Poly-protein G-expressing bacteria enhance the sensitivity of immunoassays

Sci Rep. 2017 Apr 20;7(1):989. doi: 10.1038/s41598-017-01022-w.

Abstract

The sensitivities of solid-phase immunoassays are limited by the quantity of detection antibodies bound to their antigens on the solid phase. Here, we developed a poly-protein G-expressing bacterium as an antibody-trapping microparticle to enhance the signals of immunoassays by increasing the accumulation of detection antibodies on the given antigen. Eight tandemly repeated fragment crystallisable (Fc) binding domains of protein G were stably expressed on the surface of Escherichia coli BL21 cells (termed BL21/8G). BL21/8G cells showed a higher avidity for trapping antibodies on their surface than monomeric protein G-expressing BL21 (BL21/1G) cells did. In the sandwich enzyme-linked immunosorbent assay (ELISA), simply mixing the detection antibody with BL21/8G provided a detection limit of 6 pg/mL for human interferon-α (IFN-α) and a limit of 30 pg/mL for polyethylene glycol (PEG)-conjugated IFN-α (Pegasys), which are better than that of the traditional ELISA (30 pg/mL for IFN-α and 100 pg/mL for Pegasys). Moreover, the sensitivity of the Western blot for low-abundance Pegasys (0.4 ng/well) was increased by 25 folds upon mixing of an anti-PEG antibody with BL21/8G cells. By simply being mixed with a detection antibody, the poly-protein G-expressing bacteria can provide a new method to sensitively detect low-abundance target molecules in solid-phase immunoassays.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / metabolism*
  • Antigens, Bacterial / chemistry
  • Antigens, Bacterial / genetics*
  • Antigens, Bacterial / immunology
  • Binding Sites
  • Enhancer Elements, Genetic
  • Enzyme-Linked Immunosorbent Assay
  • Escherichia coli / genetics
  • Escherichia coli / growth & development*
  • Escherichia coli / metabolism
  • Humans
  • Interferon-gamma / immunology
  • Limit of Detection

Substances

  • Antibodies, Monoclonal
  • Antigens, Bacterial
  • IFNG protein, human
  • Interferon-gamma