Comparison of three distinct ELLA protocols for determination of apparent affinity constants between Con A and glycoproteins

A procedure for determination of apparent affinity constants KDapp between Concanavalin A (Con A) and naturally d-mannose containing glycoproteins using enzyme-linked lectin assay (ELLA) is reported. Three distinct ELLA protocols are compared to each other with 3 different fitting models used (Liliom, Hill with and without a cooperativity factor). The glycoproteins were physisorbed on a highly charged polystyrene solid surface of immunoassay plates and the amount of lectin bound to the glycoproteins was determined by photometry. The interactions of Con A with five mannose-containing glycoproteins, invertase (INV), glucoamylase (GA), glucose oxidase (GOx), ovalbumin (OVA), and transferrin (TRF) were quantified with apparent affinity constant being in the range 2 × 10−7 to 9 × 10−6 M. The strength of interaction between Con A and glycoproteins is discussed on the basis of glycan structure/exposure on the protein backbone for each glycoprotein.

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► Adsorption of 5 mannose containing glycoproteins on a highly charged polystyrene surface.
► Development of novel ELLA (Enzyme Linked Lectin Assay) method for investigation of Con A (concanavalin A) binding to glycoproteins.
► 3 different ELLA protocols and 3 different fitting models used to get apparent affinity constants of interaction between glycoproteins and Con A.
► Validation of the ELLA method with other published analytical assay procedures.