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262 OD•mL/ng) and 25-fold decrease in the LOD for sEH (0.03 ng/mL). The streptavidin bridged double nanobody ELISA was then applied successfully to tests for recovery, cross-reactivity, and real samples. Meanwhile, we accidentally found that blocking with skim milk could severely damage the performance of the capture nanobody by an order of magnitude, compared to BSA. This work provides guideline to retain the high effectiveness of capture nanobody and thus to further develop double nanobody ELISA for various analytes.Plasmonic nanopartic