Solid Supports in Enzyme-Linked Immunosorbent Assay and Other Solid-Phase Immunoassays
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Most modern immunoassays involve the use of synthetic solid phases to immobilize one of the reactants, often by simple adsorption. These solid-phase immunoassays (SPIs) involve ligand-receptor interactions that occur within a reaction volume close to the solution/solid-phase interface. As a consequence, the immunochemistry/biochemistry of these ligand-receptor interactions differ from their counterparts in solution. Nevertheless, mass law equations can be derived for measuring the antigen capture of solid-phase antibodies, for determining the affinity of solid phases for protein adsorption, and for estimating antibody affinity.
Many proteins adsorbed on polystyrene or silicone suffer adsorption-induced conformational changes (ACC) and are partially or largely denatured. Alternative methods for immobilizing proteins and virus, while preserving antigenicity, may yield only a modest increase in functional reactant concentration. Peptides and small recombinant proteins appear to benefit especially from nonadsorptive immobilization. Not all solid phases commonly used in SPIs have the same properties, the same capacity for reactant immobilization, cause the same level of denaturation, or experience the same level of nonspecific binding. Empiricism, adherence to a few practical rules of thumb, and avoidance of certain “old wives tales” can be valuable in the successful development of SPIs.
