Analysis of pectic epitopes recognised by hybridoma and phage display monoclonal antibodies using defined oligosaccharides, polysaccharides, and enzymatic degradation.
The structure of epitopes recognised by anti-pectin monoclonal antibodies (mAbs) has been investigated using a series of model lime-pectin samples with defined degrees and patterns of methyl esterification, a range of defined oligogalacturonides and enzymatic degradation of pectic polysaccharides. In immuno-dot-assays, the anti-homogalacturonan (HG) mAbs JIM5 and JIM7 both bound to samples with a wide range of degrees of methyl esterification in preference to fully de-esterified samples. In contrast, the anti-HG phage display mAb PAM1 bound most effectively to fully de-esterified pectin. In competitive inhibition ELISAs using fully methyl-esterified or fully de-esterified oligogalacturonides with 3-9 galacturonic acid residues, JIM5 bound weakly to a fully de-esterified nonagalacturonide but JIM7 did not bind to any of the oligogalacturonides tested. Therefore, optimal JIM5 and JIM7 binding occurs where specific but undefined methyl-esterification patterns are present on HG domains, although fully de-esterified HG samples contain sub-optimal JIM5 epitopes. The persistence of mAb binding to epitopes in pectic antigens, with 41% blockwise esterification (P41) and 43% random esterification (F43) subject to fragmentation by endo-polygalacturonase II (PG II) and endo-pectin lyase (PL), was also studied. Time course analysis of PG II digestion of P41 revealed that JIM5 epitopes were rapidly degraded, but a low level of PAM1 and JIM7 epitopes existed even after extensive digestion, indicating that some HG domains were more resistant to cleavage by PG II. The chromatographic separation of fragments produced by the complete digestion of P41 by pectin lyase indicated that a very restricted population of fragments contained the PAM1 epitope while a (1-->4)-beta-D-galactan epitope occurring on the side chains of pectic polysaccharides was recovered in a broad range of fractions.
Willats
W G
WG
Centre for Plant Sciences, Leeds Institute of Plant Biotechnology and Agriculture, University of Leeds, UK. pab5wgtw@bmb.leeds.ac.uk
Limberg
G
G
Buchholt
H C
HC
van Alebeek
G J
GJ
Benen
J
J
Christensen
T M
TM
Visser
J
J
Voragen
A
A
Mikkelsen
J D
JD
Knox
J P
JP
D000911:Antibodies, Monoclonal; D020650:Combinatorial Chemistry Techniques; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000032:analysis*; D006825:Hybridomas; D009844:Oligosaccharides / Q000032:analysis* / Q000276:immunology; D010368:Pectins / Q000032:analysis / Q000737:chemistry* / Q000276:immunology*; D019151:Peptide Library; D011134:Polysaccharides / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009844:Oligosaccharides; D019151:Peptide Library; D011134:Polysaccharides; D010368:Pectins