IMMUNOLOGICAL APPROACH TO INVESTIGATING MEMBRANE CELL DAMAGES INDUCEDBY LIPOPEROXIDATIVE STRESS - APPLICATION TO FAR UV-IRRADIATED ERYTHROCYTES

Oxygen-reactive species are being described as agents responsible for cell degeneration mechanisms resulting from membrane, enzyme, and nucl ear alterations. Lipid peroxidation on its own is considered to be one of the consequences of the free radicals attack, and among the differ ent reactive aldehydes that can be formed from the decomposition of li pid peroxides, the most extensively assayed have been malondialdehyde (MDA). However, the different techniques currently used for MDA assay (HPLC, GLC) are barely sensitive enough to follow its production at th e cellular level. In order to develop an immunofluorescent technique a ble to detect cellular damages provoked by lipoperoxidation, polyclona l antibodies against lysozyme modified by MDA treatment have been rais ed in rabbits. We show that this immunserum recognizes specifically al l the MDA-treated proteins tested, but not the intact proteins or the proteins treated by other aldehydes. Moreover, we demonstrate using an ELISA technique that the amount of immunoreactive proteins in MDA-tre ated membrane erythrocytes is proportional to the concentration of MDA applied, suggesting that this assay may represent a quantitative meth od of determination of lipoperoxidative alterations. In addition, when coupled to an indirect fluorophore antibody (FITC), the immunserum al lows a precise location of these modified proteins within the membrane s of erythrocytes in which lipid peroxidation was initiated by far UV irradiation. In summary, the interest of this work is to provide an im munological probe that can precociously detect membrane damages induce d by MDA, regardless of the cell type and pro-oxidant (physiological o r pathological) conditions.