Development and Validation of Monoclonal and Polyclonal Antibodies for the Detection of Immunoglobulin G of Bottlenose Dolphins (Tursiops Truncatus)

Abstract

Abstract. Antibodies directed against species-specific immunoglobulin G (IgG) have a broad range of applications in serologic and immunologic research and in the development of clinical assays. Validated antiIgG antibodies for marine mammal species are in short supply. The objective of this study was to produce and validate antibodies with specificity for IgG of the common bottlenose dolphin (Tursiops truncatus). Bottlenose dolphin IgG was purified using protein G. Two mouse monoclonal antibodies and a rabbit polyclonal antibody were developed from mice and rabbits immunized with bottlenose dolphin IgG. The specificity of the monoclonal antibodies and the polyclonal antibody for bottlenose dolphin IgG was first verified by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). For further validation, both monoclonal antibodies and the polyclonal antibody were incorporated in an indirect ELISA for the detection of the immune response of bottlenose dolphins to a vaccine antigen. Three bottlenose dolphins were immunized with a commercial Erysipelothrix rhusiopathiae vaccine, and serial blood samples were collected from all dolphins for measurement of levels of circulating antibodies. Seroconversion was observed in all 3 dolphins by use of both monoclonal antibodies and the polyclonal antibody. Circulating antibodies were detectable as early as 6 days after immunization in 1 dolphin. Peak antibody levels were detected 14 days after the immunization. The ability to detect seroconversion in all 3 immunized bottlenose dolphins firmly establishes the specificity of the monoclonal antibodies and the polyclonal antibody for IgG of the common bottlenose dolphin.

Cross-Reactivity between Immunoglobulin G Antibodies of Whales and Dolphins Correlates with Evolutionary Distance

“…Two MAbs and one polyAb raised against IgG of the bottlenose dolphin (Tursiops truncatus) were used. Their derivation, evaluation, and the validation of their specificities for T. truncatus IgG molecules have been described in detail previously (27). The biotinylated MAb HL1912 is specific for the ␥ heavy chain of T. truncatus.…”

“…One difficulty with many serodiagnostic tests in diverse groups of wildlife is the relative lack of reagents, such as specific secondary antibodies. While antibottlenose dolphin immunoglobulin G (IgG) antibodies have been developed, there are limited options in other cetacean species (27). Protein A and protein G are bacterial proteins that bind to IgG and have been used in immunoassays to detect antibodies in various cetacean species (18,20,35,38).…”

“…However, since the polyAb mixture binds multiple epitopes, a broader spectrum of cross-reactivity would be expected with polyAb than with a MAb. We previously purified IgG from bottlenose dolphins and developed MAbs and polyAbs with specificities for either bottlenose dolphin IgG heavy chain, IgG light chain, or the whole IgG molecule (27). Using these reagents, we then developed and validated a competitive enzyme-linked immunosorbent assay (cELISA) for the quantification of circulating dolphin IgG concentrations, using the MAb specific for the IgG ␥ heavy chain (32a).…”

Primary bacterial pathogens in bottlenose dolphins Tursiops truncatus: needles in haystacks of commensal and environmental microbes

“…During our 20 yr study period, we report one dolphin with disseminated E. rhusiopathiae infection. Similar infections have occurred in other facilities housing bottlenose dolphins (Ridgway, 1972, Terasawa et al 2001) and have presented a risk high enough to warrant vaccinations (Nollens et al 2007). Interestingly, despite similar fish food supplies among facilities housing bottlenose dolphins, the MMP population has not appeared to be susceptible to E, rhusiopathiaeassociated epizootics.…”

Exposure to Novel Parainfluenza Virus and Clinical Relevance in 2 Bottlenose Dolphin (Tursiops truncatus) Populations

“…Archived TtPIV-1 was propagated in BSC40 cells as previously described (15). Uninfected BSC40 cells were cultured as negative controls.…”