Use of nuclear antigen-coated red cells in hemolytic plaque assays

By studying antinuclear antibody production at the cellular level, we can better understand the problems of immunoregulation in individuals with systemic lupus erythematosus. To date, the use of hemolytic plaque assays to detect B cells secreting antinuclear antibodies has been hampered by an inability to achieve reliable coating of red cells by nuclear antigens. Because the chromic chloride technique has proved ineffective for coupling nucleic acids and/or nuclear antigens to sheep red blood cells (SRBC) in our laboratory, we have developed a method of coupling SS DNA, DS DNA, poly(I).poly(C), Sm, and ENA to red cells pretreated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (ECDI) and poly(L-lysine) (PLL). Coated red cells were agglutinated by specific antisera and not by normal sera and were then used in hemolytic plaque assays to detect antinuclear plaque-forming cells (PFC) in spleens from various strains of mice with lupus-like syndromes. PFC specific for SS DNA, DS DNA, poly(I).poly(C), Sm, and ENA were found in MRL/lpr and NZB x W mice, and the number of anti-SS DNA and anti-DS DNA PFC correlated with the age of the animals. Indirect (IgG) PFC specific for nuclear antigens increased dramatically in female NZB x W mice between 11 and 13 months, a time when more than 50% of the animals usually die. Preliminary studies have shown that PFC specific for nuclear antigens can be detected in peripheral blood from patients with lupus erythematosus. Pretreatment of sheep red cells with ECDI and PLL thus allowed the coupling of selected nuclear antigens to these cells and provided the first demonstration of IgM and IgG PFC specific for a variety of nuclear antigens.