DETECTION OF FACTOR-IX ANTIBODIES BY RADIOIMMUNOASSAY - EFFECT OF CALCIUM ON ANTIBODY-FACTOR-IX INTERACTION

Abstract

A radioimmunoassay [RIA] for alloantibodies (inhibitors) and heteroantibodies to human factor [F] IX was developed using radioiodinated human FIX and formalin-fixed, heat-killed Staphylococcus aureus cells (Staph A). Staph A was used as a solid-phase adsorbent for immune complexes. The assay is specific, shows excellent correlation with FIX coagulant neutralization assays in detecting alloantibodies (r = 0.98), and is 60 times more sensitive. The Staph A method allows rapid separation of immune complexes (within 10 min of addition) and binds Ig with equivalent efficiency in the presence and absence of Ca. These characteristics have made the Staph A binding method useful for observing the Ca++ effect on the antigenicity of FIX as detected by hetero- and alloantibodies. All antisera ivestigated showed increase in antibody titer when measured in the presence of Ca++. One particular alloantibody showed the greatest increase in titer (> 2-fold). The reversibility of the Ca effect by excess EDTA indicates that is was not caused by Ca++-dependent proteolysis of the FIX molecule. The Staph A procedure can also be used as a sensitive competitive RIA for human FIX using alloantibody. The assay could detect FIX antigen to a dilution of 1:1280 of normal plasma and correctly classified hemophilia B plasmas as cross-reacting material positive or negative.