ANTI-BSA ANTIBODIES DO NOT CROSS-REACT WITH THE 69-KDA ISLET-CELL AUTOANTIGEN ICA69

In contrast to several previously published reports, we demonstrate by a variety of antibody assays that bovine serum albumin (BSA) is not a ntigenically cross-reactive with the 69-kDa islet cell autoantigen (IC A69). Fast protein liquid chromatography purified BSA and highly purif ied recombinant human ICA69 were used to establish sensitive Western b lot and ELISA assays in order to detect antibodies against these two p roteins. The assays excluded BSA or powdered milk as blocking agents, since these would interfere with antibody binding. A panel of sera fro m diabetic individuals, first degree relatives, and normal controls sh owed that the majority (similar to 70%) of individuals from each group had antibodies against ICA69 as assayed by Western blots, whereas con siderably fewer (similar to 13%) had anti-BSA antibodies on Western bl ots, and individuals with antibodies to both proteins occurred only ra rely (2-3%). To explore this issue further we immunized a total of 16 individual rats, representing four different strains (bio-breeding dia betes resistant and diabetes prone, Wistar-Furth, and Sprague-Dawley), with either BSA (n=2 of each strain) or with recombinant ICA69 (n=2 o f each strain), and for each animal pre- and postimmune antibody titre s against BSA and against ICA69 were assayed separately by enzyme-link ed immunoabsorbent assay. In rats immunized with BSA, anti-BSA titres increased about 100,000-fold over preimmune levels, whereas anti-ICA69 reactive antibodies were unchanged over preimmune levels. Similarly, in rats immunized with ICA69, anti-ICA69 titres rose about 100,000-fol d over preimmune levels, whereas anti-BSA antibodies were unchanged ov er preimmune levels. Thus we find no evidence for the existence of ant ibody cross-reactivity between ICA69 and BSA, either in humans or in i mmunized rats. When our rat anti-BSA antisera were used to probe Weste rn blots made from rat islets isolated in the strict absence of fetal calf serum, we were unable to detect a 69-kDa band, even when the isle ts were preincubated with gamma-interferon, a treatment which has been reported to induce the BSA cross-reactive islet antigen. We conclude that BSA is not antigenically cross-reactive with ICA69 at the antibod y level, and it is highly unlikely that BSA is antigenically cross-rea ctive with some other 69 kD islet cell antigen. (C) 1998 Academic Pres s.