Abstract
The streptavidin-biotin interaction has long been a cornerstone of immunoassays, valued for its exceptionally high affinity and specificity, and has been widely used in routine diagnostic assays for decades. However, elevated biotin levels in plasma, often resulting from oral supplements, can interfere with streptavidin-biotin binding, compromising signal detection in these assays. To address this limitation, we present a biotin-insensitive antigen enrichment system based on the ultra-strong binding pair barnase (Bn) and barstar (Bs) for effective antigen capture. This high-affinity system enables efficient detection of nanobody-antigen complexes with picomolar sensitivity, as demonstrated by the detection of carcinoembryonic antigen (CEA, CEACAM5). When paired with a fluorescent secondary nanobody reporter, the system achieved a detection limit as low as 98.9 pM, with a limit of detection (LOD) of 17.8 ng/mL, representing a four-order-of-magnitude improvement in sensitivity compared to light interferometry-based binding analysis. Both Bn and Bs are small (<15 kDa), single-chain proteins that can be generically incorporated into immunoactive molecules. Compared to the traditional biotin-streptavidin system, the Bn-Bs system offers enhanced specificity, improved stability, and reduced cost and complexity.
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Abstract
The streptavidin-biotin interaction has long been a cornerstone of immunoassays, valued for its exceptionally high affinity and specificity, and has been widely used in routine diagnostic assays for decades. However, elevated biotin levels in plasma, often resulting from oral supplements, can interfere with streptavidin-biotin binding, compromising signal detection in these assays. To address this limitation, we present a biotin-insensitive antigen enrichment system based on the ultra-strong binding pair barnase (Bn) and barstar (Bs) for effective antigen capture. This high-affinity system enables efficient detection of nanobody-antigen complexes with picomolar sensitivity, as demonstrated by the detection of carcinoembryonic antigen (CEA, CEACAM5). When paired with a fluorescent secondary nanobody reporter, the system achieved a detection limit as low as 98.9 pM, with a limit of detection (LOD) of 17.8 ng/mL, representing a four-order-of-magnitude improvement in sensitivity compared to light interferometry-based binding analysis. Both Bn and Bs are small (<15 kDa), single-chain proteins that can be generically incorporated into immunoactive molecules. Compared to the traditional biotin-streptavidin system, the Bn-Bs system offers enhanced specificity, improved stability, and reduced cost and complexity.
Competing Interest Statement
C.L. and L.H. have filed a patent CN113721023 (A) for this technology.
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