The mechanism underlying the prothrombotic state that characterizes the primary antiphospholipid syndrome proves to be difficult to define mainly because of the variety of the phospholipid and protein targets of antiphospholipid antibodies that have been described. Much of the debate is related to the use of polyclonal antibodies during the different antiphospholipid assays. To better describe the antiphospholipid antibodies, a strategy was designed to analyze the reactivity of each one antibody making up the polyclonal anticardiolipin activity, breaking down this reactivity at the clonal level. This was performed in a single patient with primary antiphospholipid syndrome by combining (1) the antigen-specific selection of single cells sorted by flow cytometry using structurally bilayered labeled anionic phospholipids and (2)
IntroductionAntiphospholipid autoantibodies (aPLs) 1 are associated with thrombosis, recurrent fetal loss, and thrombocytopenia in patients with antiphospholipid syndrome (APS). [2][3][4] The mechanism of the characteristic prothrombotic state related to aPLs during APS is still under debate. 5 Much of it is related to the definition of the precise nature of aPL specificity, which remains unclear because of the number of possible antigenic targets discovered over the last few years. It is now accepted that plasma proteins play an important role in aPL reactivity. 6,7 The most common protein cofactors are 2GP1 8,9 and prothrombin. [10][11][12] Other phospholipid (PL)-binding proteins such as protein C9, protein S9, and annexin V 13 have also been found to have cofactor properties. On one hand, studies have shown that the binding of aPLs to cardiolipin (CL) is enhanced by the addition of one cofactor (2GP1, prothombin, protein C, protein S), which suggests that aPLs react to a complex compounded of anionic PLs and a cofactor 8,10,11 or to an epitope that becomes exposed when a protein is bound to a PL. [14][15][16] On the other hand, other studies have shown that aPLs are able to bind directly to immobilized 2GP1 [17][18][19] or PT 20,21 in the absence of PL. In addition, some studies have shown that anti-2GP1 antibodies (Abs) are primarily associated with thrombosis in APS 22-24 although other reports have involved others Abs as anti-PT 25 or lupus anticoagulant. 26 It appears that most of these difficulties are related to the use of polyclonal Abs (serum or purified immunoglobulin [Ig]) 7,8,10,11 or a few monoclonal IgM 15,27-29 or IgG 30-33 aPLs originating from different patients. We therefore considered it necessary to concentrate our attention on aPL reactivity of each Ab making up the polyclonal aPL reactivity, breaking down this reactivity at the clonal level in a single patient.The major methods currently in use for the production of human monoclonal antibodies (mAbs) are Epstein-Barr virus transformation, 34 mouse-human hybridomas, 35 or human-human hybridomas 36 and fusion of Epstein-Barr virus-transformed B lymphocytes with a malignant cell line. 37 These methods have their dr...