Identifier

etd-03152004-113133

Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

A phage collection (M1114), which is a mixture of two collections, M710-3R and Silica 426 each derived from a human synthetic phage antibody library (Fab 2lox), was used further enriched for dextran binding using enzyme-linked immunosorbent assays (ELISA) screening. The effects of dextran concentration on phage binding affinity were tested using indirect sandwich ELISA on phage collections, M1114-m74 and AE-M1114-m74-2R. Most of the phage bound to dextran (T2000) coated on a sandwich ELISA. The combination of ELISA screening and a sephadex columns enriched dextran binding 7 fold over enrichment by a single ELISA screen. Phage collection (M1114-m74) produced by combination screening showed the greatest binding on ELISA. The color intensity produced by phage collection (AE-M1114-m74-2R) obtained after the 2nd round of selection was 3.5 fold higher than that of phage collections, AE-M1114-m74-1R after the 1st round. Dextran binding by phage collection (AE-M1114-m74-2R) was illustrated using image analysis of transmission electron micrographs. Sephadex bead agarose electrophoresis (SBAE) screening produced phage collections (AE-M1114-m74-1R and 2R) which were used in a paper-dip assay. A dip stick assay using a protein blocked paper with adsorbed high molecular size dextran (T10,000, 107) produced the most color (59 ±5) using anti-dextran phage enzyme linked assays. Low molecular size dextran (T40, 4x104) produced significantly lower color (15 ±1). Phage collection (AE-M1114-m74-2R) was tested for specificity against dextran (T2000), corn starch, sucrose, dextrose, and chitin. Dextran produced up to 18 fold the normalized intensity of the other carbohydrates. The presence of Fab inserts in the phage collections was confirmed using PCR, and the presence of the same insert in the host E.coli was checked using a â-galactosidase linked assay. DNA sequencing of phage collection (AE-M1114-m74-2R) confirmed that human origin antibody was present. The PCR products of ë, ê light chains and heavy chain from phage collection (AE-M1114-m74-2R) were approximately 420 bp, 550 bp and 600 bp. This research used various selection methods to isolate anti-dextran phages from a library. These were used to develop a paper-dip stick method for dextran detection used for routine screening of sugar juices.

Date

2004

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

Donal F. Day

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