Characterisation of epitopes of pan-IgG/anti-G3m(u) and anti-Fc monoclonal antibodies.
The characterisation of monoclonal antibodies (MAbs) and their epitopes is important prior to their application as molecular probes. In this study, Western blotting using IgG1 Fc and pFc' fragments was employed to screen seven MAbs before pepscan analysis to determine their reactivity to potentially linear epitopes. MAbs PNF69C, PNF110A, X1A11 and MAbs WC2, G7C, JD312, 1A1 detected epitopes within the C(H)3 and C(H)2 domains, respectively. However, only four MAbs showed pepscan profiles that highlighted likely target residues. In particular, MAbs PNF69C and PNF110A that have previously been characterised with pan-IgG and anti-G3m(u) specificity, detected the peptide motif 338-KAKGQPR-344 which was located within the N-terminal region of the C(H)3 domain. Furthermore the majority of residues were present in all four IgG subclasses. Consequently the peptide identified was consistent with the pan-IgG nature of these antibodies. By using PCImdad, a molecular display programme, this sequence was visualised as surface accessible, located in the C(H)2/C(H)3 inter-domain region and proximal to the residue arginine(435). It is speculated that this residue may be important for phenotypic expression of G3m(u) and specificity of these reagents. Pepscan analysis of MAbs G7C and JD312 (both pan-IgG) highlighted the core peptide sequence 290-KPREE-294, which was present in the C(H)2 domain and was common to all four IgG subclasses. PCImdad also showed this region to be highly accessible and was consistent with previous bioinformatic and autoimmune analysis of IgG. Overall these MAbs may serve as useful anti-IgG or anti-G3m(u) reagents and probes of immunoglobulin structure.
Nelson
Paul N
PN
Molecular Immunology Research Group, Division of Biomedical Sciences, University of Wolverhampton, Wulfruna Street, Wolverhampton, WV1 1SB, UK. p.n.nelson@wlv.ac.uk
Westwood
Olwyn M R
OM
Soltys
Andy
A
Jefferis
Roy
R
Goodall
Margaret
M
Baumforth
Karl R N
KR
Frampton
Geoff
G
Tribbick
Gordon
G
Roden
Denise
D
Hay
Frank C
FC
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000145:classification / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology*; D006385:Hemagglutination Inhibition Tests; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000145:classification / Q000276:immunology*; D007074:Immunoglobulin G / Q000145:classification / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D066298:In Vitro Techniques
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D000939:Epitopes; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains