DEVELOPMENT OF A MONOCLONAL-ANTIBODY TO IMMUNO-CYTOCHEMICAL ANALYSIS OF THE CELLULAR-LOCALIZATION OF THE PERIPHERAL BENZODIAZEPINE RECEPTOR

Based on the amino acid sequence deduced from the cloned human periphe ral benzodiazepine receptor (PER) gene, monoclonal antibody (Mab 8D7) was produced against the C-terminal fragment of the receptor, Immunobl ot experiments, performed against purified PER, indicated that the ant ipeptide antibody recognized, under denaturing conditions, the corresp onding amino acid sequence of the PER. When mitochondrial membranes fr om PER transfected yeast or from THP1 and U937 cells were used on immu noblot analysis, a high level of immunoreactivity was observed at 18 k Da, the PER molecular mass deduced from cDNA, establishing the specifi city of the antibody for the receptor. Moreover, binding experiments r ealized with intact mitochondria demonstrated that the immunogenic seq uence was accessible to the antibody indicating that the C-terminal fr agment of the PER faces the cytosol, Using this Mab we developed a tec hnique which allowed precise quantification of PER density per cell, F urthermore, cellular localization studies by now cytometric analysis a nd confocal microscopy on cell Lines displaying different levels of PE R showed that Mab 8D7 was entirely colocalized with an antimitochondri a Mab. (C) 1996 Wiley-Liss, Inc.