COMPARISON OF AN INDIRECT IMMUNOFLUORESCENCE ASSAY AND A MODIFIED SENSITIVE IMMUNOBLOT ASSAY FOR THE STUDY OF THE AUTOANTIBODY IN PEMPHIGUS-VULGARIS

Some patients with pemphigus vulgaris (PV) have positive direct immuno fluorescence (DIF) but are negative by indirect immunofluorescence (II F). 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 t he IB assay in PV patients in whom the clinical picture and DIF were c onsistent, 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 com parison was made of the titers of PV autoantibody in the IIF assay usi ng monkey esophagus as substrate and the modified sensitive IB assay u sing preabsorbed normal human skin lysate and COLO-16 lysate as a subs trate 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 act ive generalized disease, the titers of the autoantibody in the IB assa y were 2-4-fold higher than in the IIF assay. This difference was high ly significant (P = 0.0001). In group 2, comprising 10 patients with l imited 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 th e IB assay. An additional two such patients who had low titers in the LIF assay had significantly higher titers in the IB assay. In the IB a ssay 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 l ysate and a 105 kDa protein on COLO-16 lysate. The availability of thi s modified sensitive IB assay will have significant clinical benefit i n the diagnosis of PV patients when IIF is negative, and in the study of autoantibody production.