Comparison of an indirect immunofluorescence assay and a modified sensitive immunoblot assay for the study of the autoantibody in pemphigus vulgaris

Abstract

Some patients with pemphigus vulgaris (PV) have positive direct immunofluorescence (DIF) but are negative by indirect immunofluorescence (IIF). The purpose of this study was (1) to compare the sensitivity of an IIF assay with an immunoblot (IB) assay, (2) to compare the IIF and the IB assay in PV patients in whom the clinical picture and DIF were consistent, but the IIF was negative and (3) to compare the IIF and the IB assay in patients in clinical remission for 3 years or more. A comparison was made of the titers of PV autoantibody in the IIF assay using monkey esophagus as substrate and the modified sensitive IB assay using preabsorbed normal human skin lysate and COLO-16 lysate as a substrate in the three groups of patients. The sensitivity of the Western blot was enhanced by modifications in the extraction procedure of the lysate, by absorption of lysate with normal human serum and by the use of an enzygraphic web. In group 1, comprising 23 PV patients with active generalized disease, the titers of the autoantibody in the IB assay were 2–4-fold higher than in the IIF assay. This difference was highly significant (P=0.0001). In group 2, comprising 10 patients with limited or minimal PV who were positive on DIF and negative on IIF, all the patients were positive in the IB assay. In group 3, comprising 9 patients clinically free of disease and off all therapy for at least 3 years and negative in IIF assay, all the patients were positive in the IB assay. An additional two such patients who had low titers in the IIF assay had significantly higher titers in the IB assay. In the IB assay normal human skin and COLO-16 cell lines produced similar results even though PV sera bound to a 130 kDa protein on normal human skin lysate and a 105 kDa protein on COLO-16 lysate. The availability of this modified sensitive IB assay will have significant clinical benefit in the diagnosis of PV patients when IIF is negative, and in the study of autoantibody production.