Bobby L. Middlebrooks
BSC 483/583 Viral Ecology
BSC 485/L/585/L Viral Pathogenesis and Diagnosis
BSC 486/L/586/L Immunology and Serology
BSC 780/L Principles of Immunochemistry
The primary areas of research in my laboratory are comparative immunology and immunodiagnostics. One major project presently underway is a study of the humoral immune system of dolphins and other cetaceans, funded primarily by the Office of Naval Research. The project includes identification, purification and characterization of the various classes (isotypes) of immunoglobulins in cetaceans and investigation of the dynamics of the antibody response to protein immunogens. We are using preparations of the purified immunoglobulins to produce mouse monoclonal antibodies specific for individual cetacean isotypes. These monoclonal antibodies are required for detailed study of the role of immunoglobulin isotypes in the cetacean immune system. Other aspects of this project include a preliminary study of the mucosal immune response in dolphins and the production of dolphin/mouse heterohybridomas (secreting dolphin immunoglobulins in vitro).
A series of projects, funded through the Alaska Sea Life Center, involves studies of the humoral immune systems of several arctic species, populations of which are endangered or threatened. The species include two marine mammals (Steller sea lions and harbor seals), and two avian species (Steller eiders and spectacled eiders). We are identifying, characterizing, and purifying the major immunoglobulin isotypes from these species, and we are producing hybridomas secreting monoclonal antibodies specific for these isotypes. These antibodies will ultimately be used in ELISA assays to monitor immunoglobulin isotype levels in these species as one of several measures of health status. These projects are being conducted in collaboration with Dr. Shannon Atkinson at the Alaska Sea Life Center.
Another area of effort is development of an enzyme immunoassay (ELISA) for determination of the prevalence in captive dolphins of antibodies against Erysipelothrix rhusiopathiae, a common and potentially serious bacterial pathogen in both captive and wild dolphins. We have identified the most appropriate antigenic component (a 64 kDa protein) for use as the capture antigen in this assay, and have employed molecular biology techniques to develop a plasmid expression system to facilitate production of large amounts of the antigen in relatively pure form. The resulting plasmid construct has permitted the use of microbial cultures to produce relatively large amounts of soluble 64 kDa protein, eliminating the need to extract and purify the protein from E. rhusipoathiae for use in ELISA's.
My laboratory has also been active in the development and characterization of cell lines from available cetacean tissues. Presently the laboratory has six well-characterized cell lines from three species of cetaceans (bottlenose dolphin, beluga whale, and pantropical spotted dolphin). Lines from other tissues from these species, from other cetacean species, and from a pinniped (the harbor seal) have been initiated as well, and are in early stages of development and characterization.
My laboratory has recently reactivated a research thrust centered around the use of secretory IgA associated with fecal particulates as an indicator of the species source of fecal pollution in environmental waters. Through intensive concentration protocols followed by elution of IgA from concentrated particulates in concert with application of extremely sensitive ELISA indicator systems, we have demonstrated that we can detect and identify the species of origin of IgA associated with human or animal fecal pollutants. My laboratory is collaborating with the laboratories of Biological Sciences colleagues R.D. Ellender and Shiao Wang, who are investigating the application of bacterial source tracking using molecular biology tools to identify the probable host source of strains of bacteria found in environmental waters.
Middlebrooks, B.L., J. C. Jones, & R.A. Patterson. 2002. Application of ELISA methodology for detection of Erysipelothrix rhusiopathiae antibody titers in cetaceans. pp. 193-204 In Cell and Molecular Biology of Marine Mammals. C. J. Pfeiffer ed. Krieger Publishing Co., Inc.
Patterson, R.A. & B.L. Middlebrooks. 2002. Methods for purification and study of cetacean immunoglobulins. pp 245-252 In Cell and Molecular Biology of Marine Mammals. C. J. Pfeiffer ed. Krieger Publishing Co., Inc.
Jones, J.C., R.A. Patterson, & B.L. Middlebrooks. 2000. Evaluation and refinement of an ELISA assay designed to detect antibodies against Erysipelothrix rhusiopathiae in cetaceans. Infectious Disease Review. 2: 218-222.
Osgood, R.C., B.L. Middlebrooks, & R.A. Patterson. 2002. Cloning and expression of the gene sequence of a 66kD surface protein of Erysipelothrix rhusiopathiae. Proceedings of the 33nd Annual Conference of the International Association for Aquatic Animal Medicine, 33: 7.