Immunological approach to investigating membrane cell damages induced by 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 nuclear alterations. Lipid peroxidation on its own is considered to be one of the consequences of the free radicals attack, and among the different reactive aldehydes that can be formed from the decomposition of lipid 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 the cellular level. In order to develop an immunofluorescent technique able to detect cellular damages provoked by lipoperoxidation, polyclonal antibodies against lysozyme modified by MDA treatment have been raised in rabbits. We show that this immunserum recognizes specifically all 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-treated membrane erythrocytes is proportional to the concentration of MDA applied, suggesting that this assay may represent a quantitative method of determination of lipoperoxidative alterations. In addition, when coupled to an indirect fluorophore antibody (FITC), the immunserum allows a precise location of these modified proteins within the membranes of erythrocytes in which lipid peroxidation was initiated by far UV irradiation. In summary, the interest of this work is to provide an immunological probe that can precociously detect membrane damages induced by MDA, regardless of the cell type and pro-oxidant (physiological or pathological) conditions.
Petit
E
E
Laboratoire de Neurosciences, URA 1829-CNRS, Caen.
Divoux
D
D
Chancerelle
Y
Y
Kergonou
J F
JF
Nouvelot
A
A
D000818:Animals; D000906:Antibodies; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004910:Erythrocyte Membrane / Q000502:physiology* / Q000528:radiation effects / Q000648:ultrastructure; D004912:Erythrocytes / Q000502:physiology / Q000528:radiation effects / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D015227:Lipid Peroxidation / Q000528:radiation effects*; D008315:Malondialdehyde / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis / Q000097:blood* / Q000528:radiation effects; D009113:Muramidase / Q000032:analysis / Q000276:immunology; D018384:Oxidative Stress; D011817:Rabbits / Q000276:immunology; D012680:Sensitivity and Specificity; D014466:Ultraviolet Rays
D000906:Antibodies; D008565:Membrane Proteins; D008315:Malondialdehyde; D009113:Muramidase