A LECTIN METHOD FOR INVESTIGATING THE GLYCOSYLATION OF NANOGRAM AMOUNTS OF PURIFIED GLYCOPROTEIN

To unravel the complexities of the glycosylation of a protein is a sub stantial task, which requires considerable effort and resources. Howev er, in many situations this is unnecessary, because only a limited amo unt of information is required. A new lectin-binding assay is describe d which is rapid, cheap and versatile. A purified glycoprotein is abso rbed on to the plastic surface of a microtitre plate. After removing u nbound protein by washing, uncoated sites on the plate are blocked and digoxigenin or biotin-labelled lectin is added. The degree of lectin binding is measured using either an anti-DIG antibody or streptavidin conjugated enzyme, which is subsequently used to develop a colour reac tion. Using this method it is possible to screen multiple specimens wi th high sensitivity and excellent precision. In addition, very small a mounts of lectin are used, background absorbances are low, and the pro cedure does not require a high degree of technical skill. Because very small amounts of glycoprotein are needed, a glycoprotein can often be rapidly purified by batch affinity chromatography. The method has bee n successfully applied to several purified proteins using the lectins, Con A, LCA, LTA, MAA, and SNA, and the information obtained agrees wi th that produced by more sophisticated approaches, eg Dionex Carbohydr ate Analyser. Using a panel of lectins, a carbohydrate structural prof ile is quickly built-up, and subtle differences in glycosylation ident ified. This method should be particularly useful for screening glycosy lation in multiple clinical specimens; in specimens where very small a mounts of material are available, such as membrane molecules; and in t he screening of recombinant proteins produced commercially.