荧光素与9-羟基苯基荧光酮（HPF）与鼠单克隆抗荧光素抗体的相对结合特性。

Relative binding properties of fluorescein and 9-hydroxyphenylfluoron (HPF) with murine monoclonal anti-fluorescein antibodies.

作者信息

Bedzyk W D, Swindlehurst C A, Voss E W

机构信息

Department of Microbiology, University of Illinois, Urbana-Champaign 61801.

出版信息

Biochim Biophys Acta. 1992 Feb 13;1119(1):27-34. doi: 10.1016/0167-4838(92)90229-7.

DOI:10.1016/0167-4838(92)90229-7

PMID:1540631

Abstract

Contribution of the fluorescein (F1) carboxyl group to hapten binding by idiotypically related murine monoclonal anti-F1 antibodies 4-4-20, 9-40 and 12-40 was studied by comparing relative liganded active site properties with bound Fl or 9-hydroxyphenylfluoron (HPF). Kinetic studies revealed similar association rate constants between Fl and HPF to 4-4-20 (approximately 1.1 x 10(7) M-1 s-1); however, the 4-4-20 dissociation rate for Fl was approximately 200 times slower, relative to HPF, which resulted in relative intrinsic affinity values of 1.2 x 10(10) and 6.5 x 10(7) M-1, respectively. Mabs 9-40 and 12-40 also displayed a reduced affinity for HPF and affinity constants of 5.5 x 10(5) M-1 and 6.7 x 10(5) M-1 were obtained from a competitive ELISA. Additionally, previous studies revealed that upon binding Fl, Mabs 4-4-20 (92.1%), 9-40 (44.7%) and 12-40 (73.4%) quenched Fl fluorescence. Similar analyses with HPF resulted in 64.4% and 2.0% fluorescence quenching by 4-4-20 and 12-40, respectively; however, 9-40 increased HPF fluorescence by approximately 24%. Steady-state fluorescence polarization experiments revealed that in solution, Fl (P = 0.019) and HPF (P = 0.048) were polarized to different degrees. When bound, however, Fl and HPF expressed similar polarization values (P approximately 0.455), except 9-40 bound HPF which was significantly depolarized (P = 0.428). Fluorescence lifetime experiments revealed Fl to possess two discrete lifetimes: a 3.96 ns component (free Fl) and either a 0.52 ns (4-4-20), 2.23 ns (9-40) or 0.96 ns (12-40) short component that corresponded to bound Fl. HPF, however, when bound by 4-4-20 or 9-40, was best fit by three discrete exponentials: a relatively long 4.0 ns component, a 1.11 ns lifetime (free HPF) and either a 0.52 ns (4-4-20) or 2.23 ns (9-40) component. Finally, HPF bound by Mab 12-40 exhibited a single lorenzian distributed lifetime of 1.36 ns (+/- 0.43 ns). Results are discussed in terms of Mab active site structure and conformational state dynamics.

摘要

通过比较与结合的荧光素（Fl）或9-羟基苯基荧光酮（HPF）相关的相对配体活性位点特性，研究了荧光素（F1）羧基对独特型相关的鼠单克隆抗F1抗体4-4-20、9-40和12-40的半抗原结合的贡献。动力学研究表明，Fl和HPF与4-4-20的缔合速率常数相似（约1.1×10⁷ M⁻¹ s⁻¹）；然而，相对于HPF，Fl与4-4-20的解离速率约慢200倍，这导致相对固有亲和力值分别为1.2×10¹⁰和6.5×10⁷ M⁻¹。单克隆抗体9-40和12-40对HPF的亲和力也降低，通过竞争性ELISA获得的亲和常数分别为5.5×10⁵ M⁻¹和6.7×10⁵ M⁻¹。此外，先前的研究表明，结合Fl后，单克隆抗体4-4-20（92.1%）、9-40（44.7%）和12-40（73.4%）会淬灭Fl荧光。用HPF进行的类似分析分别导致4-4-20和12-40淬灭荧光64.4%和2.0%；然而，9-40使HPF荧光增加了约24%。稳态荧光偏振实验表明，在溶液中，Fl（P = 0.019）和HPF（P = 0.048）的偏振程度不同。然而，结合后，Fl和HPF表现出相似的偏振值（P约为0.455），但9-40结合的HPF明显去偏振（P = 0.428）。荧光寿命实验表明，Fl具有两个离散的寿命：一个3.96 ns的成分（游离Fl）和一个对应于结合Fl的0.52 ns（4-4-20）、2.23 ns（9-40）或0.96 ns（12-40）的短成分。然而，当HPF与4-4-20或9-40结合时，最好用三个离散的指数拟合：一个相对较长的4.0 ns成分、一个1.11 ns寿命（游离HPF）和一个0.52 ns（4-4-20）或2.23 ns（9-40）成分。最后，12-40单克隆抗体结合的HPF表现出1.36 ns（±0.43 ns）的单洛伦兹分布寿命。根据单克隆抗体活性位点结构和构象状态动力学对结果进行了讨论。