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Optimal Conditions for the Use of Sulphydryl Compounds in Dissociating Red Cell Antibodies
Author(s) -
Freedman J.,
Masters C. A.,
Newlands M.,
Mollison P. L.
Publication year - 1976
Publication title -
vox sanguinis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.68
H-Index - 83
eISSN - 1423-0410
pISSN - 0042-9007
DOI - 10.1111/j.1423-0410.1976.tb02821.x
Subject(s) - dithiothreitol , iodoacetamide , incubation , serial dilution , chemistry , antibody , chromatography , dilution , saline , incubation period , red cell , phosphate buffered saline , 2 mercaptoethanol , biochemistry , immunology , medicine , cysteine , enzyme , physics , alternative medicine , pathology , thermodynamics
. 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 two substances could not conveniently be used because of gel formation. Incubation at 37°C led to far more rapid inactivation than incubation at 22°C; with 2‐ME a period of 15 min at 37°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.