Optimal conditions for the use of sulphydryl compounds in dissociating red cell antibodies.
Optimal conditions for the use of 2-mercaptoethanol (2-ME) and dithiothreitol (DTT) in the inactivation of IgM red cell antibodies were investigated. 0.2 M 2-ME was better than 0.01 M DTT; higher concentrations of these 2 substances could not conveniently be used because of gel formation. Incubation at 37 degrees C led to far more rapid inactivation than incubation at 22 degrees C; with 2-ME a period of 15 min at 37 degrees C was sufficient except with very potent antibodies, which were not inactivated even after 3 h incubation. When indirect antiglobulin tests were carried out on 2-ME-treated serum which had not been dialysed, false-positive results were obtained, although only with the mixture of equal volumes of undiluted serum and of 2-ME; results were negative in tests on dilutions of the mixture in saline. False-positive results could also be avoided by incubating the serum for 3 h with iodoacetamide, but this method also involves dilution of the serum. The only method whereby 2-ME-treated serum can be tested without diluting it and without obtaining false-positive reactions is to dialyse it overnight against saline. Since this method also gets rid of the noxious smell of 2-ME it is to be preferred for most purposes.