The primary objective of this project was the development of affinity selection techniques that can be used with mass spectral detection for the direct identification of trace level target antigens from complex matrices. Mouse monoclonal antibody IgG1 specific for the human c-myc epitope---EQKLISEEDLN---was covalently immobilized on recombinant protein G beads using chemical crosslinking. This immunomatrix was then used to selectively enrich both the wild-type and mutant single chain (scFv) antibodies (containing the c-myc epitope) from the periplasmic extract of Escherichia coli (E. coli). To identify the point mutation within the scFv mutant, a tryptic digest was performed followed by LC-MS of the tryptic peptides. This novel approach was applied to a number of scFv proteins containing single point mutations. In particular, mass spectrometry was used to confirm the site of mutation L46N on one specific mutant using LC-MS/MS. Immunoaffinity enrichment was also used to select the c-myc peptide from a spiked sample of human serum at a concentration of 20 ng/ml. LC-MS/MS was performed to confirm the identity of the selected peptide. (Abstract shortened by UMI.)
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/9303 |
| Date | January 2000 |
| Creators | LeRiche, Tammy G. |
| Contributors | Kaplan, Harvey, |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Detected Language | English |
| Type | Thesis |
| Format | 134 p. |
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