Pitfalls in determining IgG and IgG subclass antibodies to food antigens

Several variables were found to influence enzyme-linked immunosorbent assay (ELISA) measurements of IgG and IgG subclass antibodies to food antigens. Two polyclonal rabbit antibody reagents to human IgG, and two sets of murine monoclonal antibodies to human IgG subclasses, were compared as secondary reagents. The choice of both polyclonal and monoclonal reagents affected significantly the results. High levels of IgA to an antigen depressed the measurements of comparable total IgG antibodies but did not influence the percentages of the four IgG subclass activities. Different ways of expressing the IgG subclass results were compared and the validity of the reference measurements used to obtain them was examined. Information from previous studies, together with the present data, suggests that absolute values cannot be reliably determined. We have therefore chosen to express the results for IgG subclass activities on a relative basis with reference to units of total IgG activity against the same antigen in each subject. This approach is practical and appears scientifically acceptable but limits the use of such determinations to comparisons between groups of subjects studied in the same laboratory.