Activity-based assessment of an engineered hyperthermophilic protein as a capture agent in paper-based diagnostic tests

Abstract

Antibodies have traditionally served as the affinity reagents of choice in point-of-care diagnostic biosensors. However, this class of proteins is not ideally suited for this use, being poorly characterized and prone to thermal denaturation. Here, we present an activity-based assessment of an alternative engineered binding protein in a cellulose-based assay.

Functional comparison of paper-based immunoassays based on antibodies and engineered binding proteins

“…1A). We tested a range of ZNS1 concentrations in buffer (PBSA: 1× PBS with 1% bovine serum albumin) by incubating 10 μL of the samples for 30 minutes, following conditions established previously 27,[29][30][31] (Fig. 1B, yellow diamonds).…”

“…14,15 In recent years, studies have begun reporting the use of nonantibody scaffolds in full sandwich assays using pairs of nanobodies, [16][17][18][19] affimers, 20,21 DARPins, 22 and the reducedcharge Sso7d variant (rcSso7d). [23][24][25][26][27][28][29][30][31] However, a direct functional comparison of antibodies and non-antibody scaffolds in an identical full sandwich immunoassay test format has yet to be conducted.…”

Engineering hyperthermostable rcSso7d as reporter molecule forin vitrodiagnostic tests

“…We expressed all of the Sso.TB constructs in BL21(DE3) E. coli and purified the proteins using IMAC columns as previously described and as detailed further in the ESI. † 11,12 We quantified the protein concentrations using a bicinchoninic acid (BCA) assay and visualized purified proteins via SDS-PAGE to ensure product purity (Fig. 2).…”

“…Previous studies for diagnostic applications often used the alternate scaffold as the capture protein but used a reporter antibody [6][7][8][9] or a phage-displayed variant of the scaffold with a reporter antibody 9,10 to quantify target proteins in solution, or a biotinylated antigen to quantify capture efficiency and tolerance for thermal challenges. [11][12][13] Soluble reporter proteins were investigated with sdAb by chemically conjugating biotins, 6 genetically adding tags, 7,14,15 or fusing the scaffold to a reporter enzyme. [14][15][16] Although sdAb clones with high in vitro stability and excellent yields upon bacterial expression can be identified, these desirable properties are not universal, thus requiring additional screening during production of new clones.…”

“…The reduced-charged variant of Sso7d (rcSso7d)-a 7 kDa DNA-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus-fulfills our design criteria for an antibody replacement protein due to its intrinsic stability (wildtype melting temperature of 98°C; engineered variants retained their thermostability with melting temperatures >60°C and generally >90°C); facile and high-yield bacterial expression; isolated binding face that has demonstrated highaffinity binding to various target molecules; and ease of genetic modification (Table S1 †). 4,[9][10][11][12][13]17 Although studies have already demonstrated the use of rcSso7d as a soluble capture molecule, 9,[11][12][13] analogous studies have not yet been reported for the use of soluble rcSso7d as the reporter molecule. Further engineering is required to enable the protein to associate a detectable signal with the presence of a target antigen.…”