Degree Granting Department
Biology (Cell Biology, Microbiology, Molecular Biology)
Daniel V. Lim, Ph.D.
ELISA, RAPTOR, Biosensor, Waveguide, Immobilization
A novel capture matrix utilizing human serum albumin (HSA) and streptococcal Protein G (PG), which possesses an albumin binding domain (ABD), was used to immobilize antibodies for improved bacterial capture efficiency in immunoassays. Enzyme linked immunosorbent assays (ELISA) were used to characterize and optimize a specific protocol for the HSA-PG capture matrix; which revealed several critical factors that should be considered. The Fc binding domain, on PG, should have high affinity for the species of capture antibody used in the assay. Goat and rabbit species antibodies bound strongly to the Fc binding domain of PG. Displacement of the capture antibody, by the detector antibody should be avoided to reduce background signals. The Fc binding domain on PG should have equivalent or lower affinity for the detector antibody, when compared to the capture antibody. Goat species antibody, used as a detector antibody, did not displace the same-species capture antibody. ELISA analysis showed detection of Escherichia coli O157:H7 cells at 1.0 x 104 CFU/ml using HSA-PG and goat antibody raised against Escherichia coli O157:H7; unlabeled antibody was used for capture while HRP labeled antibody was used for detection. Studies were performed on an automated fiber optic biosensor, RAPTOR, which was used for the rapid detection of pathogens. Biosensor assays showed detection of E. coli O157:H7 at 1.0 x 10³ CFU/ml in PBS and 1.0 x 105 CFU/ml in homogenized ground beef supernatant. Capture efficiency of the HSA-PG capture matrix was studied using the biosensor and GFP-E. coli O157:H7. The amount of cells captured was less than one percent of the sample concentration. This limit of detection and capture efficiency was comparable to the streptavidin-biotin capture matrix.
Scholar Commons Citation
McCabe, Christie Renee, "A novel antibody based capture matrix utilizing human serum albumin and streptococcal Protein G to increase capture efficiency of bacteria" (2009). USF Tampa Graduate Theses and Dissertations.