Affinity maturation of anti-NT-proBNP single-chain variable fragment (scFv) and biophysical characterization for heart failure biosensor application

Affinity maturation of anti-NT-proBNP single-chain variable fragment (scFv) and biophysical characterization for heart failure biosensor application | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 16 September 2025 V1 Latest version Share on Affinity maturation of anti-NT-proBNP single-chain variable fragment (scFv) and biophysical characterization for heart failure biosensor application Authors : Kanchana Monkhang , Jarumas Jitwasinkul , Lueacha Tabtimmai , Kiattawee Choowongkomon , and Napachanok Mongkoldhumrongkul Swainson 0000-0001-6005-4316 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175802243.32591328/v1 186 views 154 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Rapid, specific detection of N-terminal pro-B-type natriuretic peptide (NT-proBNP) enhances heart-failure diagnosis. An NT-proBNP–binding single-chain variable fragment (scFv) bearing a gold-assembling linker (C12AP) was engineered and affinity-matured to serve as a biosensor recognition element. Docking of C12AP–NT-proBNP complexes guided semi-rational mutagenesis at D110 (VH-CDR3) and Y174 (VL-CDR1). Twenty-two variants were expressed and screened by ELISA against NT-proBNP with human serum albumin as a specificity control and C12AP for normalization. Eight candidates were purified for quantitative analyses. Microscale thermophoresis (MST) identified variant D110-57 as significantly improved over C12AP ( p = 0.0174). Docking predicted a more favorable binding energy for D110-57 (−254 kcal·mol -1 ) versus C12AP (−209.6 kcal·mol -1 ), with additional contacts in VH-CDR2 and VL-CDR3. To emulate biosensor conditions, surface plasmon resonance imaging (SPRi) employed a self-assembling histidine/serine-rich linker and a C-terminal His tag to orient scFvs on gold. D110-57 exhibited concentration-dependent binding with concordance between steady-state and kinetic analyses (affinity K D = 5.167 nM; kinetic K D = 6.5 nM; k a = 5.396 × 10^6 M -1 ·s -1 ; k d = 0.0351 s -1 ). Specificity was retained, with minimal binding to A- and C-type natriuretic peptides. Overall, D110-57 shows enhanced affinity and specificity and is compatible with gold-surface immobilization, supporting application in NT-proBNP point-of-care biosensors. Supplementary Material File (manuscript_nms_biotechnology journal.docx) Download 2.79 MB Information & Authors Information Version history V1 Version 1 16 September 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords affinity maturation biochemical engineering biomolecular engineering biosensors nt-probnp protein expression protein purification single-chain variable fragment (scfv) synthetic biology Authors Affiliations Kanchana Monkhang Kasetsart University Department of Biochemistry View all articles by this author Jarumas Jitwasinkul King Mongkut's University of Technology North Bangkok Faculty of Applied Science View all articles by this author Lueacha Tabtimmai King Mongkut's University of Technology North Bangkok Faculty of Applied Science View all articles by this author Kiattawee Choowongkomon Kasetsart University Department of Biochemistry View all articles by this author Napachanok Mongkoldhumrongkul Swainson 0000-0001-6005-4316 [email protected] Kasetsart University Department of Biochemistry View all articles by this author Metrics & Citations Metrics Article Usage 186 views 154 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Kanchana Monkhang, Jarumas Jitwasinkul, Lueacha Tabtimmai, et al. Affinity maturation of anti-NT-proBNP single-chain variable fragment (scFv) and biophysical characterization for heart failure biosensor application. Authorea . 16 September 2025. DOI: https://doi.org/10.22541/au.175802243.32591328/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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