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.
Highlights
The development of rapid, accurate and low cost diagnostics for point-of-care applications in developing countries is a major challenge that needs to be overcome if the morbidity and mortality from diseases is to be reduced in these countries
We have developed in particular a model system based around the 12Y-2 family of variable new antigen receptor domain antibody fragments (VNARs) that target the apical membrane antigen 1 (AMA-1) of malarial (Plasmodium falciparum) parasites [5]
Our results are clear evidence of how the activity of monoclonal antibodies (mAbs) onto nitrocellulose membranes can be impaired by temperatures commonly found in endemic areas (>40 °C), and that VNARs are an attractive option for the improvement of rapid diagnostic tests (RDTs) for malaria
Summary
The development of rapid, accurate and low cost diagnostics for point-of-care applications in developing countries is a major challenge that needs to be overcome if the morbidity and mortality from diseases is to be reduced in these countries. Monoclonal antibodies (mAbs) have a variety of features that make them well suited to this task, such as high specificity and high affinity for the target antigen Despite these favorable characteristics, diagnostic tests based upon such antibodies may be adversely affected by high temperatures or by storage for extended periods under conditions of high humidity, when the test must be conducted in developing countries where constant access to refrigeration is not always practicable [1]. We hypothesized that individual VNAR domains would display high stability under conditions that would otherwise compromise the integrity of traditional monoclonal antibodies Such stability, if demonstrable as a consistent feature of these binding reagents, would be advantageous for use in diagnostic assays where the reagents are likely to be exposed to extreme conditions. We have identified a set of conditions which allows one VNAR to retain significant activity for up to 4 weeks at 50 °C
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