Surface immunoglobulin of rabbit peripheral blood lymphocytes: detection of allotypes by fluorescence and rosetting

Specific anti-allotype reagents were prepared to detect heavy and light chain allotypes on surface immunoglobulin-positive (sIg+) cells from a3a3b4b4, a2a2b5b5 homozygous and a2a2b4b5 heterozygous rabbits. Total sIg+ peripheral blood lymphocytes (PBL) were detected with fluorescein-labeled goat anti-light chain and sheep anti-rabbit Ig reagents. The total sIg+ cells detected with these reagents and the sum of a and b allotypes individually measured with specific fluorescent anti-allotype reagents (a2 + a3 or b4 + b5) were measured more or ess the same when measured by fluorescence-activated cell sorter (FACS) II flow microfluorometry. The data provided no evidence for single cells expressing both allelic allotypes (allelic inclusion). We found that FACS and resetting techniques were generally equally sensitive. However, we detected a greater proportion of total b4+ plus b5+ cells by rosetting than by fluorescence in some PBL preparations from heterozygous b4b5 rabbits. This was not seen with artificial mixtures of b4b4 and b5b5 cells. The nature of these cells is not yet known. Conceivably, they were not scored as lymphocytes by light-scatter analysis on the FACS and hence were not counted, but were indistinguishable from lymphocytes by microscopy.