Neutralizing anti-diphtheria toxin scFv produced by phage display

IF 2 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Biotechnology Letters Pub Date : 2024-04-12 DOI:10.1007/s10529-024-03476-1

Ehsan Khalili, Mostafa Lakzaei, Mahdi Aminian

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Abstract

Background

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.

Materials and methods

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.

Results

The size of the constructed scFv library was calculated to be 1.3 × 10⁶ 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 10⁷ M⁻¹.

Conclusion

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.

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通过噬菌体展示产生中和性抗白喉毒素 scFv

背景白喉可以通过接种疫苗来预防，但有些流行病会在多个地方发生，白喉的威胁相当大。最常见的治疗方法是注射由超免疫动物生产的白喉抗毒素（DAT）。重组人类抗体片段，如通过噬菌体展示文库产生的单链可变片段（scFv），可能会为克服传统抗体疗法的局限性带来一种有趣的方法。本研究利用免疫志愿者的 B 细胞构建了人类单链片段（HuscFv）文库。以白喉类毒素（DTd）为抗原，通过四轮噬菌体生物筛选，从库中筛选出能显示与 DTd 结合的 HuscFv 的噬菌体克隆。筛选后，再选出单个 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 中以表达 scFv，然后通过 Ni 金属离子亲和层析进行纯化。毒素中和试验在 Vero 细胞上进行。结果经计算，构建的 scFv 库的大小为 1.3 × 106 个成员。经过四轮筛选，分离出 40 个抗体克隆，在 ELISA 检测中与 DTd 呈阳性反应。在 Vero 细胞实验中，有五个克隆能够中和 DTd。这些中和克隆被用于可溶性表达和纯化 scFv 片段。其中一些可溶性 scFv 片段对两倍细胞毒性剂量的白喉毒素具有 0.6 至 1.2 µg 的中和活性。本研究描述了从免疫库中成功构建和分离出的 scFv，它能特异性中和白喉毒素。本研究制备的 HuscFv 有可能替代动物抗体用于治疗白喉和检测毒素。

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来源期刊

Biotechnology Letters 工程技术-生物工程与应用微生物

CiteScore

5.90

自引率

3.70%

发文量

108

审稿时长

1.2 months

期刊介绍： Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them. All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included. Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields. The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories. Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.