Differentiation between neutrophil-bound antibodies and immune complexes.

It is often unknown whether neutrophil-bound immunoglobulins (NBIg) in patients with chronic diseases consist of bound immune complexes (IC) or of anti-neutrophil antibodies. Until now, the detection of IC in serum by the C1q-binding test has been used to define the nature of NBIg. Here we present studies on methods for characterization of NBIg using a bivalent IC and heat-aggregated IgG as IC models and neutrophil alloantibodies as antibody models. Heat-aggregated IgG was readily detected in the C1q-binding test. However, the bivalent IC, which is capable of binding to both low-affinity Fc receptors (FcRII and FcRIII) on neutrophils was not detected in this test. This was due to failure of the bivalent IC to bind to C1q, as was demonstrated by studies with immobilized C1q. However, the bivalent IC as well as heat-aggregated IgG, when bound to neutrophils from healthy donors, were not or only marginally eluted from the cell surface by lowering the pH, in contrast to a number of different cell-bound antibodies. This led to the observation that eluates, prepared from sensitized cells, showed reactivity with donor neutrophils, whereas eluates, prepared from cells to which immune complexes had bound, did not. Thus, a negative result in the C1q-binding test does not prove that a serum does not contain neutrophil-binding IC, but the reactivity of an eluate can distinguish between anti-neutrophil antibodies and bound IC.