Title

Purification and Preliminary Qualitative and Quantitative Evaluation of Immunoglobulin Isotypes of the Harbor Seal (Phoca vitulina)

Date of Award

2006

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Bobby Middlebrooks

Advisor Department

Biological Sciences

Abstract

Various affinity chromatography and size exclusion chromatography methods were employed to purify IgG, IgA, and IgM from serum pooled from several individual harbor seals held at the Alaska SeaLife Center. Commercially available antisera against immunoglobulin isotypes of other species were tested with each purified harbor seal isotype to determine cross-reactivity with the isotype. Similarly, the bacterial proteins Protein A and Protein G, which bind IgG from many species, and the lectin Jacalin, which binds the IgA of many species, were tested for cross-reactivity with each harbor seal isotype. Antisera or proteins showing cross-reactivity with one of the harbor seal immunoglobulins were evaluated for use in the development of enzyme-linked immunosorbant assays (ELISA). In these ELISA's, immunoglobulin concentrations in individual harbor seal serum samples (taken from both captive animals and wild animals that were in rehabilitation at the Alaska SeaLife Center) were determined and compared to standards having known concentrations of the harbor seal isotype in question in order to measure isotype levels in the serum sample. Anti-human IgA, which was found to be cross-reactive with harbor seal IgA, and Protein A, which was found to cross-react with harbor seal IgG and IgM, were used as specific ligands in these ELISA assays. The sensitivity of the ELISA format used to determine harbor seal IgA levels permitted determination only of IgA levels of 2.2mg/ml or above. Thus, in this study, the average IgA level was 9.79mg/ml for those samples having concentrations greater than 2.2 mg/ml. The average combined harbor seal IgG/IgM level of the samples tested was found to be 26.84mg/ml. It was determined statistically that the average IgG/IgM levels in rehab harbor seals were significantly lower than that of the captive harbor seals. Three monoclonal antibodies were produced during the course of this study, two that showed specificity for harbor seal IgA and one that showed specificity for harbor seal IgG when tested in a western blot. Further development and characterization of these monoclonal antibodies will have to continue to be available for continued evaluation of the humoral immune system of the harbor seal.