Quantification of streptavidin adsorption in microtitration wells

Streptavidin-coated microtitration plates have an important role as a solid phase in clinical diagnostics. We have designed techniques for evaluating quantitative and functional aspects of streptavidin adsorbed in microtitration wells. The theoretical monolayer adsorption capacity was modeled based on the molecular dimensions of the protein. Adsorbed streptavidin was quantified by direct labeling of protein with terbium chelate and with a sensitive bicinchoninic acid-based protein assay. A new small molecular weight (1037 Da) reporter molecule, a europium-labeled biotin (Eu-biotin), was synthesized and used for monitoring adsorption and for determination of biotin-binding capacities of the streptavidin-coated wells. The theoretical monolayer adsorption of streptavidin yielded 6.20 pmol/cm 2 (370 ng) and consequently the theoretical adsorption capacity of a C12-format microtitration well (200 μl liquid, coated area 1.54 cm 2) was 9.55 pmol/well (570 ng). Adsorption properties of streptavidin from two suppliers were tested, one of which yielded 350–380 ng/well while the other yielded over 500 ng/well. The biotin binding capacities were about 11 and 14 pmol/well, respectively. We managed to quantify surface-adsorbed streptavidin with sensitive fluorescence and protein measurement methods in the microtitration well. The new Eu-biotin reporter molecule enabled an exact and convenient determination of the biotin-binding capacities of streptavidin surfaces.