Laricchia-Robbio L, Liedberg B, Platou-Vikinge T, Rovero P, Beffy P, Revoltella R P
Institute of Mutagenesis and Differentiation, C.N.R., Pisa, Italy.
Hybridoma. 1996 Oct;15(5):343-50. doi: 10.1089/hyb.1996.15.343.
An automated surface plasmon resonance (SPR)-based biosensor system has been used for mapping antibody and receptor-binding regions on the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) molecule. A rabbit antimouse IgG1-Fc antibody (RAM.Fc) was coupled to an extended carboxymethylated-hydrogel matrix attached to a gold surface in order to capture an anti-rhGM-CSF monoclonal antibody (MAb) injected over the sensing layer. rhGM-CSF was subsequently injected and allowed to bind to this antibody. Multisite binding assays were then performed, by flowing sequentially other antibodies and peptides over the surface, and the capacity of the latter to interact with the entrapped rhGM-CSF in a multimolecular complex was monitored in real time with SPR. Eleven MAb (all IgG1K), were analyzed: respectively, four antipeptide MAb raised against three distinct epitopes of the cytokine (two clones against residues 14-24, that includes part of the first alpha-helix toward the N-terminal region; one clone against peptide 30-41, an intrahelical loop; and one clone against residues 79-91, including part of the third alpha-helix) and seven antiprotein MAbs raised against the entire rhGM-CSF, whose target native epitopes are still undetermined. In addition, the binding capacity to rhGM-CSF of a synthetic peptide, corresponding to residues 238-254 of the extracellular human GM-CSF receptor alpha-chain, endowed with rhGM-CSF binding activity, was tested. The results from experiments performed with the biosensor were compared with those obtained by a sandwich enzyme-linked immunosorbent assay (ELISA), using the same reagents. The features of the biosensor technology (fully automated, measure in real time, sharpened yes/no response, less background disturbances, no need for washing step or labeling of the reagent) offered several advantages in these studies of MAb immunoreactivity and epitope mapping, giving a much better resolution and enabling more distinct epitopes to be identified over ELISA.
一种基于自动表面等离子体共振（SPR）的生物传感器系统已被用于绘制重组人粒细胞巨噬细胞集落刺激因子（rhGM-CSF）分子上的抗体和受体结合区域。将兔抗小鼠IgG1-Fc抗体（RAM.Fc）偶联到附着在金表面的延伸羧甲基化水凝胶基质上，以捕获注射到传感层上的抗rhGM-CSF单克隆抗体（MAb）。随后注射rhGM-CSF并使其与该抗体结合。然后进行多位点结合测定，通过依次使其他抗体和肽流过表面，并利用SPR实时监测后者与多分子复合物中捕获的rhGM-CSF相互作用的能力。分析了11种单克隆抗体（均为IgG1K）：分别为针对细胞因子三个不同表位产生的四种抗肽单克隆抗体（两种克隆针对残基14-24，包括朝向N端区域的第一个α螺旋的一部分；一种克隆针对肽30-41，一个螺旋内环；一种克隆针对残基79-91，包括第三个α螺旋的一部分）和针对整个rhGM-CSF产生的七种抗蛋白单克隆抗体，其靶向天然表位仍未确定。此外，还测试了与细胞外人类GM-CSF受体α链残基238-254相对应的具有rhGM-CSF结合活性的合成肽与rhGM-CSF的结合能力。使用相同的试剂，将生物传感器实验结果与夹心酶联免疫吸附测定（ELISA）结果进行比较。生物传感器技术的特点（全自动、实时测量、清晰的是/否响应、背景干扰少、无需洗涤步骤或试剂标记）在这些单克隆抗体免疫反应性和表位图谱研究中具有多个优势，与ELISA相比具有更好的分辨率，能够识别更多不同的表位。
