Cupit P M, Lorenzen N, Strachan G, Kemp G J, Secombes C J, Cunningham C
Sars International Centre for Marine Molecular Biology, High Technology Centre, 5008, Bergen, Norway.
Virus Res. 2001 Dec 4;81(1-2):47-56. doi: 10.1016/s0168-1702(01)00354-9.
We have previously reported the cloning and characterisation of the heavy and light chain variable domain genes encoding three monoclonal antibodies (Mabs) that bind viral haemorrhagic septicaemia virus (VHSV). Two of these antibodies, 3F1H10 and 3F1A2 both neutralised the virus though 3F1A2 appeared to recognise a broader range of virus isolates. The variable domains of these two antibodies differ by only four residues (Lorenzen et al., 2000a. Fish Shellfish Immunol. 10, 129-142). To further study the mechanism of neutralisation, Fab fragments as well as a series of recombinant bacterial single chain antibody (scAb) fragments were generated from the three anti-VHSV Mabs and their variable domain genes, respectively. Fabs and scAbs derived from the neutralising Mabs were both able to neutralise the VHSV type 1 isolate DK-F1. In addition, a series of scAb fragments were produced using the 3F1H10 variable heavy (VH) chain and variable light (Vkappa) chain domains but containing, either alone or in dual combination, each of the four different residues present in 3F1A2. The dissociation constants of Mabs 3F1H10 and 3F1A2 and their respective Fab and scAb fragments were measured by BIAcore analysis and found to correlate with the capacity of each molecule to neutralise DK-F1. These investigations, together with computer assisted molecular analysis of the theoretical influence of each mutation on antigen binding, led to the identification of a single mutation at position 35a in the VH domain as having the most marked impact on viral neutralisation.
我们之前报道过编码三种结合病毒性出血性败血症病毒（VHSV）的单克隆抗体（Mab）的重链和轻链可变结构域基因的克隆及特性分析。其中两种抗体，3F1H10和3F1A2都能中和该病毒，不过3F1A2似乎能识别更广泛的病毒分离株。这两种抗体的可变结构域仅相差四个残基（Lorenzen等人，2000a。《鱼类和贝类免疫学》10，129 - 142）。为了进一步研究中和机制，分别从三种抗VHSV单克隆抗体及其可变结构域基因产生了Fab片段以及一系列重组细菌单链抗体（scAb）片段。源自中和性单克隆抗体的Fab和scAb都能够中和1型VHSV分离株DK - F1。此外，使用3F1H10可变重链（VH）结构域和可变轻链（Vκ）结构域产生了一系列scAb片段，但单独或双重组合包含3F1A2中存在的四个不同残基中的每一个。通过BIAcore分析测量了单克隆抗体3F1H10和3F1A2及其各自的Fab和scAb片段的解离常数，发现其与每个分子中和DK - F1的能力相关。这些研究，连同对每个突变对抗原结合的理论影响的计算机辅助分子分析，导致鉴定出VH结构域中35a位置的单个突变对病毒中和具有最显著的影响。
