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#1 Posted : 05 July 2017 11:29:59(UTC)

Joined: 30/06/2017(UTC)
Posts: 3

In a recent post I described the benefits of buffer optimization for improving assay sensitivity. What I did not include in this post was our finding around immobilization levels and the impact this parameter have on assay sensitivity. Beside salt and pH, we also included immobilization level as a variable in our buffer optimization study . In study we immobilized antibodies and we varied the immobilization level between 2000 and 12000 RU. What we found was that high immobilization levels, > 10000 RU, resulted in significantly lower sensitivity. Optimum, that is the highest sensitivity, was found for immobilization levels around 4000-6000 RU. Immobilization levels lower than 4000 RU resulted in lower assay sensitivity. We also found that there was an interaction effect between buffer pH (range 7.0-9.0) and immobilization level, but only at high immobilization levels. Thus, the assay was sensitive to buffer pH if the ligand was immobilized at high levels. Low immobilization levels, on the other hand, made the assay insensitive to pH changes. So, to improve assay sensitivity for crude samples when working with Biacore there are two simple rules to follow: 1) Use high salt concentration, 0.5 M NaCl in your buffer 2) Use low/medium immobilization levels (around 5000 RU for an IgG antibody) For more info see reference below: J Pharm Biomed Anal. 2013 May 5;78-79:224-32. doi: 10.1016/j.jpba.2013.02.018. Epub 2013 Feb 21. /Anna M, GEHC
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