An automated Dengue virus microneutralization plaque assay performed in human Fc{gamma} receptor-expressing CV-1 cells.
We describe microneutralization assays that used automated 96-well enzyme-linked immunospot (ELI-SPOT) readout instrumentation to measure human anti-dengue virus (DENV) antibodies in CV-1 cells that were stably transfected to express human FcgammaRIIA (CD32) using conventional Vero cells as a comparator. Classic plaque reduction neutralization test (PRNT) end-point titers were determined by probit analysis. Neutralization titers against DENV measured in CV-1 transfectants were expressed in terms of both conventional 50% to 90% PRNT end-point titers and differential infectivity of antibody-treated virus in control and CD32-expressing CV-1 cells. Significantly reduced PRNT titers and strikingly heightened infectivity (up to 100-fold) of antibody-treated DENV was observed in CV-1 CD32 transfectants compared with that observed in control CV-1 or Vero cells. Because DENVs may preferentially replicate in CD32-expressing monocytes/macrophages and dendritic cells, in vivo, it is possible that CD32 introduced into a conventional DENV neutralization assay might provide results that better correlate with protection.
Rodrigo
W W Shanaka I
WW
Departments of Pathology and Laboratory Medicine, Microbiology and Immunology, and Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Alcena
Danielle C
DC
Rose
Robert C
RC
Jin
Xia
X
Schlesinger
Jacob J
JJ
D000328:Adult; D000330:Aedes; D000818:Animals; D001331:Automation; D002522:Chlorocebus aethiops; D003716:Dengue Virus / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D009500:Neutralization Tests; D017452:Receptors, IgG / Q000032:analysis; D014709:Vero Cells; D010948:Viral Plaque Assay
C080568:Fc gamma receptor IIA; D017452:Receptors, IgG