Application of the Farr assay to the analysis of antibodies specific for UV irradiated DNA.

Abstract

In order to determine the optimum conditions for reactivity in the ammonium sulphate precipitation (Farr) assay was have studied the DNA binding properties of two antibodies raised against ultraviolet single stranded DNA (UVssDNA) complexed with methylated bovine serum albumin. In general the buffer composition, pH, temperature, and ionic strength conditions described for binding to undamaged DNA were found to be appropriate for binding to UV-irradiated DNA. However, some differences in detail were noted which indicate the necessity for checking the physical conditions of binding of individual antibodies. Mouse monoclonal antibody and rabbit polyclonal antisera bound to UVssDNA very rapidly, even when DNA and ammonium sulphate were added simultaneously, whereas this procedure prevented binding of rabbit antisera to UV-irradiated double stranded DNA. Incubation at 45 degrees C for 30 min inhibited binding by mouse antibody, and incubation at 37 degrees C for 60 min caused reversible dissociation of the DNA-antibody complex. The optimised Farr assay was used to define the antigen specificities of the antibodies. The mouse antibody specifically bound to UVssDNA, but not to ssDNA, double stranded (ds) DNA, or UVdsDNA, whereas the rabbit antisera bound to UVssDNA, ssDNA or UVdsDNA, but not dsDNA. The extent of binding of the mouse antibody was dependent on the UV dose to the antigen, as well as the antigen concentration, indicating that the Farr assay can form the basis of a quantitative assay for photoproducts in DNA.