4.7 Article

Development of a sandwich ELISA for the specific quantitation of hemagglutinin (HA)-tagged proteins during their inducible expression in Escherichia coli

Journal

ANALYTICAL AND BIOANALYTICAL CHEMISTRY
Volume 415, Issue 23, Pages 5563-5574

Publisher

SPRINGER HEIDELBERG
DOI: 10.1007/s00216-023-04846-w

Keywords

Nanobody; VHH; Protein expression; Sandwich ELISA

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A simple, sensitive, and low-cost sandwich ELISA method was developed to quantify heavy single-chain antibodies (VHHs). The method had a sensitivity of 0.149 OD·mL/ng and a limit of detection of 0.029 ng/mL. The method was applied to detect high levels of VHHs in bacterial cell media, raising attention to protein losses in cell culture supernatants and providing a continuous detection method for optimizing expression and purification protocols for nanobodies.
Heavy single-chain antibodies (VHH or nanobodies) are popular in the medical and analytical fields due to its small size, high solubility, stability, and other advantageous features. However, the usage of VHHs is limited by the low yield of its production and purification. In order to determine the optimal purification strategy for VHH to improve the yield, a method to monitor purification at the intermediate steps is needed. In this study, a simple, sensitive, low-cost sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantitate VHHs throughout the purification steps. Under optimized conditions, the assay has a sensitivity of 0.149 OD center dot mL/ng and a limit of detection (LOD) of 0.029 ng/mL. The average recoveries of the assay against the spiked samples were 101.9-106.0% and 100.7-108.0%. The method was applied to a variety of real samples for the detection of different VHHs in bacterial cell media. High amount of VHHs (up to 41.3 mg/ mL), which are comparable to the average yield of VHH in standard production protocols, were detected in the media. This study raises attention to the problem of protein losses in cell culture supernatants and provides a method for the continuous detection of the protein abundance to optimize the expression and purification protocols especially for nanobodies.

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