Isolation and characterization of malaria PfHRP2 specific VNAR antibody fragments from immunized shark phage display library

• Published in: Malaria journal Vol. 17; no. 1; p. 383
• Main Authors: Leow, Chiuan Herng, Fischer, Katja, Leow, Chiuan Yee, Braet, Katleen, Cheng, Qin, McCarthy, James
• Format: Journal Article
• Language: English
• Published: London BioMed Central Ltd 24.10.2018; BioMed Central; BMC
• Subjects: Immunization; Malaria; Medical tests; Monoclonal antibodies
• ISSN: 1475-2875; 1475-2875
• DOI: 10.1186/s12936-018-2531-y

Background 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.sub.NAR) from shark has a potential as alternative to monoclonal antibodies in RDTs due to high thermal stability. Methods In this study, new binders derived from shark V.sub.NAR 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.sub.NAR phage were isolated by panning. One specific clone was selected for expression in Escherichia coli expression system, and study of binding reactivity undertaken. Results The primary V.sub.NAR domain library possessed a titre of 1.16 x 10.sup.6 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. Conclusions Target-specific bacteriophage V.sub.NARs 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.sub.NAR antibodies that specifically recognize the malaria biomarkers represents an appealing approach to improve the performance of RDTs.