Date of Award

1-1-2015

Language

English

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (viii, 76 pages) : color illustrations.

Dissertation/Thesis Chair

Dr. Nicholas J Mantis

Committee Members

Dr. Janice Pata, Dr. William Lee

Keywords

Infectious diseases, Ricin, Antigenic determinants, Apoptosis

Subject Categories

Immunology of Infectious Disease

Abstract

Ricin toxin is a glycoprotein produced by the castor bean plant, Ricinus communis. Ricin is an extraordinarily potent inducer of cell death and inflammation, especially following inhalation. The toxin’s enzymatic subunit (RTA) is transported via retrograde transport into the cytoplasm of mammalian cells by the toxin’s B subunit (RTB). Once in the cytoplasm, RTA inactivates ribosomes through cleavage of ribosomal RNA. In this study, I characterized a ricin-specific monoclonal IgA antibody (mAb) known as 23D7. I confirmed that 23D7 reacts with RTA and is effective at neutralizing ricin in a Vero cell cytotoxicity assay in vitro. To localize the epitope recognized by 23D7, I performed Pepscan analysis, comparative binding assays with ricin/RCA-I/RTA/RVEc and competitive ELISA with mouse IgG mAbs directed against known epitopes on RTA. 23D7 partially inhibited cluster II group of IgG’s, indicating that the epitope overlaps with but is not identical to, the residues recognized by mAbs SyH7 and TB12. Comparative assays revealed that 23D7 was more reactive to ricin than to RCA-I, which followed by sequence alignment pinpointed residues on RTA that may contribute towards antibody recognition. My results suggest that 23D7 recognizes residues 13-25 and another region which needs to be determined as evidenced by differential competition and comparative assays. I next examined the protective efficacy of 23D7 in mice using the ‘backpack tumor model’. 23D7 was sufficient to protect mice against an intra-peritoneal ricin challenge, demonstrating for the first time that it imparts protection against the toxin in a systemic challenge model. Preliminary mucosal protection studies using an intra-nasal ricin challenge, however, revealed no evidence of protection, even though mice had the same serum titers as the systemic challenge model. These data suggest that higher amounts of Ab are required in mucosal secretions to provide immunity. Further studies will be needed to evaluate 23D7’s potential to protect mucosal surfaces. I conclude from this study that 23D7 binds to an epitope overlapping cluster II Abs, SyH7 and TB12 and provides systemic immunity against ricin in vivo.

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