Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Biotechnol Lett. 2024 Jun;46(3):385-398. doi: 10.1007/s10529-024-03476-1. Epub 2024 Apr 12.
Diphtheria can be prevented by vaccination, but some epidemics occur in several places, and diphtheria's threat is considerable. Administration of diphtheria antitoxin (DAT) produced from hyperimmunized animals is the most common treatment. Recombinant human antibody fragments such as single-chain variable fragments (scFv) produced by phage display library may introduce an interesting approach to overcome the limitations of the traditional antibody therapy. In the present study, B cells of immunized volunteers were used to construct a human single-chain fragment (HuscFv) library.
The library was constructed with the maximum combination of heavy and light chains. As an antigen, Diphtheria toxoid (DTd) was used in four-round phage bio-panning to select phage clones that display DTd bound HuscFv from the library. After panning, individual scFv clones were selected. Clones that were able to detect DTd in an initial screening assay were transferred to Escherichia coli HB2151 to express the scFvs and purification was followed by Ni metal ion affinity chromatography. Toxin neutralization test was performed on Vero cells. The reactivity of the soluble scFv with diphtheria toxin were done and affinity calculation based on Beatty method was calculated.
The size of the constructed scFv library was calculated to be 1.3 × 10 members. Following four rounds of selection, 40 antibody clones were isolated which showed positive reactivity with DTd in an ELISA assay. Five clones were able to neutralize DTd in Vero cell assay. These neutralizing clones were used for soluble expression and purification of scFv fragments. Some of these soluble scFv fragments show neutralizing activity ranging from 0.6 to 1.2 µg against twofold cytotoxic dose of diphtheria toxin. The affinity constant of the selected scFv antibody was determined almost 10 M.
This study describes the prosperous construction and isolation of scFv from the immune library, which specifically neutralizes diphtheria toxin. The HuscFv produced in this study can be a potential candidate to substitute the animal antibody for treating diphtheria and detecting toxins.
白喉可以通过接种疫苗来预防，但在一些地方仍会发生一些疫情，白喉的威胁仍然相当大。使用从高度免疫动物中制备的白喉抗毒素（DAT）进行治疗是最常见的治疗方法。噬菌体展示文库产生的重组人抗体片段，如单链可变片段（scFv），可能为克服传统抗体治疗的局限性带来一种有趣的方法。在本研究中，我们使用免疫志愿者的 B 细胞构建了一个人源单链片段（HuscFv）文库。
使用最大重链和轻链组合构建文库。将白喉类毒素（DTd）作为抗原，在四轮噬菌体生物淘选中从文库中筛选出能结合 DTd 的展示 HuscFv 的噬菌体克隆。淘选后，选择单个 scFv 克隆。在初步筛选试验中能够检测到 DTd 的克隆被转移到大肠杆菌 HB2151 中表达 scFv，并通过 Ni 金属离子亲和层析进行纯化。在 Vero 细胞上进行毒素中和试验。对可溶性 scFv 与白喉毒素的反应性进行检测，并根据 Beatty 法计算亲和力。
构建的 scFv 文库大小估计为 1.3×10个成员。经过四轮筛选，从文库中分离出 40 个与 DTd 在 ELISA 试验中呈阳性反应的抗体克隆。有 5 个克隆能够在 Vero 细胞试验中中和 DTd。这些中和克隆被用于可溶性 scFv 片段的表达和纯化。其中一些可溶性 scFv 片段对二倍细胞毒性剂量的白喉毒素具有 0.6 至 1.2µg 的中和活性。所选 scFv 抗体的亲和常数几乎为 10M。
本研究描述了 scFv 从免疫文库中的成功构建和分离，该文库能特异性中和白喉毒素。本研究中产生的 HuscFv 可替代动物抗体用于治疗白喉和检测毒素，具有潜在的应用价值。
