Quantitative radioimmunohistochemical method using [125I]-protein A to measure the content of methionine enkephalin in discrete rat brain areas.

Abstract

We report a quantitative radioimmunohistochemical method, using [125I]-protein A in combination with a specific antibody to methionine enkephalin (Met-enk), for determination of the content of this peptide in discrete areas of rat brain. After paraformaldehyde fixation, rat brain sections were incubated with a Met-enk polyclonal antibody, followed by incubation with [125I]-protein A. After autoradiography with 3H-sensitive Ultrofilm, optical densities (OD) were quantified by computerized microdensitometry. The OD obtained were compared to a standard curve, constructed after determination by radioimmunoassay of the Met-enk content in corresponding brain areas from adjacent tissue sections. After comparing 15 different brain areas over a ninetyfold range of concentrations, we found a linear relationship between the content of Met-enk, as determined by radioimmunoassay, and the OD generated by autoradiography. The content of Met-enk in other discrete brain areas can be quantified by interpolation of the OD determined by autoradiography in the standard curve. The method allows, for the first time, precise quantification of peptide concentrations in discrete areas and nuclei from thin sections of rat brain. This technique has a more than 100-fold higher sensitivity than classical radioimmunoassays, with the additional advantage of neuroanatomical localization. It also has the potential for application to the quantification of many other antigens present in brain and other tissues.