The multipoint competitive ELISA then evaluates binding of the sub-saturating rAB concentration to immobilized antigen competed by pre-incubation with serial dilutions of antigen in solution to produce an inhibition curve from which the IC 50 value can be determined. The quantitation of rAB EC 50 is required to establish an accurate sub-saturating concentration of rAB for conducting the competitive binding experiment.
It does however, allow for determination of a sub-saturating concentration of rAB that can provide for more accurate determination of rAB affinity in competitive ELISA.Ī similar, yet more accurate determination of rAB affinity can be obtained through direct binding of rAB to immobilized target protein in the presence of series of concentrations of soluble target antigen in a competitive ELISA format. EC 50 measurement allows for rank ordering of many unique clones, but may not provide an accurate measure of affinity. Since most antibodies will, at sufficiently high concentrations, begin to bind non-specifically to non-target proteins, this assay will enable determination of a that provides maximal specific to non-specific signals when conducted using the appropriate non-specific control. The assay can be done in a high-throughput manner using 3-5 fold dilutions of rAB of a stock antibody concentration in binding buffer. An estimate of affinity is interpreted from one-half the concentration at which rAB binding first achieves saturation. In general, two related methods are used to estimate rAB affinity but can also provide an indication of the optimal antibody concentration to be used in other applications and the specificity via direct comparison of binding to non-specific control proteins.ĮC 50 or the concentration of antibody that gives half-maximal binding is determined by direct and saturable binding of a rAB dilution series to both target antigen and a non-specific control protein. Following expression and purification of soluble recombinant antibodies fragments (rABs), ELISA-based assays may be useful to verify reagent binding to target antigen.
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