Abstract
BackgroundMalaria 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 (VNAR) from shark has a potential as alternative to monoclonal antibodies in RDTs due to high thermal stability.MethodsIn this study, new binders derived from shark VNAR 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 VNAR phage were isolated by panning. One specific clone was selected for expression in Escherichia coli expression system, and study of binding reactivity undertaken.ResultsThe primary VNAR domain library possessed a titre of 1.16 × 106 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.ConclusionsTarget-specific bacteriophage VNARs 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 VNAR antibodies that specifically recognize the malaria biomarkers represents an appealing approach to improve the performance of RDTs.
Highlights
Malaria rapid diagnostic tests (RDTs) represent an important antibody based immunoassay platform
Similar to VHH in the camelid family, variable domain of new antigen receptor (VNAR) domains can function as soluble single domains which are capable of antigen binding [15]
Immunization of wobbegong shark and immunoglobulin new antigen receptor (IgNAR) ELISA screening A wobbegong shark was immunized with three malaria antigens, rPfHRP2, rPfLDH, and rPvAldolase
Summary
Malaria rapid diagnostic tests (RDTs) represent an important antibody based immunoassay platform. Conventional monoclonal antibodies are subject to degradation shortening shelf lives of RDTs. The variable region of the receptor (VNAR) from shark has a potential as alternative to monoclonal antibodies in RDTs due to high thermal stability. Most of the P. falciparum-detecting rapid diagnostic tests (RDTs) target histidine-rich protein 2 (PfHRP2) [2]. Similar to VHH in the camelid family, VNAR domains can function as soluble single domains which are capable of antigen binding [15]. These single domain fragments display excellent solubility and high thermostability due to substitutions of amino acids at VH–VL interaction, making the interface more hydrophilic compared to the hydrophobic interface present in conventional antibodies [14, 16]
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