The presence of antibodies directed against carbohydrate determinants in anti-trout immunoglobulin (Ig) and anti-keyhole limpet hemocyanin (KLH) antisera raised the possibility that these reagents might react with Ig and non-Ig membrane determinants. In order to discriminate between these two specificities, trout splenocytes and thymocytes were radioiodinated by the lactoperoxidase procedure and immunoprecipitated with anti-trout Ig and anti-KLH. With splenocyte membrane proteins, reduced anti-trout Ig immunoprecipitates, when analyzed on SDS-gels, resolved into a 100,000 m.w. component as well as the heavy and light chain constituents from membrane Ig. The anti-KLH, on the other hand, reacted preferentially with a 100,000 m.w. component. The binding to the 100,000 m.w. component could be removed from the anti-Ig sera by absorption of the reagent with KLH such that only the membrane Ig was precipitated. The lower reactivity of anti-KLH to membrane Ig, when compared with the 100,000 m.w. component, was probably due to a difference in carbohydrate content because specifically purified antibodies eluted with l-fucose were capable of reacting with both glycoproteins from splenocytes.

On thymocytes, anti-Ig and anti-KLH reacted with membrane proteins that gave qualitatively similar SDS-gel patterns. The anti-Ig sera absorbed with KLH failed to react with any thymocyte membrane proteins indicating an absence of Ig-specific determinants on these cells. By immunofluorescence, 100% of the thymocytes were stained with anti-Ig and anti-KLH reagents. Some faint residual fluorescence was found when anti-Ig was absorbed with erythrocytes, KLH, and trout Ig glycopeptides. Thus, although some minor reactivity of anti-Ig on thymocytes cannot be accounted for by cross-reactive determinants by immunofluorescence, most of the reactivity was due to carbohydrate moieties. It does not appear that conventional Ig is found on thymocytes, at least in amounts that are detectable by the lactoperoxidase labeling method.

1

This work was supported by National Institutes of Health Postdoctoral Fellowships AI-05343, AI-05334, National Institutes of Health Grants AI-12136, AI-09758, and American Heart Association Grant 74856.

2

Some of the data were presented in summary form in Immune System: Genetics and Regulation, p 297, Academic Press, New York, 1977.

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