Technical NoteAn iBody-based lateral flow assay for semi-quantitative determination of His-tagged protein concentration
Introduction
Since its introduction in 1988 (Hochuli et al., 1988), the polyhistidine tag (His-tag) has become one of the most commonly used epitope tags for production and purification of recombinant proteins. His-tag commonly consists of six to ten consecutive histidine residues, which can be fused to accessible regions of proteins to facilitate their purification and detection. Polyhistidine peptides bind bivalent metal ions, and the most commonly used for protein purification are Ni2+ and Co2+ immobilized with chelation agents such as nitrilotriacetic acid (NTA, Hochuli et al., 1987) and its multivalent derivatives (Lata et al., 2005). In our previous work, we demonstrated that an iBody polymer conjugated with TrisNTA-targeting ligands can be used to detect His-tagged proteins in complex biological matrices (Sacha et al., 2016).
Due to the common use of this tag in recombinant protein expression, various techniques have been employed for detection and quantitation of His-tagged proteins. In addition to accurate but time- and equipment-intensive methods such as ELISA and Western blot, there is a need for rapid methods for preliminary identification and relative quantitation of His-tagged proteins. To address this need, we present a rapid lateral flow assay for detection of His-tagged proteins based on polymer antibody-mimetics.
Section snippets
Reagents
Polyvinyl alcohol (PVA, 8148940101) was purchased from Merck Millipore (Burlington, MA, USA). Tween-20 (20605) and PEG-8000 (19966) were purchased from Affymetrix (Santa Clara, CA, USA). The other reagents used in this work were purchased from Sigma (St. Louis, MO, USA). The proteins used for assay validation are listed in Table 1.
Gold nanoparticle stock solution was kindly supplied by Jitka Neburková. Colloidal gold was prepared according to the procedure described by (Frens, 1973). Briefly,
Assay optimization
The first step in assay design was to determine the optimal pH for iBody-colloid conjugation. The performance of the conjugate was verified in a half-strip format (direct application of conjugate onto a test strip lacking a sample pad). Conjugate performance was consistent across the pH range 6.5–9.1, with a carbonate buffer system at pH 8.6 being only marginally better than other conditions tested. The polymer concentration in the conjugation reaction was also optimized using the half-strip
Conclusion
In the present study, a lateral flow assay based on iBody antibody mimetics was developed, optimized, and shown to have sensitivity suitable for preliminary protein concentration estimation and qualitative testing. The test is able to detect multiple His-tagged proteins with different tag attachment strategies with a visual detection limit of 1 μM and densitometric detection limit of 0.5 μM.
Our test was compared with two commercially available antibody-based assays in terms of reagent
Acknowledgment
The authors would like to thank Jitka Neburková for preparation of stock gold nanoparticle solution, Jakub Staníček for supplying recombinant His-tagged proteins for testing, as well as Vladimír Šubr and Libor Kostka for preparation of anti-His-tag polymer conjugate. The study was supported by the Czech Science Foundation (No. 19-10280S), by Ministry of Industry and Trade of the Czech Republic (No. FV10490) and by the European Regional Development Fund; OP RDE (No.
Declaration of Competing Interest
The authors declare no competing financial interest.
References (11)
- et al.
New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues
J. Chromatogr. A
(1987) His-Tag Check&Go! Protein Expression and Purification
(2018)Pro-Detect™ Rapid His Competitive Assay Kit
Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions
Nat. Phys. Sci.
(1973)Bioconjugate Techniques
(1996)