Millar A L, Jackson N A, Dalton H, Jennings K R, Levi M, Wahren B, Dimmock N J
Department of Biological Sciences, University of Warwick, Coventry, UK.
Eur J Biochem. 1998 Nov 15;258(1):164-9. doi: 10.1046/j.1432-1327.1998.2580164.x.
Progress in therapeutic or prophylactic immune intervention in HIV-1 infections may only come about with a detailed understanding at the molecular/atomic level of how antibodies neutralize (inactivate) virus infectivity. Currently information on the molecular aspects of antibody-virus interaction comes predominantly from X-ray crystallography, a process that is dependent on the production of suitable crystals. NMR can also be valuable but is complex and time consuming, while mass spectrometry has been limited to matrix-assisted laser-desorption ionization (MALDI) analysis of peptides eluted from the cognate antibody. Here, we have used electrospray ionization mass spectrometry (ESI-MS) to detect directly the interactions of a novel 17-amino-acid microantibody (MicroAb) that has HIV-1-inhibitory activity, and peptides representing the V3 regions of primary HIV-1 strains isolated from Brazil (clade B) and Africa (clade A). The MicroAb is based on the third complementarity-determining region of the heavy chain (CDR-H3) of a murine monoclonal IGGI (F58) specific for the V3 loop of the gp120 envelope glycoprotein of HIV-1. ESI-MS proved to be rapid (taking < 3 h for the entire analysis), sensitive (analytes at 2 mmol/ml), and accurate (RMM estimation to 0.01-0.1%). With it, we showed that the MicroAb forms complexes with the V3 peptides, implying that its antiviral activity is mediated by binding directly to the virus particle. In addition, through controlled protease digestion of the V3 peptides, we concluded that the CDR-H3 MicroAb bound to RKXXXIGPGR, a region similar to the epitope of the whole IgG as determined by ELISA. We believe that the approach exemplified here will be applicable generally to the identification of groups involved in receptor-ligand interactions.
在HIV-1感染的治疗性或预防性免疫干预方面取得进展，可能只有在分子/原子水平上详细了解抗体如何中和（灭活）病毒感染性之后才能实现。目前，关于抗体-病毒相互作用分子层面的信息主要来自X射线晶体学，这一过程依赖于合适晶体的产生。核磁共振（NMR）也可能有价值，但复杂且耗时，而质谱分析一直局限于对从同源抗体洗脱的肽段进行基质辅助激光解吸电离（MALDI）分析。在此，我们使用电喷雾电离质谱（ESI-MS）直接检测一种具有HIV-1抑制活性的新型17氨基酸微型抗体（微型抗体）与代表从巴西（B亚型）和非洲（A亚型）分离的原发性HIV-1毒株V3区的肽段之间的相互作用。该微型抗体基于鼠单克隆IGGI（F58）重链的第三个互补决定区（CDR-H3），该单克隆抗体对HIV-1包膜糖蛋白gp120的V3环具有特异性。ESI-MS被证明是快速的（整个分析过程耗时不到3小时）、灵敏的（分析物浓度为2 mmol/ml）且准确的（相对分子质量（RMM）估计误差为0.01 - 0.1%）。通过它，我们表明微型抗体与V3肽段形成复合物，这意味着其抗病毒活性是通过直接与病毒颗粒结合来介导的。此外，通过对V3肽段进行可控的蛋白酶消化，我们得出结论，CDR-H3微型抗体与RKXXXIGPGR结合，该区域类似于通过酶联免疫吸附测定（ELISA）确定的整个IgG的表位。我们相信这里所举例的方法将普遍适用于鉴定参与受体-配体相互作用的基团。
