E. coli -produced nanobody fusions as a pan-serotype platform for AAV purification, quantification, and detection

Research, production and application of the recombinant adeno-associated virus (rAAV) require versatile tools for rapid and cost-effective advances. We developed fusion constructs with the pan-serotype, AAV-binding VHH-antibody-fragment named nanobody AAVX and devised procedures for AAV purification, quantification, and detection. Starting from an amino acid sequence, we cytosolically expressed in E. coli nanobody AAVX carrying various tags and fusion partners. Very efficient soluble production followed by a one-step immobilized metal-ion affinity chromatography (IMAC) yielded mostly >90% pure protein. Crosslinking AAVX with an agarose matrix enabled efficient purification of intact AAV2 and AAV5 capsids from cell lysates, as assessed by qPCR and ELISA. AAVX also supported effective AAV quantification by ELISA, as capture reagent and, when fused to luciferase, as detection reagent. Using tagged AAVX and streptavidin biosensors, we developed a rapid and label-free quantification assay of AAV capsids by biolayer interferometry (BLI). Finally, immunostaining with an AAVX-eGFP fusion visualized AAV capsids in fixed HEK-293 cells by fluorescence microscopy. Collectively, these results establish nanobody AAVX fusion proteins as versatile and readily customizable tools for AAV research and bioprocessing.