Flow cytometric immunoassay using E. coli with autodisplayed Z-domains

Ultrasonic isolation of the outer membrane of Escherichia coli with autodisplayed Z-domains

“…When antibodies were applied to the outer membrane layer on the solid supports, the F c region of the antibodies bound to the autodisplayed Z-domains, and the F c -bound antibodies developed an active orientation to bind antigens [21,22,11]. This control of the orientation of antibodies through the autodisplayed Z-domains has been reported to improve the sensitivity of immunoassays [3][4][5][6][7][8][9][10][11][12].…”

“…The flow cytometry was conducted as previously described [10][11][12]. After the freshly prepared E. coli was washed three times with PBS, the E. coli was resuspended in PBS/3% fetal calf serum to a final OD600nm of 1.0 at a wavelength of 600 nm.…”

“…The antibodies that bound to the Z-domains always had an active orientation that could bind antigens because the F ab region was exposed to antigens; thus, the sensitivity of immunoassays with orientation-controlled antibodies was reported to be greatly improved compared with that of the immunoassays that used randomly oriented antibodies [6]. The entire E. coli cell with autodisplayed Z-domains could be used for immunoassays with various assay configurations, such as the sandwich-format assay on a microplate [7][8][9] and FACS-based assays [10][11][12]. Another configuration of immunoassays was also reported where only the outer membrane of E. coli was isolated, and the membrane was then layered on solid supports for immunoassays, such as those on microplates [4], the metal surface of SPR biosensors [5] and microbeads [13].…”

A magnetite suspension-based washing method for immunoassays using Escherichia coli cells with autodisplayed Z-domains

“…Each bead, which has a diameter of 3-10 m, binds to several E. coli cells [15,16]. Although compared with centrifugation, magnetic separation decreases the loss of E. coli cells during washing steps in immunoassays, cells are still lost owing to their weak electrostatic interactions with the beads.…”

“…1 These authors contributed equally to this study E. coli cells with autodisplayed Z-domains are used for immunoassays after the immobilization of IgGs. Because immunoassays exploit the highly specific interactions between IgGs and target analytes (antigens), the nonspecific binding of other proteins must be avoided through effective washing processes with detergents such as Tween 20 and sodium dodecyl sulfate [12,13]. Immunoassays incorporating E. coli cells with autodisplayed Z-domains have been performed with either of the following washing steps: (1) centrifugation of E. coli cells to a pellet [14], or (2) magnetic separation of E. coli cells via immobilization on large superparamagnetic beads [15][16][17][18][19][20].…”