Patients with systemic lupus erythematosus (SLE) spontaneously produce antibodies to nuclear antigens, particularly DNA. Normal individuals produce similar antibodies only after certain in vitro stimulation. We propose to compare the antibodies to DNA made by lymphocytes from normals and SLE patients in vitro to determine if they differ in specificity. Monoclonal antibodies will be derived by using the parallel techniques of Epstein-Barr virus (EBV) transformation of B cells and by cell hybridization. Lymphocytes from patients with SLE and from normals will be stimulated in vitro, transformed with EBV and/or fused to the human myelomas, LSM 2.7, which has been used to derive human monoclonal antibodies to pneumococcus, and GM 4672, which has been used to produce human monoclonal antibodies to DNA from patients with SLE. Hybrid cells will be grown in selective medium (HAT), cloned and screened for production of anti DNA antibodies using an enzyme linked immunoassay (ELISA). EBV transformed B cell lines will be cloned, using a new cloning method we have developed, and screened. The resulting positive clones will be expanded and the antibodies produced by normal and SLE cells characterized and compared. Immunoglobulin class and IgG subclass will be determined. Isoelectric focusing followed by incubation with radiolabelled DNA will be performed and binding patterns determined. The specificities of the antibodies will be studied by determining the ability of various nucleic acid antigens including single and double stranded DNA, DNA homopolymers, DNA duplexes and copolymers, RNA, RNA copolymers, RNA duplexes, oligonucleotides, mononucleotides and nucleosides to inhibit the anti DNA reactivity of the supernatants. Antibody affinities will be evaluated by measuring dissociation rates of bound labelled DNA over time. We will characterize whether the monoclonal antibodies produced by normals and patients with SLE are the same or whether patients with SLE produce antibodies directed against a unique epitope or have a unique pattern immunochemically. If the antibodies derived from normals and patients with SLE are different, this work could suggest possible clues to the nature of the inciting antigen. If similar antibodies are found, this would suggest that the abrogation of control of production of an antibody made by both normals and SLE patients may be more important than the nature of the antibody itself.
| Hoch, S; Schwaber, J (1987) Identification and sequence of the VH gene elements encoding a human anti-DNA antibody. J Immunol 139:1689-93 |
| Hoch, S; Schwaber, J (1986) Specificity analysis of human anti-DNA antibodies. J Immunol 136:892-7 |
