Molecular evolution of single chain fragment variable (scFv) for diagnosis of lymphatic filariasis
Endemic countries with lymphatic filariasis are striving towards the Global Program to Eliminate Lymphatic Filariasis (GPELF) by 2020. Efficient and cost-effective diagnostic tools to assess active filarial infection are critical to eradicate lymphatic filariasis. Detection of circulating filarial antigens in sera is one of the precise methods to identify this infection. Monoclonal antibodies and single chain fragment variable (scFv) against Wuchereria bancrofti antigen SXP1 have been developed for antigen detection. Molecular cloning of scFv for recombinant expression has laid a platform for developing novel genetic constructs with enhanced reactivity. In this study, a simple procedure is developed to create diverse libraries of scFv based on a single DNA framework with all the requisites for an in vitro protein synthesis and ribosomal display. Error Prone-PCR was performed to incorporate random mutations and screened by ribosome display technique to isolate evolved scFv. Evolved scFv with six mutations showed tenfold increase in affinity compared to wild-type scFv for rWbSXP1. In silico studies showed that four mutations introduced unique molecular interactions between the evolved scFv and SXP1. Reactivity with asserted clinical samples of endemic normals (EN), microfilariaemic (MF), chronic pathology (CP) and non-endemic normals (NEN) showed significant augment (59.69%, p < 0.0001) in reactivity to MF samples with evolved scFv in comparison to wild-type scFv. Sensitivity of scFv was increased from 15.62 ng to 195 pg by evolved scFv in serum samples. This evolutionary method coupled with ribosome display has facilitated us to improve the reactivity of the ScFv without diminishing the specificity.
KeywordsscFv Detection Lymphatic filariasis Ribosome display
We acknowledge the support from DBT-BUILDER Programme (BT/PR12153/INF/22/200/2014) and Indian Council for Medical Research for Emeritus Medical Scientist award to Dr. P. Kaliraj, Centre for Biotechnology, Anna University, Chennai. We are grateful to Ms. Naveena Kandasamy for her valuable inputs in computational analysis.
SM and PK conceived and supervised the study; NM and RR designed the experiments; NM and RK performed the experiments; NM and NS analysed data; NM and NS wrote the manuscript; RR, PK and SM corrected the manuscript.
Compliance with ethical standards
Conflict of interest
All the authors declared that they have no conflict of interest.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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