Chromatographic Purification of Tetramethylrhodamine-Immune Globulin Conjugates and Their Use in the Cellular Localization of Rabbit γ-Globulin Polypeptide Chains
Summary: Rabbit spleen imprints were double stained with pairs of fluorescein and tetramethylrhodamine conjugates derived from the following goat reagent globulins: anti-Fragment III (anti-γG heavy chain), anti-light chain and anti-Fragment I. To obtain specific staining of the component chains of rabbit immunoglobulin it was necessary to develop procedures for the chromatographic fractionation of tetramethylrhodamine-immune globulin conjugate to supplement existing procedures for the preparation of specific staining fluorescein-conjugates. Bright specific staining could be obtained using the subfractionated conjugates at concentrations as low as 32 to 43 µg of antibody/ml. A series of simple Kodak Wratten filters, K2, 23A and 57A, were used to differentiate between singly and doubly stained fluorescent cells. These filters permitted detection and proper classification of fluorescent cells even when the optimal ratio of reagent concentrations was varied over a 16-fold range. Staining with anti-light chain and anti-III, conjugated with contrasting fluorochromes, resulted in double labeling of 63 to 78% of all fluorescent cells. These cells presumably contained all component chains of γG-globulin. A distinct minority of the fluorescent cells, 21 to 34% of the population, stained only with the anti-light chain reagent. A similar class of cells, comprising 14 to 26% of all fluorescent cells, stained only with the anti-I reagent when it was used as a counterstain with anti-III reagent. Anti-light chain reagents detected more immunoglobulin-containing cells than did anti-I reagents. Double staining with anti-I and anti-light chain, conjugated with contrasting fluorochromes, yielded 63 to 85% doubly stained cells, 15 to 32% of cells stained with the anti-light chain alone, and 0 to 10% of cells stained with anti-I alone.