Fluorescence immunoassay of E. coli using anti-lipopolysaccharide antibodies isolated from human serum
- Fluorescence immunoassay of E. coli using anti-lipopolysaccharide antibodies isolated from human serum
- 강민정; 봉지홍; 김지윤; 이가연; 박준희; 김태훈; 변재철
- LPS; Anti-LPS antibody; Separation; Human Serum; ClearColi
- Issue Date
- Biosensors and bioelectronics
- VOL 126-528
- In this work, the gram-negative bacterium Escherichia coli strain BL21(DE3) (with lipopolysaccharide (LPS) in its outer membrane) and its modified ClearColi™ strain (lacking LPS) were used for the separation of anti-LPS antibodies from human serum by the following steps: (1) binding of the serum proteins to BL21(DE3); (2) dissociation of the bound proteins (including anti-LPS antibodies) from BL21(DE3) with acid; (3) filtering of the dissociated proteins using ClearColi to remove unwanted proteins; and (4) separation of the antibody fraction by protein-A column chromatography. The binding properties of the separated antibodies were analyzed by fluorescence-activated cell sorting to confirm their selective binding to LPS on the outer membrane of BL21(DE3), and by thermophoretic immunoassay to estimate their dissociation constant. The in vitro applicability of the separated anti-LPS antibodies was demonstrated through a fluorescence assay of BL21(DE3), after immobilizing the antibodies onto a modified microplate surface. The electrochemical detection of BL21(DE3) was also achieved after immobilizing the anti-LPS antibodies onto a gold electrode.
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