The clonal analysis of anticardiolipin antibodies in a single patient withprimary antiphospholipid syndrome reveals an extreme antibody heterogeneity

The mechanism underlying the prothrombotic state that characterizes the pri mary antiphospholipid syndrome proves to be difficult to define mainly beca use of the variety of the phospholipid and protein targets of antiphospholi pid antibodies that have been described. Much of the debate is related to t he use of polyclonal antibodies during the different antiphospholipid assay s. To better describe the antiphospholipid antibodies, a strategy was desig ned to analyze the reactivity of each one antibody making up the polyclonal anticardiolipin activity, breaking down this reactivity at the clonal leve l. This was performed in a single patient with primary antiphospholipid syn drome by combining (1) the antigen-specific selection of single cells sorte d by flow cytometry using structurally bilayered labeled anionic phospholip ids and (2) the cloning of immunoglobulin tig) variable (V) region genes or iginating from individual IgG anticardiolipin-specific B cells by a single- cell polymerase chain reaction technique. The corresponding V regions were cloned in order to express human recombinant antibodies in insect cells by a baculovirus expression system. The molecular analysis, the fine specifici ty, and the protein cofactor dependency of the first 5 monoclonal IgG antic ardiolipins are reported here. This clonal analysis reveals the extreme het erogeneity of these antibodies, which could account for the difficulties in the previous attempts to define the pathogenic antiphospholipid response. This approach should help to unravel the complex antiphospholipid immune re sponse and the mechanism of the prothrombotic state associated with these a ntibodies, but it could also shed some light on their possible origins.