Subnucleosome structures as substrates in enzyme-linked immunosorbent assays.
Histone preparations preserving the tertiary and quaternary structure of histone-histone complexes and histone-DNA complexes, as well as individual histones, were isolated or reconstituted. Various parameters were tested in order to determine the suitability of these complexes for use as substrates in enzyme-linked immunosorbent assays. The protein concentration required to saturate the solid phase was determined, and the amount of bound protein was quantified by the micro-bicinchoninic acid protein assay. In addition, the relative DNA content of solid phase antigen was measured by the binding of monoclonal anti-native DNA antibodies. Prototype sera representing different disease groups produced reproducible and unique patterns of reactivity on the panel of antigens, demonstrating the lack of substantial assay bias. Two substrates, the H2A-H2B dimer and the H2A-H2B-DNA complex, both appear to be oriented in a random manner on the solid phase, leaving a number of different epitopes exposed to the solution. This novel set of histone antigens can now be used to define the specificity of anti-histone antibodies in relation to the quaternary structure of chromatin.
W.M. Keck Autoimmune Disease Center, Research Institute of Scripps Clinic, La Jolla, CA 92037.
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Burlingame
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Rubin
D000818:Animals; D000974:Antibodies, Antinuclear / Q000032:analysis*; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002417:Cattle; D002843:Chromatin / Q000276:immunology*; D004247:DNA / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D006657:Histones / Q000276:immunology*; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D009707:Nucleosomes / Q000276:immunology*
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D002843:Chromatin; D000939:Epitopes; D006657:Histones; D046911:Macromolecular Substances; D009707:Nucleosomes; D004247:DNA