Verification of ELISA results by immunomagnetic isolation of antigens from extracts and analysis with SDS-PAGE and western blotting, demonstrated for Erwinia spp. in potatoes.
Isolation of antigens on immunomagnetic beads and subsequent analysis with SDS-PAGE and Western blotting (immunomagnetic isolation-Western blotting (IMI-WB)) was used to verify positive ELISA results for Erwinia chrysanthemi and Erw. carotovora subsp. atroseptica in potato peel extracts. Direct analysis of highly contaminated extracts by Western blotting without previous immuno-isolation resulted in background reactions, whereas immunomagnetic isolation resulted in distinct bands of specific antigens. Target cells as well as antigenic cell products were captured in IMI-WB. Band patterns on IMI-WB of cell-free culture filtrates and cell suspensions were highly similar, but the removal of cells lowered the detection level by 10- to 100-fold. Threshold levels of IMI-WB were generally comparable with those of ELISA. No differences in threshold levels and band patterns were found between a direct format and an indirect format of immuno-isolation. In IMI-WB, blotting patterns differed between Erw. chrysanthemi and Erw. carotovora subsp. atroseptica. The patterns were identical for 15 Erw. chrysanthemi strains, isolated from potato peel extracts in The Netherlands. However, one of 15 strains of Erw, carotovora subsp. atroseptica from potato peel extracts in The Netherlands gave an aberrant pattern. Target bacteria could be easily distinguished from those of cross-reacting strains on the basis of band patterns. Potato peel extracts naturally contaminated with Erw. chrysanthemi gave IMI-WB patterns that were similar to pure cultures of the homologous strains.
DLO Research Institute for Plant Protection (IPO-DLO), Wageningen, The Netherlands.
J M
JM
van der Wolf
J R
JR
van Beckhoven
P M
PM
de Vries
J W
JW
van Vuurde
D000942:Antigens, Bacterial / Q000032:analysis*; D015153:Blotting, Western; D016974:Dickeya chrysanthemi / Q000276:immunology / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007106:Immune Sera; D018189:Immunomagnetic Separation / Q000379:methods*; D016973:Pectobacterium carotovorum / Q000276:immunology / Q000302:isolation & purification*; D011198:Solanum tuberosum / Q000382:microbiology*
D000942:Antigens, Bacterial; D007106:Immune Sera