Poster

There are five isotypes of immunoglobulins (Igs) that are commonly found within most mammalian species, designated IgG, IgA, IgM, IgD, and IgE. Each isotype is characterized by its heavy chain: gamma (γ), alpha (α), mu (µ), delta (Δ) or epsilon (ε), respectively. All of the immunoglobulin isotypes have one of two isotypes of light chains: kappa (κ) or lambda (λ). Cetaceans are similar to other mammals in that IgG, IgA, and IgM are the most prevalent immunoglobulins in their serum (Nash and Mach, 1971). However, knowledge of the structure and of the functions of these immunoglobulins has remained limited, primarily because of the lack of availability of techniques for preparation of purified immunoglobulin isotypes from cetaceans. Even though effective techniques for purifying IgG from cetacean serum have been determined, techniques for purifying IgA and IgM from cetacean serum are still necessary. With this in mind a technique, the Jacalin affinity chromatography column, that has been highly effective at purifying IgA from human serum, tears, and colostrum was attempted to purify IgA from cetacean serum. The serum tested was from three different species of cetaceans: Atlantic bottlenose dolphin, beluga whale, and Pacific white-sided dolphin.

An Immobilized Jacalin (Pierce) affinity chromatography column was setup and run at room temperature (although further testing showed that the column could also be run at 4°C with the same results). One ml of ammonium sulfate precipitated serum, from one of the three cetacean species, was diluted 1:2 in binding buffer, pH 7.4 (0.1 M NaPO₄ and 0.15 M NaCl) and applied to the column. Two ml fractions were collected with the binding buffer and the absorbance readings of the fractions were monitored at 280 nm. Once a peak was obtained and the absorbance readings had returned to baseline values the elution buffer, pH 7.4 (0.1 M melibiose in binding buffer) was applied to the column and a series of 2.0 ml fractions were collected and monitored for protein. The elution buffer was passed through the column until the presumptive IgA peak was eluted, then binding buffer was passed through the column for re-equilibration and storage. This same procedure was followed for precipitated serum samples for all three species of cetaceans in question. The purified, dialyzed, and concentrated protein samples from the three species of cetaceans from the Immobilized Jacalin column were tested by Grabar-Williams immunoelectrophoresis to determine the effectiveness of this technique for purifying IgA. Each sample produced a single arc compatible with the expected arc for pure IgA; thus, indicating that the Jacalin affinity column is an effective technique for purification of IgA from Atlantic bottlenose dolphin, beluga whale, and Pacific white-sided dolphin serum.