NON-SPECIFIC AGGLUTINATION OF BROMELIN-TREATED RED BLOOD CELLS BY NORMAL HUMAN SERUM : REACTION OF IMMUNOGLOBULINS TO MEMBRANE PROTEINS EXPOSED BY BROMELIN-DIGESTION

Abstract

Although it is known that agglutinins specific for bromelin-treated red blood cell (BrRBC) exist in normal human serum, their immunological and biological characteristics have not yet been elucidated. We have found two different types of agglutinins specific for BrRBC in the normal serum. The one reacts specifically with red blood cell (RBC) pretreated for 15 minutes with lower concentration of bromelin (Br) (1.25 U/ml to 25 U/ml in terms of casein-digestion activity), and thus, it is responsible for the non-specific agglutination encountered in routine works. The other is detected with RBC pretreated with Br of higher activities (>125 U/ml). The former agglutinin was proved to be IgM and the latter to be IgG on the basis of biochemical and immunological criteria, and they are termed NSA-BrRBC-Ⅰ (non-specific agglutinin for BrRBC) and NSA-BrRBC-Ⅱ, respectively. The incidences of NSA-BrRBC-Ⅰ and -Ⅱ in normal human sera were 2.3% and 99.9%, respectively. The binding site for NSA-BrRBC-Ⅰ on RBC was specifically elicited by mild Br-treatment but not by other proteinase digestions. From the Br-digestion products of RBC membrane, we partially purified the protein fraction which exhibited a high inhibitory activity against the agglutination by NSA-BrRBC-Ⅰ without suppressing the agglutination by most of the blood group antibodies examined. This fraction was therefore assumed to contain the target protein of NSA-BrRBC-Ⅰ. Four fragments with different molecular weights (26.000, 61.000, 74.000 and 160.000 dalton) could be detected by Western blotting of the fractions after immunoprecipitation and electrophoresis in the reduced condition. This fraction successfully inhibited non-specific agglutination by NSA-BrRBC-Ⅰ in two-stage Br-method but not in one-stage method. These results indicate that one of the non-specific agglutinations of BrRBC encountered in routine works is caused by the interaction between NSA-BrRBC-Ⅰ and Br-modified membrane protein(s).