Anti-DNA antibody: Avidity and valence measurements, studies using sera from immunized rabbits and from two systemic lupus erythematosus patients

Abstract

We have developed methods to characterize the relationship of single strand DNA and anti-DNA antibodies in terms of the immunochemical parameters of avidity and antigenic valence. In this report we present methods for the purification of anti-DNA antibodies obtained from the sera of hyperimmunized rabbits and patients with systemic lupus erythematosus. Three methods of avidity measurement using purified anti-DNA antibodies are compared. These are: (1) sedimentation of mixtures of DNA and anti-DNA antibody on sucrose gradients; (2) binding of anti-DNA antibody to DNA which is linked to Sepharose 4B followed by filtration; and (3) precipitation of DNA-anti-DNA complexes by polyethylene glycol. The values of avidity obtained by these direct methods are compared with values obtained indirectly from Farr tests using ammonium sulphate to precipitate labelled DNA which is antibody bound. The concentrations of free and antibody bound DNA at various total DNA concentrations are determined to utilize this method. The value for valence, as determined from work with the purified antibody, permits calculation of avidity from the Farr test data. To determine if purification of antibody alters the results of avidity determinations in the Farr assay, specimens from various stages of antibody purification were tested. These specimens were also utilized in determinations of avidity index from antibody dilution results in the Farr assay. Our results showed that all techniques give values of avidity in the range of 1–9 × 10 8 L/M for the rabbit antibodies, and significantly lower avidity of approximately 10 7 L/M for the human SLE sera. Whole sera as well as all of the more purified antibody fractions give similar results. A DNA valence of two antibody binding sites per molecule of mol. wt. 55,000 is found by all techniques for the rabbit antibodies, while the valence for SLE patient No. 1 is approximately 3, and for SLE patient No. 2 approximately 10. From this data it appears that the methods used are applicable' for determinations of DNA-anti-DNA avidity and antigenic valence. Such determinations may be important in determining which types of anti-DNA antibodies are prone to form DNA-anti-DNA complexes in vitro or in vivo which have a capacity to lodge at various tissue sites and produce tissue injury in SLE patients.