OBJECTIVE: To analyse the phospholipid binding specificity, functional characteristics and idiotype expression of human hybridoma derived monoclonal autoantibodies (MAb) derived from the spleens of two patients with active systemic lupus erythematosus (SLE). METHODS: The IgM MAbs binding to phospholipids were generated from spleen cells of two patients (RSP and RT) with active SLE and their specificity of binding to neutral phospholipids (phosphatidyl ethanolamine, phosphatidyl choline, platelet activating factor, sphingomyelin) and negatively charged phospholipids (phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, phosphatidyl inositol and cardiolipin (CL)) analysed. Binding specificity of cross reactive antibodies (those binding to CL and DNA) was confirmed by fluid phase inhibition assays. Lupus anticoagulant activity and beta 2-glycoprotein-1 (beta 2 GP-1) requirement for the antigen binding of these MAbs were detected using the modified dilute Russell's viper venom test and modified anti-CL enzyme linked immunosorbent assay (ELISA), respectively. Expression of idiotypes (Id) Id RT-84 and Id H3 was analysed using rabbit polyclonal and murine monoclonal anti-idiotype reagents, respectively. RESULTS: Twelve clones from the patient RSP and eight clones from patient RT were reactive with phospholipids. Marked differences in phospholipid binding of these MAbs were noted, varying from truly polyreactive (RT-72 bound to most phospholipids tested) to monospecific (RT-84 bound only to CL). Furthermore, MAbs RT-84, RT-129, and RSP-57 had lupus anticoagulant activity and required beta 2 GP-1 for CL binding. It was found that 75% of phospholipid binding antibodies from RT clones expressed RT-84 Id, but none from RSP clones did so, and that Id H3 was expressed only by the RT-83 antibody. CONCLUSION: These results show that human anti-phospholipid MAbs are heterogeneous with respect to phospholipid binding, functional characteristics, and Id expression.
OBJECTIVE: To analyse the phospholipid binding specificity, functional characteristics and idiotype expression of human hybridoma derived monoclonal autoantibodies (MAb) derived from the spleens of two patients with active systemic lupus erythematosus (SLE). METHODS: The IgM MAbs binding to phospholipids were generated from spleen cells of two patients (RSP and RT) with active SLE and their specificity of binding to neutral phospholipids (phosphatidyl ethanolamine, phosphatidyl choline, platelet activating factor, sphingomyelin) and negatively charged phospholipids (phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, phosphatidyl inositol and cardiolipin (CL)) analysed. Binding specificity of cross reactive antibodies (those binding to CL and DNA) was confirmed by fluid phase inhibition assays. Lupus anticoagulant activity and beta 2-glycoprotein-1 (beta 2 GP-1) requirement for the antigen binding of these MAbs were detected using the modified dilute Russell's viper venom test and modified anti-CL enzyme linked immunosorbent assay (ELISA), respectively. Expression of idiotypes (Id) Id RT-84 and Id H3 was analysed using rabbit polyclonal and murine monoclonal anti-idiotype reagents, respectively. RESULTS: Twelve clones from the patient RSP and eight clones from patient RT were reactive with phospholipids. Marked differences in phospholipid binding of these MAbs were noted, varying from truly polyreactive (RT-72 bound to most phospholipids tested) to monospecific (RT-84 bound only to CL). Furthermore, MAbs RT-84, RT-129, and RSP-57 had lupus anticoagulant activity and required beta 2 GP-1 for CL binding. It was found that 75% of phospholipid binding antibodies from RT clones expressed RT-84 Id, but none from RSP clones did so, and that Id H3 was expressed only by the RT-83 antibody. CONCLUSION: These results show that human anti-phospholipid MAbs are heterogeneous with respect to phospholipid binding, functional characteristics, and Id expression.