REAL-SPACE DIFFERENTIATION OF IGG AND IGM ANTIBODIES DEPOSITED ON MICROTITER WELLS BY SCANNING FORCE MICROSCOPY

Enzyme-linked immunosorbent assay as carried out in microtiter wells i s a common method of identifying the presence of specific biomolecules in human samples to allow diagnosis of medical conditions. The presen ce, in a minority of samples, of human antibodies reactive with the im munoassay-specific antibodies is a well-known phenomenon (Maxey, K. M. ; Maddipati, K. R.; Birkmeir, B. J. Clin. Immunoassay 1992, 15, 116). These antibodies are often referred to as heterophilic antibodies or r ogue samples and are commonly of the IgM isotype (Levinson, S. S. Clin . Biochem. 1992, 25, 77). The presence of such antibody species within a sample can result in incorrect analyte determination and clinical m isclassification. We have employed scanning force microscopy (SFM) to study microtiter wells coated with a passively adsorbed IgG antibody t hat is specific for human IgM in order to determine if SFM is an appro priate technology to discriminate between these two biomolecular types and hence useful in the evaluation of interactions between IgG coated on microtiter wells and IgM in human sample fluids. It should be note d that in this model system, IgG that specifically binds human IgM has been employed. In a true immunoassay system where interference occurs , the IgM normally binds to IgG specific for the analyte being measure d. SFM data clearly differentiate between IgM bound by IgG and any fre e IgG. The observed molecular diameter for the IgG antibody was approx imately 33 nm, and that for the IgG-IgM antibody complex was approxima tely 53 nm. While these observations are made from traditional raw SFM data images, we also demonstrate here the potential value of removing tip profile information from the data to allow more accurate determin ation of molecular dimensions (28 nm for the IgG and 47 nm for the IgG -IgM complex).