Fingerprinting complex pectins by chromatographic separation combined with ELISA detection.
Enzyme-resistant pectin or modified hairy regions were subjected to size exclusion (HPSEC) and weak anion exchange (WAX) chromatography. Fractions collected after separation were tested for the presence of different pectic epitopes using the monoclonal antibodies LM2, LM5, LM6, and JIM7. Separation by HPSEC showed that based on molecular weight the different epitopes were restricted to distinct molecular weight populations. WAX chromatography resulted in an even better separation of the different pectic epitopes present. A clear separation between arabino galactan type II epitopes and the RG I side chains, (1,5)-alpha-l-arabinan and (1,4)-beta-d-galactan, could be established. Arabinogalactan type II was found in the first populations eluting off the WAX column. The observations made within the ELISA assays of the collected fractions could be confirmed by determination of the sugar composition of the individual populations obtained. The sugar composition of the AGII positive populations eluting off the WAX column shows the presence of significant amounts of rhamnose and galacturonic acid. Together with the delay on an anion exchanger, this observation indicates a possible linkage between RGI and AGII. The volume of the individual fractions collected provides enough material for a maximum of 20 different antibodies to be tested from one analytical separation.
Verhoef
René
R
Wageningen University, The Netherlands.
Lu
Yu
Y
Knox
J Paul
JP
Voragen
Alphons G J
AG
Schols
Henk A
HA
D000911:Antibodies, Monoclonal; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004797:Enzyme-Linked Immunosorbent Assay; D005685:Galactans / Q000032:analysis; D006603:Hexuronic Acids / Q000032:analysis; D010368:Pectins / Q000737:chemistry* / Q000302:isolation & purification; D012210:Rhamnose / Q000032:analysis
D000911:Antibodies, Monoclonal; D005685:Galactans; D006603:Hexuronic Acids; C007819:galacturonic acid; D010368:Pectins; D012210:Rhamnose; C005653:arabinogalactan