Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia.
QIMR Berghofer Medical Research Institute, Brisbane, Australia.
Malar J. 2018 Oct 24;17(1):383. doi: 10.1186/s12936-018-2531-y.
Malaria rapid diagnostic tests (RDTs) represent an important antibody based immunoassay platform. Unfortunately, conventional monoclonal antibodies are subject to degradation shortening shelf lives of RDTs. The variable region of the receptor (V) from shark has a potential as alternative to monoclonal antibodies in RDTs due to high thermal stability.
In this study, new binders derived from shark V domains library were investigated. Following immunization of a wobbegong shark (Orectolobus ornatus) with three recombinant malaria biomarker proteins (PfHRP2, PfpLDH and Pvaldolase), a single domain antibody (sdAb) library was constructed from splenocytes. Target-specific V phage were isolated by panning. One specific clone was selected for expression in Escherichia coli expression system, and study of binding reactivity undertaken.
The primary V domain library possessed a titre of 1.16 × 10 pfu/mL. DNA sequence analysis showed 82.5% of isolated fragments appearing to contain an in-frame sequence. After multiple rounds of biopanning, a highly dominant clone specific to PfHRP2 was identified and selected for protein production in an E. coli expression system. Biological characterization showed the recombinant protein expressed in periplasmic has better detection sensitivity than that of cytoplasmic proteins. Assays of binding activity indicated that its reactivity was inferior to the positive control mAb C1-13.
Target-specific bacteriophage Vs were successfully isolated after a series of immunization, demonstrating that phage display technology is a useful tool for selection of antigen binders. Generation of new binding reagents such as V antibodies that specifically recognize the malaria biomarkers represents an appealing approach to improve the performance of RDTs.
疟疾快速诊断检测（RDT）是一种重要的基于抗体的免疫分析平台。不幸的是，传统的单克隆抗体容易降解，从而缩短了 RDT 的保质期。鲨鱼的受体（V）可变区由于具有高热稳定性，因此有可能替代 RDT 中的单克隆抗体。
本研究中，研究了来自鲨鱼 V 结构域文库的新型结合物。在用三种重组疟疾生物标志物蛋白（PfHRP2、PfPldh 和 PvAldolase）对皱唇鲨（Orectolobus ornatus）进行免疫后，从脾细胞中构建了单域抗体（sdAb）文库。通过淘选分离出针对靶标的 V 噬菌体。选择一个特异性克隆进行在大肠杆菌表达系统中的表达，并进行结合反应性研究。
初级 V 结构域文库的效价为 1.16×10 pfu/mL。DNA 序列分析表明，82.5%的分离片段似乎含有一个框内序列。经过多轮生物淘选，鉴定出一种针对 PfHRP2 的高度优势克隆，并选择其在大肠杆菌表达系统中进行蛋白生产。生物学特性研究表明，表达在周质中的重组蛋白比细胞质蛋白具有更好的检测灵敏度。结合活性测定表明，其反应性低于阳性对照 mAb C1-13。
经过一系列免疫，成功分离出针对靶标的噬菌体 V，表明噬菌体展示技术是筛选抗原结合物的有用工具。生成专门识别疟疾生物标志物的新型结合试剂，如 V 抗体，代表了改善 RDT 性能的一种有吸引力的方法。
