An improved ELISA for the determination of sialyl Lewis(x) structures on purified glycoconjugates

The membrane carbohydrate antigen, sialyl Lewis x (sLe(x)), is involved in cellular adhesive interactions in many diseases, such as cancer, inflammation and thrombosis. This antigen is also found on soluble macromolecules, such as serum glycoproteins, but the precise role of soluble sLe(x) in modifying disease processes, or reflecting the pathological changes is still unclear. Although methods were previously reported for the measurement of soluble sLe(x), many of these were not well characterised, measurements were mainly made on mixtures of molecules, and the anti-sLe(x) antibodies were used at concentrations that made the assay expensive. In this study an ELISA has been devised that detects sLe(x) in purified soluble glycoconjugates using the anti-sLe(x) antibody, CSLEX I. Commercially-available haptoglobin (Hp) and synthetic complexes of Lewis antigens with polyacrylamide were used as model substances in developing the procedure. Key steps were washing the antibody/antigen complex with ten times diluted salt solution to prevent dissociation of the complex and the use of bovine serum albumin for blocking non-specific interactions. The assay was shown to be very specific, its precision was in the range 6-12%, and it could detect less than a pmol of sLe(x). It could also distinguish between different densities of sLe(x) on the same amount of glycoconjugate. Determination of sLe(x) in Hp isolated from small groups of healthy individuals, cancer patients, and rheumatoid arthritis sufferers suggested that the antigen expression is increased in disease. This method, which is an improvement on those previously described will be useful for determining sLe(x) in many different types of soluble glycoconjugate, and used in combination with synthetic carbohydrate polyacrylamide complexes, will help to standardize measurements of soluble sLe(x) in the future.