Identification of a nanobody specific to a heart failure biomarker, B-type natriuretic peptide, from a synthetic library using a combination of yeast surface display and FLI-TRAP methods

Heart failure (HF) is both a symptom and a sign of an abnormal heart, either in structure or function. In 2013, there were 5.1 million HF patients and treatment incurred a direct cost of $32 billion. Predictions indicate that by 2030, this cost is expected to triple. N-terminal Pro-B-type Natriuretic Peptide (NT-proBNP) is one of the biomarkers proposed for the diagnosis of HF, as it is produced in response to myocardial stretching. Current diagnostic kits for HF using specific biomarkers typically involve costly monoclonal or polyclonal antibodies, requiring mammalian cell cultivation. This study aims to isolate nanobodies, a form of the variable domain of heavy-chain-only antibodies found in the serum of the Camelidae family, that are specific to NT-proBNP and could be used to develop an affordable HF detection kit. The study employed Yeast Surface Display (YSD) and the Functional Ligand-binding Identification by Tat-based Recognition of Associating Proteins (FLI-TRAP) techniques to isolate nanobodies (Nb) specific to NT-proBNP. After FLI-TRAP selection, 40 Nb candidates were isolated and subjected to DNA sequencing. Seven unique sequences were identified, and they were confirmed to grow at a single colony level by spot plating, suggesting successful Tat export of the antibody-antigen complex. Nb6, the only candidate that only showed cell resistance in the presence of the inducer, was selected for other in vivo and in vitro analysis, which helped demonstrating its specific binding to NT-proBNP.