Immunological-chromatographic analysis of lysozyme variants

Immunological-chromatographic analysis (ICA) was used to evaluate the cross-reactivities of eight lysozyme variants with five different immobilized monoclonal anti-hen-egg-white lysozyme antibodies. ICA is a dual-column high-performance liquid chromatography-based method in which an immunoaffinity and a conventional analytical column are coupled with a switching valve. Antigens are first captured on the affinity column and then desorbed and concentrated on the second column, where they are separated further. This arrangement permits antigen-antibody interactions occurring on the affinity column to be monitored on-line with the second column. The ICA system was used to perform direct and competitive inhibition binding immunoassays with unlabeled antigens. Seven of the eight lysozymes tested bound to all five immobilized monoclonal antibodies. Competitive inhibition of binding of hen egg white lysozyme to the monoclonal antibody, HyHel-5, was measured by using the variants Japanese quail and bobwhite quail lysozymes as inhibitors. The ratio of the amount of bobwhite quail to Japanese quail lysozyme required to give 50% inhibition of binding of hen egg white determined by ICA compares well with the results obtained by other investigators who used an enzyme-linked immunosorbent assay plate binding assay.