Determination of Brain Enolase Isozymes with an Enzyme Immunoassay at the Level of Single Neurons

Abstract

Ultrasensitive enzyme immunoassay systems for the assay of rat brain enolase isozymes (αα, αγ, and γγ forms) were prepared by use of β-d-galactosidase from Escherichia coli as label and the purified rabbit antibodies to αα and γγ enolases. The antibodies were purified from the immunoglobulin G (IgG) fractions of antisera by immunoaffinity chromatography with a column of the corresponding antigen-coupled Sepharose. Sandwich-type immunoassay systems with the galactosidase-labeled antibody Fab’fragments and the antibody F(abapos;)₂-immobilized polystyrene beads could determine amounts as small as 1 amol (10⁻¹⁸ mol) of each isozyme. Purkinje cell bodies picked up from the bulk-separated fraction by means of a nylon loop were subjected to the assay at the level of single cells. In contrast to previous report, this neuron contained not only the γγ but also the αγ and αα enolases at a level of amol per cell body, although the concentration of γγ was the highest. Immunohistochemical experiments on the cerebellum with the peroxidase-labeled antirabbit IgG antibody and the unlabeled antibody method confirmed the above results, and indicated that both α and γ subunits of the enolase were stained intensely in axons.