Abstract

Cetacean placentation restricts transfer of maternal antibody to the neonate. Healthy young are born without humoral immunity and obtain maternal antibody via colostrum in the first few days of life; failure to do so can typically become a life-threatening event. In domestic species, colostrum can be collected for storage but this is not practical for cetaceans. Concentrated serum-derived, species-specific immunoglobulin (Ig) can serve as an alternative to colostrum.

Immunoglobulin was purified from pools of serum obtained from multiple individuals of a given species in a two-step procedure. Caprylic acid treatment first precipitates non-immunoglobulin serum proteins that can be discarded; immunoglobulin is subsequently precipitated using saturated ammonium sulfate. All reagents used were certified to contain low levels of endotoxin. Purity of the final product was tested via polyacrylamide gel electrophoresis (PAGE) analysis and immunoglobulin concentrations were determined using a BCA kit with Bovine IgG standards (Pierce Chemical Co., Rockford, IL). Ig was concentrated to 20-25mg/ml; greater concentration can result in protein aggregation. Purified Ig was lyophilized for long-term storage.

Bacteria-derived proteins A, G and L are widely used to purify immunoglobulins but exhibit varying affinities between different Ig isotypes and between species. The caprylic acid/saturated ammonium sulfate methodology increases the probability that all Ig isotypes will be retained in the purification process.

Stability of lyophilized antibody was tested over time as follows. Rabbits were immunized with tetanus toxoid using three intramuscular vaccinations, three weeks apart, with serum being collected three weeks after the final immunization. This serum was processed using the same protocol as described above and the lyophilized product stored at -20C. Antibody titers specific for the tetanus toxoid were determined by indirect ELISA. Anti-tetanus ELISA assays were performed on serum prior to purification and on lyophilized samples at random intervals; tetanus antibody titers continue to remain stable after eight years of storage, the last time point tested.

Lyophilized immunoglobulin product for multiple marine mammal species has been successfully prepared. Preliminary data on successes/failures following administration to neonates is currently being accumulated.

Acknowledgements

We would like to acknowledge Sea World Adventure Parks, Kewalo Basin Marine Mammal Laboratory, John G. Shedd Aquarium and Indianapolis Zoo for providing serum samples for Ig purification.