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 human antibody (Ab) reactive with bovine and marsupial Fn, but not with human Fn. Indirect immunofluorescence, wet cleaving and protein adherence assays, immunoblotting, blot-affinity purification, a cell adhesion inhibition assay, and competitive experiments with synthetic peptides were used to characterize the anti-Fn Ab in serum from a patient with an undifferentiated connective tissue disease. A characteristic Fn-like network was observed by indirect immunofluorescence on bovine MDBK and marsupial PtK2 cells, but not on various human cell lines. Double immunofluorescence revealed colocalization of the Ab with a mouse monoclonal anti-Fn Ab. A reactive polypeptide of 240 kDa corresponding to 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 bovine Fn with an endpoint titer of 1:64,000, while no reactivity was observed with human cellular or plasma Fn. Blot-affinity purification of the Ab from the 240-kDa PtK2 region confirmed that the Fn-like fluorescent pattern observed was due to reactivity with the 240-kDa band and not with other regions of the blot. The Ab affinity-purified from the 240-kDa region also reacted with purified bovine Fn by immunoblotting. Functional analysis disclosed specific inhibition of PtK2 and MDBK cell adhesion by the affinity-purified anti-Fn Ab. Competitive experiments 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 years. Bovine and marsupial Fn can be the focus of a highly specific and persistent human immune response. Reactivity of a human Ab with bovine Fn does not imply cross-reactivity with human Fn. In light of recent reports using bovine Fn to characterize human anti-Fn "autoantibodies," future studies on human anti-Fn should specifically employ purified human Fn as antigen.
Girard
D
D
Division of Rheumatology, Hôpital Notre-Dame, Montréal, Québec, Canada.
Raymond
Y
Y
Labbé
P
P
Senécal
J L
JL
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification; D002417:Cattle; D003240:Connective Tissue Diseases / Q000276:immunology*; D003429:Cross Reactions; D005260:Female; D005353:Fibronectins / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology
D000906:Antibodies; D005353:Fibronectins; D010455:Peptides