Factors which govern the sensitivity of direct and indirect rosetting reactions and reverse passive haemagglutination in the identification of cell surface and free macromolecules.

A series of immunoassays have recently been elaborated in which the red cell is used as a label or marker of interacting antibody, often anti-immunoglobulin (anti-Ig). This paper considers and investigates quantifiably, the different variables which affect the sensitivity of direct and indirect antiglobulin rosetting reactions (DARR and IARR) and of reverse passive haemagglutination (RPH). Sensitivity is governed by the surface properties and amount of antibody on the indicator red cell and, in detection of cell-bound antigen, by the antigen density. By varying the antibody:Ig ratio on the red cell using affinity purified anti-Ig, cells with more than 1:32–64 antibody:Ig showed similar sensitivity in detection of lymphocyte sIg in DARR and IARR tests and of serum Ig by RPH. Using these indicator cells coupled with different Ab:Ig ratios to detect lymphocytes sensitized with different levels of anti-Ig in a model IARR test, it was clear that studying cells with a high density of determinants on the lymphocyte surface it is not necessary to have `strong' anti-immunoglobulin on the indicator cell, but increasingly sparse determinants require increasingly strong anti-Ig on the red cell to detect all positively reacting cells. Red cells of different species afford carriers of varing sensitivity in all three reactions—DARR, IARR and RPH. Preliminary trypsin treatment may greatly further enhance the sensitivity of some species red blood cells (e.g. bovine and sheep), but leaves others (e.g. pig and donkey) unchanged. Use of I-labelled Ig and anti-Ig showed that the increased sensitivity of trypsinized bovine and sheep RBC is not due to increased uptake of Ig during chromic chloride linkage or of antibody in agglutinations.