Double electrophoretic separation and lectin analyses of the component chains of secretory immunoglobulin A from human saliva

A new method is presented for the separation of secretory immunoglobulin A (SIgA) from salivary samples. Salivary proteins (from parotid or stimulated whole mouth saliva) were precipitated with methanol to concentrate SIgA fr om salivary samples whilst removing other salivary proteins. SIgA purified from breast milk and salivary proteins was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing con ditions. Following completion of electrophoresis the top strip of gel was r emoved and the proteins present reduced with dithiothreitol. The gel strip was then applied to the top of a second 10% SDS gel, and the proteins were electrophoresed and then stained by Coomassle Brilliant Blue R-250. Three m ajor protein bands were stained in all samples corresponding in molecular m ass to secretory component, x-heavy chain and light chains of SIgA. Separat ed proteins were also electroblotted onto nitrocellulose and stained by flu orescein isothiocyanate (FITC). Lectin analysis was then used to detect the O-glycans present on IgA1. Lectins from Helix aspersa and Arachis hypogaea were used to determine the amount of terminal N-acetyl galactosamine and n onsialylated O-glycans, respectively. Maclura pomifera lectin was used to d etermine the total amount of IgA1 present on the blots. The results indicat e that SIgA in stimulated whole mouth saliva, stimulated parotid saliva and purified from breast milk contain similar O-glycans.