Determination of Terminal Sugars in Transferrin by Radio-lectin Immunoassay (RLIA)—A New Microanalytical Procedure

Abstract

A new method for the determination of terminal sugars in immunologically defined glycoproteins with microheterogeneity in the sugar residue has been developed. The method has been elaborated for transferrin and involves the following three steps: 1. Binding of antitransferrin antibodies to cyanogen bromide activated Sepharose. 2. Adsorption to the antitransferrin gel of transferrin from serum or from standard solutions of defined composition of terminal glycoprotein sugars. 3. Adsorption of a ¹²⁵I - labelled lectin to the Sepharose-antitransferrin-transferrin complex. A galactose-binding lectin from Crotalaria juncea and a sialic acid-binding lectin from Limulus polyphemus have been successfully labelled with the Bolton-Hunter reagent and used in the radio-lectin immunoassay determinations. Care must be taken that significant amounts of lectins are not lost from the transferrin complex during washing. The resolving power of the method was at best 20 pmoles of asialotransferrin, a figure that probably can be improved significantly by optimizing the assay conditions.