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

Abstract

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.

Verification of immunofluorescence colony‐staining of Erwinia carotovora subsp. atroseptica by reisolation and immunodiffusion or PCR

“…PCR has also shown its value for verification of bacteria sampled from IFC preparations (Van der Wolf et al, 1994). PCR detection of E.c.…”

Magnetically responsive microparticles for targeted drug and radionuclide delivery.

“…The use of magnetic iron oxide in polymer matrices (magnetic nano-and microencapsulation) to impart a magnetic moment to the particles began in the 1970s in the field of biotechnology. Most of these studies focused on enzyme immobilization and isolation of a wide range of specific cell binding agents (Robinson et al 1973, Kronick et al 1978, Olsvik et al 1994, Thomas and Landsdorp 1994, Van der Wolf et al 1994, hyperthermia (Chen et al 1992, Jordan et al 1997, Shinkai et al 1999, magnetic resonance imaging contrast enhancement (Weissleder et al 1995, Jung and Jacobs 1995, Bulte et al 1999, tissue specific release of therapeutic agents (Gupta and Hung 1994, Lübbe et al 1996a, Kumar 2000, radioimmunoassay (Nye et al 1976), and magnetic targeting of therapeutic radio isotopes (Hafeli et al 1994).…”

The application of magnetic separations in applied microbiology

“…carotovora subsp. atroseptica can be detected in potato peel extracts with BioMag immuno-magnetic beads to capture the target cells and antigenic cell products prior to SDS-PAGE and Western blotting analysis (van der Wolf et al 1994). The immunocapture was necessary to reduce background reactions and lowered the detection level by 10-to 100-fold.…”