title: Development of a high-throughput assay to measure the neutralization capability of anti-cytomegalovirus antibodies.
abstract: Infection by human cytomegalovirus (CMV) elicits a strong humoral immune response and robust anti-CMV antibody production. Diagnosis of virus infection can be carried out by using a variety of serological assays; however, quantification of serum antibodies against CMV may not present an accurate measure of a patient's ability to control a virus infection. CMV strains that express green fluorescent protein (GFP) fusion proteins can be used as screening tools for evaluating characteristics of CMV infection in vitro. In this study, we employed a CMV virus strain, AD169, that ectopically expresses a yellow fluorescent protein (YFP) fused to the immediate-early 2 (IE2) protein product (AD169IE2-YFP) to quantify a CMV infection in human cells. We created a high-throughput cell-based assay that requires minimal amounts of material and provides a platform for rapid analysis of the initial phase of virus infection, including virus attachment, fusion, and immediate-early viral gene expression. The AD169IE2-YFP cell infection system was utilized to develop a neutralization assay with a monoclonal antibody against the viral surface glycoprotein gH. The high-throughput assay was extended to measure the neutralization capacity of serum from CMV-positive subjects. These findings describe a sensitive and specific assay for the quantification of a key immunological response that plays a role in limiting CMV dissemination and transmission. Collectively, we have demonstrated that a robust high-throughput infection assay can analyze the early steps of the CMV life cycle and quantify the potency of biological reagents to attenuate a virus infection.
authors: Mount Sinai School of Medicine, Department of Microbiology, New York, NY, USA.
authors: Thomas J
authors: TJ
authors: Gardner
authors: Cynthia
authors: C
authors: Bolovan-Fritts
authors: Melissa W
authors: MW
authors: Teng
authors: Veronika
authors: V
authors: Redmann
authors: Thomas A
authors: TA
authors: Kraus
authors: Rhoda
authors: R
authors: Sperling
authors: Thomas
authors: T
authors: Moran
authors: William
authors: W
authors: Britt
authors: Leor S
authors: LS
authors: Weinberger
authors: Domenico
authors: D
authors: Tortorella
mesh_terms: D057134:Antibodies, Neutralizing / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology*; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics / Q000276:immunology*; D003586:Cytomegalovirus Infections / Q000175:diagnosis* / Q000276:immunology*; D005260:Female; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D009500:Neutralization Tests / Q000379:methods; D011247:Pregnancy; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria
references: J Infect Dis. 1998 Nov;178(5):1233-43
references: 9780241
references: J Virol. 1998 Sep;72(9):7563-8
references: 9696854
references: Nat Methods. 2009 Aug;6(8):577-9
references: 19578384
references: Lancet. 1987 Oct 31;2(8566):996-9
references: 2889962
references: Prenat Diagn. 2004 Nov;24(11):861-3
references: 15565653
references: Blood. 2007 Aug 1;110(3):937-45
references: 17440050
references: J Clin Immunol. 2012 Apr;32(2):300-11
references: 22198680
references: DNA Seq. 1991;2(1):1-12
references: 1666311
references: Clin Diagn Lab Immunol. 1997 Jul;4(4):469-73
references: 9220166
references: J Virol. 2001 Feb;75(4):1870-8
references: 11160686
references: J Virol. 1991 Jul;65(7):3839-52
references: 1645794
references: Virology. 1988 Feb;162(2):478-82
references: 2829429
references: Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):12396-401
references: 14519856
references: J Virol. 1972 Oct;10(4):766-75
references: 4117347
references: J Infect Dis. 2001 Nov 15;184(10):1252-61
references: 11679913
references: Pediatr Infect Dis J. 1995 Jan;14(1):34-40
references: 7715987
references: J Virol. 2006 Jan;80(2):710-22
references: 16378974
references: J Med Virol. 2000 Mar;60(3):324-30
references: 10630965
references: Transplant Proc. 1993 Feb;25(1 Pt 2):1441-3
references: 8382875
references: J Med Virol. 1980;6(2):119-27
references: 6264020
references: J Virol. 1997 Feb;71(2):1246-55
references: 8995648
references: Cell. 2012 Dec 21;151(7):1569-80
references: 23260143
references: Expert Rev Vaccines. 2010 Nov;9(11):1303-14
references: 21087108
references: J Virol. 2008 Oct;82(19):9525-36
references: 18653451
references: J Virol. 2005 Jul;79(13):8361-73
references: 15956581
references: J Virol. 1993 Jan;67(1):489-96
references: 7677958
references: J Virol. 2000 Jul;74(13):6132-46
references: 10846097
references: J Virol. 1990 Mar;64(3):1079-85
references: 2154594
references: J Virol Methods. 2008 Jul;151(1):66-73
references: 18490063
references: J Infect Dis. 1997 Apr;175(4):944-6
references: 9086155
references: Viral Immunol. 2000;13(3):329-41
references: 11016597
references: J Virol. 1976 Jul;19(1):243-54
references: 181595
references: Nature. 2007 May 17;447(7142):326-9
references: 17507983
references: J Virol. 2002 Jan;76(2):928-32
references: 11752183
references: Clin Transplant. 1997 Oct;11(5 Pt 1):432-7
references: 9361936
references: J Virol. 2002 Mar;76(5):2316-28
references: 11836410
references: J Virol. 2006 Oct;80(20):9998-10009
references: 17005678
references: N Engl J Med. 1992 Mar 5;326(10):663-7
references: 1310525
references: Cell Host Microbe. 2008 Apr 17;3(4):253-62
references: 18407068
references: J Virol. 2005 Aug;79(16):10330-8
references: 16051825
references: J Intern Med. 2006 Mar;259(3):219-46
references: 16476101
references: PLoS One. 2010 Feb 11;5(2):e9174
references: 20161802
references: Nucleic Acids Res. 2005 Feb 24;33(4):e36
references: 15731329
references: J Immunol. 1986 Oct 1;137(7):2299-306
references: 3760563
references: Vaccine. 2011 Mar 24;29(15):2705-11
references: 21310190
references: J Virol. 2004 Sep;78(18):10023-33
references: 15331735
references: Virology. 1975 Aug;66(2):464-73
references: 168685
references: Virology. 1983 Oct 15;130(1):118-33
references: 6314643
references: Mol Immunol. 2012 Jun;51(2):245-53
references: 22497807
references: Antiviral Res. 2011 Mar;89(3):246-56
references: 21277329
references: J Virol. 2004 Feb;78(4):1817-30
references: 14747546
references: J Gen Virol. 2008 Apr;89(Pt 4):853-865
references: 18343825
references: Clin Vaccine Immunol. 2011 Sep;18(9):1536-42
references: 21795465
references: J Virol. 1989 Mar;63(3):1435-40
references: 2536844
references: J Virol. 1997 Jun;71(6):4599-613
references: 9151854
references: Immunol Res. 2012 Dec;54(1-3):140-51
references: 22454101
references: J Virol. 2004 Jul;78(14):7803-12
references: 15220454
references: Virology. 1992 Nov;191(1):387-95
references: 1329327
references: J Virol. 1999 Feb;73(2):878-86
references: 9882288
references: Rev Med Virol. 2007 Jul-Aug;17(4):253-76
references: 17579921
references: Rev Infect Dis. 1990 Sep-Oct;12 Suppl 7:S745-53
references: 2173104
references: Antimicrob Agents Chemother. 2008 Jul;52(7):2420-7
references: 18458124
references: J Virol. 2000 Aug;74(15):7108-18
references: 10888651