Detection of a Geographically Diverse Malarial Biomarker via Multi-Epitope Targeted Screening

We report on the development of high affinity macrocyclic peptides as ligands for the capture and detection of PfHRP2, a protein without a defined tertiary structure. PfHRP2 is a key biomarker of the fatal falciparum strain of malaria. This intrinsically disordered protein has repeated epitopes that have inconstant occurrence over geographic regions. Thus, current antibody based diagnostic tests that target a single epitope do not account for the sequence diversity of PfHRP2 and exhibit variable performance. Ligands selected via high throughput epitope-targeted in situ click screening with combinatorial macrocylic peptide libraries yield binders with high affinity and selectivity for their protein targets. These macrocyclic peptide binders offer greater biochemical and thermal stability than antibodies. We provide a general strategy for the amplification of sensitivity in disease detection through the development of ligands that target multiple epitopes in a single and unstructured protein biomarker. We apply our macrocyclic peptide ligands towards the development of an antibody free diagnostic test for PfHRP2.