Characterisation of epitopes of pan-IgG/anti-G3m(u) and anti-Fc monoclonal antibodies

Abstract

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 antiG3m(u) reagents and probes of immunoglobulin structure. #

Comparison of Antigenic Regions Identified on IgG1Fc Using Bioinformatics vs Pepscan Analysis

“…3). Both the G1m(a) motif, 356-DELTK-360, alanine/glycine 431 and histidine 435 residues are solvent accessible [7,26]. Overall, for all fi ve RFAb epitopes there were few amino acid variations between isotypes suggesting that these antibodies would bind to all four subclasses.…”

“…A PC interactive molecular display and design programme (PCImdad) was kindly provided by Dr. Michael Levitt and used as previously described [7]. The software utilised the Brookhaven Protein Data Bank (PDB fi les) for the coordinates of macromolecules.…”

“…But the development of pepscan methodologies, which use a series of overlapping synthetic peptides, have provided a systematic approach for identifying key amino acids in antibody recognition and binding. Table 1 summarises a number of eloquent studies [7][8][9][10][11] that have used polyclonal/monoclonal rheumatoid factors which highlight CH2 and CH3 target epitopes on IgG1 Fc. Moreover, Table 1 gives details of three antigenic regions on IgG1 Fc previously identifi ed through pepscan by monoclonal anti-IgG reagents MAbs: G7C, PNF69C and A57H) [7,9].…”

“…A molecular modelling programme (IMDAD) [7] was used to locate the potential target epitopes of these antibodies as antigenic 'hot spots' within the IgG1 Fc macromolecule. It is recognised that pepscan remains an expensive investigative tool.…”

“…X (italic) = nuance between Eu and NIE. ** reactivity of polyclonal and ++ monoclonal RFs from previous studies [7][8][9][10]. IgG proteins: Cγ1 a reference protein Eu IgG1 Fc accession number AF150959-1, Cγ1 b protein NIE (used for pepscan).…”