Further Characterization of the Alternative Protein-A Interaction of Immunoglobulins: Demonstration of an Fc-binding Fragment of Protein A Expressing the Alternative Reactivity

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Abstract
Intact IgG and fragments F(ab'')2.gamma., Fab.gamma. and Fc.gamma. from a rabbit anti-protein-A serum (RapA) and corresponding preparations from normal rabbit IgG (NRG) were tested for their inhibitory effect on the binding of protein-A-reactive 125I-IgE and 125I-Fc.gamma., respectively, to protein-A-Sepharose. Intact IgG, F(ab'')2.gamma. and Fab.gamma. of RapA inhibited the binding of protein-A-reactive 125I-IgE; only intact IgG and Fc.gamma. fragments from RapA and NRG inhibited the binding of 125I-Fc.gamma. to protein A-Sepharose. The functional relationship between RapA and human polyclonal IgG was studied in a nephelometric test system. Intact IgG or fragments of IgG from human polyclonal IgG and RapA were found to affect the precipitation between human IgG and protein A in a similar way. Thus, F(ab'')2.gamma. fragments and intact IgG enhanced the precipitation, whereas Fab.gamma. and Fc.gamma. fragments inhibited the precipitation. Protein A and an Fc-binding fragment of protein A (fragment B) were tested for their abilities to link different radiolabeled Ig preparations expressing the alternative and the classical protein-A reactivity to immobilized Fc fragments. All proteins expressing the alternative reactivity were efficiently bound both by fragment B and by protein A, indicating that fragment B, in addition to its classical Fc-binding activity, also expresses the alternative protein-A reactivity.

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