Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Department of Medical Biotechnology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
J Cell Physiol. 2019 Jun;234(6):9486-9494. doi: 10.1002/jcp.27636. Epub 2018 Nov 11.
The biopanning process is a critical step in phage display for isolating peptides or proteins with specific binding properties. Conventional panning methods are sometimes not so effective and may result in nonspecific or low-yield positive results. In this study, three different strategies including soluble antibody-capturing, pH-stepwise elution, and conventional panning were used for enrichment of specific clones against diphtheria toxoid. The reactivity of the selected clones was evaluated using an indirect enzyme-linked immunosorbent assay. The positive clones were screened using Vero cell viability assay. The neutralizing clones were expressed in HB2151 strain of Escherichia coli and soluble single-chain fragment variable (scFv) fragments were purified by nickel-nitrilotriacetic acid affinity chromatography. Finally, the ability of scFv fragments for neutralizing diphtheria toxin (DT) were evaluated again using Vero cell viability assay. After four rounds of panning, the soluble antibody-capturing method yielded 15 positive phage-scFv clones against diphtheria toxoid. Conventional panning and pH-stepwise elution model resulted from nine and five positive phage-scFv clones, respectively. Among all positive clones, three clones were able to neutralize DT in Vero cell viability assay. Two of these clones belonged to a soluble antibody-capturing method and one of them came from conventional panning. Three neutralizing clones were used for soluble expression and purification of scFvs fragments. It was found that these soluble scFv fragments possessed neutralizing activity ranging from 0.15 to 0.6 µg against two-fold cytotoxic dose 99% of DT. In conclusion, the results of our study indicate that soluble antibody-capturing method is an efficient method for isolation of specific scFv fragments.
生物淘选过程是噬菌体展示技术中分离具有特定结合特性的肽或蛋白质的关键步骤。传统的淘选方法有时并不那么有效，可能导致非特异性或低产阳性结果。在这项研究中，使用了三种不同的策略，包括可溶性抗体捕获、pH 逐步洗脱和常规淘选，来富集针对白喉类毒素的特异性克隆。使用间接酶联免疫吸附试验评估所选克隆的反应性。使用 Vero 细胞活力测定筛选阳性克隆。将中和克隆在大肠杆菌 HB2151 菌株中表达，并通过镍-亚氨基二乙酸亲和层析纯化可溶性单链片段可变 (scFv) 片段。最后，再次使用 Vero 细胞活力测定评估 scFv 片段中和白喉毒素 (DT) 的能力。经过四轮淘选，可溶性抗体捕获法得到了 15 个针对白喉类毒素的阳性噬菌体-scFv 克隆。常规淘选和 pH 逐步洗脱模型分别得到了 9 个和 5 个阳性噬菌体-scFv 克隆。在所有阳性克隆中，有 3 个克隆能够在 Vero 细胞活力测定中中和 DT。其中两个克隆属于可溶性抗体捕获法，其中一个来自常规淘选。用三个中和克隆进行可溶性表达和 scFv 片段的纯化。结果发现，这些可溶性 scFv 片段对 DT 的半数细胞毒性剂量的 2 倍具有 0.15 至 0.6μg 的中和活性。总之，我们的研究结果表明，可溶性抗体捕获法是分离特异性 scFv 片段的有效方法。
