The validity of the radial immunodiffusion method for the quantitative determination of human IgM. Evaluation of a modified method.

Three subgroups of human IgM can be distinguished on the basis of differences of slopes of the D2 versus absolute concentration plot in the radial immunodiffusion technique (Klein et al., 1973). Individual IgM fractions, whether mono- or polyclonal, always belong to only one of these groups. The differences between the subgroups disappear after reduction of the IgM to 7S subunits. These findings provide an explanation for the large discrepancies between absolute IgM determinations in different laboratories. It follows that most of the readings of individual IgM values in the Mancini technique must be wrong by any standard, including WHO reference preparations. The insertion of a simple reduction step in the assay abolishes the differences in quantitative reactivity between IgM subgroups as well as between natural 7S and 19S IgM. This allows an absolute determination of both forms together. The values thus obtained differ considerably from the estimates given by Humphrey and Batty (1974). It also appears that the International Units of the WHO do not represent the same quantity of IgM in different reference sera. The modified method allows the determination of total monoclonal as well as polyclonal human IgM by weight.