AN IMPROVED ELISA FOR THE DETERMINATION OF SIALYL LEWIS(X) STRUCTURESON PURIFIED GLYCOCONJUGATES

The membrane carbohydrate antigen, sialyl Lewis x (sLe(x)), is involve d in cellular adhesive interactions in many diseases, such as cancer, inflammation and thrombosis. This antigen is also found on soluble mac romolecules, such as serum glycoproteins, but the precise role of solu ble sLe(x) in modifying disease processes, pr reflecting the pathologi cal changes is still unclear. Although methods were previously reporte d for the measurement of soluble sLe(x), many of these were not well c haracterised, measurements were mainly made on mixtures of molecules, and the anti-sLe(x) antibodies were used at concentrations that made t he assay expensive. In this study an ELISA has been devised that detec ts sLe(x) in purified soluble glycoconjugates using the anti-sLe(x) an tibody, CSLEX 1. Commercially-available haptoglobin (Hp) and synthetic complexes of Lewis antigens with polyacrylamide were used as model su bstances in developing the procedure. Key steps were washing the antib ody/antigen complex with ten times diluted salt solution to prevent di ssociation of the complex and the use of bovine serum albumin for bloc king non-specific interactions. The assay was shown to be very specifi c, 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 densiti es of sLe(x) on the same amount of glycoconjugate. Determination of sL e(x) in Hp isolated from small groups of healthy individuals, cancer p atients, and rheumatoid arthritis sufferers suggested that the antigen expression is increased in disease. This method which is an improveme nt on those previously described, will be useful for determining sLe(x ) in many different types of soluble glycoconjugate, and used in combi nation with synthetic carbohydrate polyacrylamide complexes, will help to standardize measurements of soluble sLe(x) in the future.