A microfluorometric assay for immunoglobulin class and subclass levels in murine serum

The IgG fractions of rabbit antisera specific for the Fc part of mouse IgA, IgM, and the four subclasses of IgG (1, 2a, 2b, and 3) were coupled covalently to Sepharose beads by the cyanogen bromide method. The beads were then incubated with different dilutions of mouse serum. The mouse immunoglobulins (Ig) binding to the beads were demonstrated by the use of a fluorescent goat anti-mouse/Ig conjugate. Incubations were done in flat bottom of the plates with an inverted microfluorometer. The logarithm of the fluorescence was linearly related to the logarithm of the serum dilution for serum dilutions between 10(3) and 10(5). Ig levels of individual samples can be determined with as little as 1 mul of serum. The sensitivity of the method for IgM was estimated with MOPC 104E plasmacytoma IgM. Five ng of IgM per sample was easily detected. The method offers an attractive alternative to radioimmunoassay techniques.