Sensitive immunoassay of Legionella using multivalent conjugates of engineered VHHs

Abstract

VHH antibodies or nanobodies, which are antigen-binding domains of heavy chain antibodies from camelid species, have several advantageous characteristics, including compact molecular size, high productibility in bacteria and easy engineering for functional improvement. Focusing on these advantages of VHHs, we attempted to establish an immunoassay system for detection of Legionella, the causative pathogen of Legionnaires' disease. A VHH phage display library was constructed using cDNA from B cells of alpacas immunized with Legionella pneumophila serogroup1 (LpSG1). Through biopanning, two specific VHH clones were isolated and used to construct a Legionella detection system based on the latex agglutination assay. After engineering the VHHs and improving the assay system, the sensitive detection system was successfully established for the LpSG1 antigen. The immunoassay developed in this study should be useful in easy and sensitive detection of Legionella, the causative agent of Legionnaires' disease, which is a potentially fatal pneumonia.

Keywords: VHH.Abbreviations: Abs, antibodies; BSA, bovine serum albumin; CDR, complementarity determining region; CFU, colony forming unit; DBCO, dibenzylcyclooctyne; ELISA, enzyme-linked immunosorbent assay; FR, framework region; HcAbs, heavy chain antibodies; KLH, keyhole limpet hemocyanin; LpSG1, Legionella pneumophila serogroup1; MALDI-TOFMS, matrix assisted laser desorption/ionization time of flight mass spectrometry; NHS, N-hydroxysuccinimide; PBMC, peripheral blood mononuclear cells; PCR, polymerase chain reaction; RT-PCR, reverse transcription PCR; SDS-PAGE, sodium do-decyl sulphate-polyacrylamide gel electrophoresis; TMB, 3,3′,5,5′-tetramethylbenzidine solution; alpaca; antibody; engineering; immunoassay.