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 K-D(app) 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 x 10(-7) to 9 x 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. (C) 2012 Elsevier B.V. All rights reserved.