Purification of secretory immunoglobulin A from milk of the brushtail possum (Trichosurus vulpecula)

Abstract

AIM: To identify and purify secretory immunoglobulin A (sIgA), a key effecter molecule in mucosal immune responses, from milk of the brushtail possum (Trichosurus vulpecula).

METHODS: Milk samples were collected from female possums with pouch young, and clarified by centrifugation and precipitation methods. The clarified fraction was purified by gel filtration and affinity chromatography to yield sIgA. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques were used to assess the purity of the final product, and to identify the heavy (H) chain, light (L) chain and secretory component (SC) of possum sIgA.

RESULTS: Immunoblotting, using antibodies raised against cloned possum sIgA SC and H-chain, and a synthetic peptide fragment of the H-chain, confirmed the identity of the purified protein. The N-terminal amino acid sequence of purified possum sIgA showed strong homology to reported sequences of H-chain variable regions of marsupial immunoglobulins.

CONCLUSIONS: Milk was shown to be a convenient source of mucosal secretion containing sIgA, and a process involving 2 precipitation and 2 chromatography steps produced purified sIgA. This IgA preparation will prove useful for the generation of sIgA-specific immunological reagents for measurement of immune responses in the development of mucosal-based vaccines for biological control of possums.

KEYWORDS:

• Immunoglobulin A
• purification
• milk
• brushtail possum
• Trichosurus vulpecula
• mucosal immunity