Nieba L, Krebber A, Plückthun A
Biochemisches Institut der Universität Zürich, Switzerland.
Anal Biochem. 1996 Feb 15;234(2):155-65. doi: 10.1006/abio.1996.0067.
In attempting to use the BIAcore instrument for the determination of binding constants of several haptens or peptides to different antibodies by measuring on- and off-rates, we found that neither the absolute nor the relative values of the binding constants corresponded to the measurements in solution. Even at the lowest coupling densities useful for measurements, rebinding and bivalency effects offset the measurements by a factor of up to 500. We caution therefore about using on- and off-rates for the determination of absolute or even relative binding constants without controlling for rebinding and avidity effects. Instead, we show that binding constants in solution can be reproduced well by using on-rate determinations of antibody preincubated with antigen, and we derive the conditions under which such an approach is valid.
在尝试使用BIAcore仪器通过测量结合和解离速率来测定几种半抗原或肽与不同抗体的结合常数时，我们发现结合常数的绝对值和相对值均与溶液中的测量结果不一致。即使在可用于测量的最低偶联密度下，再结合和二价效应也会使测量结果产生高达500倍的偏差。因此，我们提醒在未控制再结合和亲和力效应的情况下，不要使用结合和解离速率来测定绝对或甚至相对结合常数。相反，我们表明，通过使用与抗原预孵育的抗体的结合速率测定，可以很好地重现溶液中的结合常数，并且我们推导了这种方法有效的条件。
