Use of a resonant mirror biosensor to characterize the interaction of carboxypeptidase A with an elicited monoclonal antibody
Use of a resonant mirror biosensor to characterize the interaction of carboxypeptidase A with an elicited monoclonal antibody
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DOI：
10.1006/abio.1996.9867
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发表时间：
1997-01-01
影响因子：
2.9
通讯作者：
Winzor, DJ
中科院分区：
文献类型：
--
作者：
Hall, DR;Winzor, DJ
脱羧羧肽酶A的固定化形式的其引发的单克隆抗体的结合已被用于探索基于共振镜技术的生物传感器仪器（IAsys）的抗体-抗原相互作用的定量表征的潜力。利用IAsys仪器的搅拌比色皿设计开发了用于相互作用热力学表征的逐步滴定程序，在研究的条件下（0.1 M Tris/HCl-0.5 M NaCl，pH 7.5，21 ℃）获得了3.3（+/-0.9）× 10（7）M（-1）的缔合平衡常数。在对生物传感器响应的时间进程进行常规伪一级动力学分析的可行性测试中，在脱羧羧肽酶A的高浓度和低浓度下遇到与这种描述不一致的结果。尽管在高抗原浓度下与朗缪尔动力学行为的偏差无疑源于与在使用BIA核心生物传感器仪器的研究中已经遇到的那些相同的来源，但在另一个浓度极限下的偏差发生在液相中游离抗原浓度的假定恒定性变得不佳近似的范围内。在这种情况下的另一种方法已经过测试，其中先前的热力学表征是通过二阶动力学分析的速率常数评价的先决条件。最后，可溶性抗羧肽酶A对生物传感器响应的伪一级动力学的影响已被用于说明用于评估溶液中抗体-抗原相互作用的亲和常数的简单动力学程序，通过这种方法获得的值为1.9（+/-0.2）× 10（8）M（-1）。(C)出版社：Academic Press，Inc.
The binding of apocarboxypeptidase A to an immobilized form of its elicited monoclonal antibody has been used to explore the potential of a biosensor instrument (IAsys) based on resonant mirror technology for the quantitative characterization of antibody-antigen interactions. Advantage has been taken of the stirred cuvette design of the IAsys instrument to develop a stepwise titration procedure for thermodynamic characterization of the interaction, an association equilibrium constant of 3.3 (+/-0.9) x 10(7) M(-1) having been obtained under the conditions studied (0.1 M Tris/HCl-0.5 M NaCl, pH 7.5, 21 degrees C). In a test of the feasibility of subjecting the time course of biosensor response to conventional pseudo-first-order kinetic Analysis, nonconformity of results with such description was encountered at high and low concentrations of apocarboxypeptidase A. Whereas the deviations from Langmuirian kinetic behavior at high antigen concentrations undoubtedly stem from the same sources as those already encountered in studies with the BIA-core biosensor instrument, the deviations at the other concentration extreme occur in a range in which the assumed constancy of free antigen concentration in the liquid phase is becoming a poor approximation. An alternative approach in such circumstances has been tested in which prior thermodynamic characterization is a prerequisite for rate constant evaluation by means of a second-order kinetic analysis. Finally, the effect of soluble anticarboxypeptidase A on the pseudo-first-order kinetics of the biosensor response has been used to illustrate a simple kinetic procedure for evaluating the affinity constant for the antibody-antigen interaction in solution a value of 1.9 (+/-0.2) X 10(8) M(-1) being Obtained by such means. (C) 1997 Academic Press, Inc.