| Assay Method Information | |
| | CD33 Competitive Binding Assay |
| Description: | This is a competitive binding assay that uses recombinant CD33 immobilized to a plate and a fluorescently-labeled multivalent sialic acid analog as a probe. Binding of drug to the site on CD33 to which sialic acid binds is detected by reduced binding of the fluorescent probe.Recombinant human CD33 protein (for example Acro Biosystems cat #CD3-H5257) was added at a concentration of 10 μg/ml in 20 mM acetate buffer (pH5.5) to a black plastic plate (Corning 4510) and allowed to incubate overnight at 4° C. The next day, the wells were washed 3 times with PBS containing 0.02% Tween-20. This was followed by a 30-minute incubation with PBS containing 0.2% Tween-20 and 0.5% BSA to block non-specific binding to the well. This buffer is removed and replaced with 10 μl of the same buffer, which is also used as the assay buffer.The fluorescent multivalent sialic acid probe was prepared as a 4× concentration as follows. A biotinylated sialic acid analog A-375 was mixed with biotin-4-Fluorescein isothiocyanate (biotin-4-FITC) at a 3:1 molar ratio with final concentrations (4× stock) of 60 μM A-375 and 20 μM biotin-4-FITC. After these were mixed, 20 μM of neutravidin was added, which should form complexes consisting of neutravidin, fluorescein and sialic acid with an average complex consisting of 1 neutravidin, 1 FITC and 3 sialic acid analogs. The multimerization of the sialic acid analog is critical to the assay and improves the affinity of the sialic acid analog several orders of magnitude.A test compound, for example A-001, is added at 5 μl to the 10 μl assay buffer already in the well. After a brief incubation, 5 μl of the 4× stock solution of sialic acid complexes was added. The final concentration of the complexes was therefore 5 μM neutravidin, 15 μM A-375 and 5 μM FITC. The plate was incubated at room temperature for 30 mins and then washed with PBS containing 0.2% Tween-20. Binding of the sialic acid complex is very stable, with an off-rate of several days. Bound complex was then detected using a standard plate reader to quantify FITC fluorescence. |
| Affinity data for this assay | |
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