in all regions of bovine central nervous system. CGA concentrations were within a narrow range, being lowest in the cerebellar white matter and highest in the putamen (17.3 and 78.1 ng/mg protein, respectively). The concentrations were extremely low compared to the concentration in the adrenal medulla (205,000 ng/mg protein). The highly sensitive EIA system should be useful for studies of materials containing very small amounts of CGA.
Kawakubo
A
A
First Department of Internal Medicine, Nagoya University School of Medicine, Japan.
Takatsuki
K
K
Yoneda
M
M
Kurokawa
M
M
Suzuki
A
A
Semba
R
R
Kato
K
K
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002852:Chromatography, Ion Exchange; D053379:Chromogranin A; D002864:Chromogranins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D007124:Immunoenzyme Techniques; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000302:isolation & purification; D008832:Microchemistry; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000032:analysis*; D009928:Organ Specificity; D013116:Spinal Cord / Q000032:analysis*
D053379:Chromogranin A; D002864:Chromogranins; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D009419:Nerve Tissue Proteins