CHARACTERIZATION OF A NOVEL HUMAN-ANTIBODY XENOREACTIVE WITH FIBRONECTIN

Recent reports have used bovine fibronectin (Fn) as source of antigen to study human anti-Fn autoantibodies. We have characterized a novel h uman antibody (Ab) reactive with bovine and marsupial Fn, but not with human Fn. Indirect immunofluorescence, wet cleaving and protein adher ence assays, immunoblotting, blot-affinity purification, a cell adhesi on inhibition assay, and competitive experiments with synthetic peptid es were used to characterize the anti-Fn Ab in serum from a patient wi th an undifferentiated connective tissue disease. A characteristic Fn- like network was observed by indirect immunofluorescence on bovine MDB K and marsupial PtK2 cells, but not on various human cell lines. Doubl e immunofluorescence revealed colocalization of the Ab with a mouse mo noclonal anti-Fn Ab. A reactive polypeptide of 240 kDa corresponding t o the M(r) of Fn was identified by immunoblotting using MDBK and PtK2 total cell lysates. The Ab reacted with the 240-kDa band of purified b ovine Fn with an endpoint titer of 1:64,000, while no reactivity was o bserved with human cellular or plasma Fn. Blot-affinity purification o f the Ab from the 240-kDa PtK, region confirmed that the Fn-like fluor escent pattern observed was due to reactivity with the 240-kDa band an d not with other regions of the blot. The Ab affinity-purified from th e 240-kDa region also reacted with purified bovine Fn by immunoblottin g. Functional analysis disclosed specific inhibition of PtK2 and MDBK cell adhesion by the affinity-purified anti-Fn Ab, Competitive experim ents with synthetic peptides demonstrated that the epitope is located in the decapeptide RGDSPASSKP containing the cell-binding domain of Fn . Longitudinal analysis of the Ab revealed its persistence over 6 year s. Bovine and marsupial Bn can be the focus of a highly specific and p ersistent human immune response. Reactivity of a human Ab with bovine Fn does not imply cross-reactivity with human Fn. In light of recent r eports using bovine Fn to characterize human anti-Fn ''autoantibodies, '' future studies on human anti-Fn should specifically employ purified human Fn as antigen. (C) 1995 Academic Press, Inc.