Structural immuno-analysis of human and porcine interferon gamma: identification of shared antigenic domain.
We examined the antigenic resemblance between human (h) and porcine (p) interferon (IFN)-gamma by binding (ELISA) and neutralization assays. The murine polyclonal antisera and sets of murine monoclonal antibodies (mAbs) raised against either IFN were tested in confrontation with recombinant IFNs of either species, and with site-specific mutants of hIFN-gamma. Several of the mAbs raised against pIFN-gamma cross-reacted in ELISA with hIFN-gamma. In contrast, none of the anti-hIFN-gamma mAbs cross-reacted. By employing site-specific mutants of recombinant hIFN-gamma as antigens in ELISA we succeeded in identifying the C-terminal portion 97-111 as the antigenic site in hIFN-gamma recognized by the cross-reactive anti-pIFn-gamma mAbs. None of the mAbs recognizing the common antigenic structure had neutralizing potency, although His111 was determined by others as the residue important for bioactivity of hIFN-gamma. Mutations in the domain 97-111 had no or little influence on homospecific reactivity of anti-hIFN-gamma mAbs, indicating that this domain, while being mouse-immunodominant in the case of pIFn-gamma was poorly immunogenic in the case of hIFN-gamma. The epitopes of three out of five anti-hIFN-gamma mAbs mapped in the N-terminal region 1-23, indicating immunodominance of this region in hIFN-gamma. Another mAb (D9D10), also directed to the N-terminus of hIFN-gamma, apparently recognized a conformational epitope. This antibody lacked ELISA-reactivity with the wild-type hIFN-gamma but strongly bound mutant protein with an engineered disulfide bridge Cys7-Cys69. Surprisingly, D9D10 showed high reactivity also with the wild type hIFN-gamma produced by baculovirus construct coding for the mature protein with signal sequence or with wild type protein possessing residues Cys-Tyr-Cys from the signal sequence.
Kontsek
P
P
Rega Institute, Catholic University Leuven, Leuven, 3000, Belgium. virupeko@savba.savba.sk
Martens
E
E
Vandenbroeck
K
K
Kontseková
E
E
Waschütza
G
G
Sareneva
T
T
Billiau
A
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D005260:Female; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D013045:Species Specificity; D013552:Swine
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007371:Interferon-gamma