Characterization of monoclonal antibody to L-homocysteic acid and its immunohistochemistry in rat hippocampus

Abstract

L-homocysteic acid (HCA) and other amino acids were conjugated to rat brain material (extracted rat brain protein) with glutaraldehyde to form HCA- and amino acids-brain material conjugates. The specificity of monoclonal antibody (McAb) was tested on serial dilution test and absorption test on enzyme-linked immunosorbent assay (ELISA) using these conjugates as antigens instead of amino acids-BSA (bovine serum albumin) conjugates used previously. The characterized McAb was applied for immunohistochemical staining using PAP (peroxidase antiperoxidase) technique in combination with silver enhancement of diamino-benzene (DAB) products. The results indicated that McAb to L-HCA reacted with L-HCA-brain material conjugates, but not with other amino acids-brain material conjugates so far tested. McAb absorbed with L-HCA-brain material abolished or decreased immunoreactivity of L-HCA-brain material with McAb. The antibody selectively stained subpopulation of cells and processes in the hippocampus fixed with glutaradehyde. Absorption of McAb with L-HCA-brain material abolished immunohistochemical staining. These results suggested that McAb was specific for L-HCA-brain materials and could be used for imuno-histocytochemistry. This would provide a new tool for immunohistochemical visualization and localization of L-HCA in the nervous system.