Abstract
A histidine (His)-tag is composed of six His residues and typically exerts little influence on the structure and solubility of expressed recombinant fusion proteins. Purification methods for recombinant proteins containing His-tags are relatively well-established, thus His-tags are widely used in protein recombination technology. We established a one-step enzyme-linked immunosorbent assay (ELISA) for His-tagged recombinant proteins. We analyzed variable heavy and light chains of the anti-His-tag monoclonal antibody 4C9 and used BLAST analyses to determine variable zones in light (VL) and heavy chains (VH). VH, VL, and alkaline phosphatase (ALP) regions were connected via a linker sequence and ligated into the pGEX-4T-1 expression vector. Different recombinant proteins with His tags were used to evaluate and detect ALP-scFv activity. Antigen and anti-His-scFv-ALP concentrations for direct ELISA were optimized using the checkerboard method. ZIKV-NS1, CHIKV-E2, SCRV-N, and other His-tag fusion proteins demonstrated specific reactions with anti-His-scFv-ALP, which were accurate and reproducible when the antigen concentration was 50 µg mL(-1) and the antibody concentration was 6.25 µg mL(-1). For competitive ELISA, we observed a good linear relationship when coating concentrations of recombinant human anti-Müllerian hormone (hAMH) were between 0.78 and 12.5 µg mL(-1). Our direct ELISA method is simple, rapid, and accurate. The scFv antibody can be purified using a prokaryotic expression system, which provides uniform product quality and reduces variations between batches.