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General, Label-Free Method for Determining Kd and Ligand Concentration Simultaneously

Jalali-Yazdi, Farzad, Takahashi, Terry T., Roberts, Richard W.
Analytical chemistry 2015 v.87 no.23 pp. 11755-11762
antibodies, immunoassays, ligands, models, proteins
Some of the most commonly used affinity reagents (e.g., antibodies) are often developed and used in conditions where their input concentrations ([L]₀) and affinities (Kd) are not known. Here, we have developed a general approach to determine both [L]₀ and Kd values simultaneously for affinity reagents (small molecules, proteins, and antibodies). To do this, we perform quantitative equilibrium exclusion immunoassays with two different concentrations of target and fit the data simultaneously to determine Kd and [L]₀. The results give accurate and reproducible measures of both values compared to established methods. By performing detailed error analysis, we demonstrate that our fitting gives unique solutions and indicates where Kd and [L]₀ measures are reliable. Furthermore, we found that a divalent model of antibody binding gives accurate Kd and [L]₀ values in both the forward (antibody immobilized) and the reverse (target immobilized) assays addressing the long-term problem of obtaining quantitative data from reverse assays.