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Shark Variable New Antigen Receptor (VNAR) Single Domain Antibody Fragments: Stability and Diagnostic Applications
The Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Plenty Road, Bundoora, VIC., 3086, Australia
AdAlta Pty. Ltd., 15/2 Park Drive, Bundoora, VIC., 3086, Australia
The CSIRO Materials Science and Engineering, 343 Royal Parade, Parkville, VIC., 3052, Australia
The Foundation for Innovative New Diagnostics (FIND), Avenue de Budé 16, 1202 Geneva, Switzerland
* Author to whom correspondence should be addressed.
Received: 29 November 2012; in revised form: 9 January 2013 / Accepted: 21 January 2013 / Published: 25 January 2013
Abstract: The single variable new antigen receptor domain antibody fragments (VNARs) derived from shark immunoglobulin new antigen receptor antibodies (IgNARs) represent some of the smallest known immunoglobulin-based protein scaffolds. As single domains, they demonstrate favorable size and cryptic epitope recognition properties, making them attractive in diagnosis and therapy of numerous disease states. Here, we examine the stability of VNAR domains with a focus on a family of VNARs specific for apical membrane antigen 1 (AMA-1) from Plasmodium falciparum. The VNARs are compared to traditional monoclonal antibodies (mAbs) in liquid, lyophilized and immobilized nitrocellulose formats. When maintained in various formats at 45 °C, VNARs have improved stability compared to mAbs for periods of up to four weeks. Using circular dichroism spectroscopy we demonstrate that VNAR domains are able to refold following heating to 80 °C. We also demonstrate that VNAR domains are stable during incubation under potential in vivo conditions such as stomach acid, but not to the protease rich environment of murine stomach scrapings. Taken together, our results demonstrate the suitability of shark VNAR domains for various diagnostic platforms and related applications.
Keywords: single-domain antibody; shark VNAR; thermal stability; pH stability; protease resistance; AMA-1; Plasmodium; malaria; diagnosis
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Griffiths, K.; Dolezal, O.; Parisi, K.; Angerosa, J.; Dogovski, C.; Barraclough, M.; Sanalla, A.; Casey, J.L.; González, I.; Perugini, M.A.; Nuttall, S.; Foley, M. Shark Variable New Antigen Receptor (VNAR) Single Domain Antibody Fragments: Stability and Diagnostic Applications. Antibodies 2013, 2, 66-81.
Griffiths K, Dolezal O, Parisi K, Angerosa J, Dogovski C, Barraclough M, Sanalla A, Casey JL, González I, Perugini MA, Nuttall S, Foley M. Shark Variable New Antigen Receptor (VNAR) Single Domain Antibody Fragments: Stability and Diagnostic Applications. Antibodies. 2013; 2(1):66-81.
Griffiths, Katherine; Dolezal, Olan; Parisi, Kathy; Angerosa, Julie; Dogovski, Con; Barraclough, Miles; Sanalla, Abdulmonem; Casey, Joanne L.; González, Iveth; Perugini, Matthew A.; Nuttall, Stewart; Foley, Michael. 2013. "Shark Variable New Antigen Receptor (VNAR) Single Domain Antibody Fragments: Stability and Diagnostic Applications." Antibodies 2, no. 1: 66-81.