In this study, sandwich chemiluminescent immunoassay (CLIA) for the detection of Staphylococcal enterotoxin B (SEB) was developed using nanobody-alkaline phosphatase (Nb-ALP) fusion protein. The SEB-binding nanobodies were obtained from a naïve phage-display library and the Nb-ALP fusion protein was constructed and obtained as a thermally stable and potentially effective substance for detecting antibodies in CLIA. The working range of the sandwich CLIA based on anti-SEB monoclonal antibodies (mAbs) and our fusion protein, Nb37-ALP, was 3.12-50.0 ng mL-1 with SC50 = 8.59 ± 0.37 ng mL-1. The limit of detection was 1.44 ng mL-1 according to the blank value plus 3 standard deviations. In order to understand the interaction of SEB and Nb37 in depth, the 3D structure of the SEB-Nb37 complex was constructed and verified by molecular modeling and the docking method. The results showed that the complementary-determining region 3 (CDR3) of Nb37 embedded itself in the opening generated by the major histocompatibility complex (MHC) and T-cell receptor- (TcR) binding sites of SEB, indicating that Nb37 may affect the recognition of SEB by MHC class Ⅱ molecules and the TcR. The arginine residue (Arg) 101, Arg102 and phenylalanine residue (Phe)103 of CDR3 in Nb37 may have contributed to specific binding to form six salt-bridges between these and SEB. In conclusion, in terms of their specificity and sensitivity, the obtained anti-SEB Nb-ALP appears to have the potential to replace chemically labeled probes for the detection of SEB.
Keywords: Alkaline phosphatase; Immunoassay; Nanobody; Staphylococcal enterotoxin B.
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