Little is known about the molecules that cause rejection of long-term tissue transplants either in man or in mouse. The diversity of responses elicited by the mouse minor histocompatibility (H) antigens, both in vivo and in vitro, suggests they are a heterogeneous group of molecules. The intent of this proposal is to identify and briefly characterize the minor H antigens of BALB/cBy mice by the coordination of electrophoretic techniques (two-dimensional electrophoresis and other types of polyacrylamide gel electrophoresis), and immunological assays (delayed-type hypersensitivity assays, skin graft rejection, and cell mediated lympholysis). By comparing the two-dimensional electrophoretic patterns of BALB/cBy proteins to C57BL/6By proteins by a new and very rapid technique, electrophoretic variants between them are to be identified. The variant proteins will be genetically mapped by using the strain distribution patterns derived from the CXB recombinant-inbred and B6.C (HW) strains. Variants that are found to have patterns of distribution identical to those of the minor H antigens will be tested for such activity by standard immunological assays. The detection of anamnestic immune responses, either as the result of primary immunization or as the result of secondary challenge with these isolated proteins (in conjunction with lymphoid cells of known H antigen types), will define whether these isolated proteins are minor H antigenic determinants. New techniques that greatly increase the sensitivity of the analysis of electrophoretic patterns (or electrophoretograms) to be used are described. These techniques include: photographic detection of two radioisotopes within the same gel, enhancement of electrophoretograms by unsharp-masking and blink comparison of these images. The completion of this proposal research will lead to a better understanding of the molecules causing the rejection of long-term tissue transplants, as well as the development and utilization of more sensitive techniques for analyses of electrophoretograms.