Novel microneutralization assay for HCMV using automated data collection and analysis

In addition to being sensitive and specific, an assay for the assessment of neutralizing antibody activity from clinical trial samples must be amenable to automation for use in high-volume screening. To that effect, we developed a 96-well microplate assay for the measurement of HCMV-neutralizing act...

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Bibliographic Details
Published in:	Journal of immunological methods Vol. 322; no. 1; pp. 82 - 93
Main Authors:	Abai, Anna Maria, Smith, Larry R., Wloch, Mary K.
Format:	Journal Article
Language:	English
Published:	Amsterdam Elsevier B.V 30-04-2007 Elsevier
Subjects:	Biological and medical sciences Blood - virology Cells, Cultured Complement System Proteins - immunology Cytomegalovirus - isolation & purification Cytomegalovirus Infections - diagnosis ELISPOT analyzer Fundamental and applied biological sciences. Psychology Fundamental immunology Human cytomegalovirus Humans Microbiology Microneutralization assay Miniaturization Molecular immunology Neutralization Tests Sensitivity and Specificity Techniques Techniques used in virology Virology NT 50 HCMV kDa GPS EGFP N Human cytomegalovirus BBS Microneutralization assay IE1 FBS GPSC ELISPOT analyzer AEC NSC PBS PSC DAB IEA TMB ELISPOT 4-PL mAb CV NA pDNA EC 50 gB ELISA Virus Data analysis Herpesviridae ELISPOT assay Betaherpesvirinae Immunological method
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Summary:	In addition to being sensitive and specific, an assay for the assessment of neutralizing antibody activity from clinical trial samples must be amenable to automation for use in high-volume screening. To that effect, we developed a 96-well microplate assay for the measurement of HCMV-neutralizing activity in human sera using the HCMV-permissive human cell line HEL-299 and the laboratory strain of HCMV AD169. The degree to which neutralizing antibodies diminish HCMV infection of cells in the assay is determined by quantifying the nuclei of infected cells based on expression of the 72 kDa IE1 viral protein. Nuclear IE1 is visualized using a highly sensitive immunoperoxidase staining and the stained nuclei are counted using an automated ELISPOT analyzer. The use of Half Area 96-well microplates, with wells in which the surface area of the well bottom is half the area of a standard 96-well microplate plate, improves signal detection compared with standard microplates and economizes on the usage of indicator cells, virus, and reagents. The staining process was also streamlined by using a microplate washer and data analysis was simplified and accelerated by employing a software program that automatically plots neutralization curves and determines NT 50 values using 4-PL curve fitting. The optimized assay is not only fast and convenient, but also specific, sensitive, precise and reproducible and thus has the characteristics necessary for use in measuring HCMV-neutralizing activity in the sera of vaccine trial subjects such as the recipients of Vical's HCMV pDNA vaccine candidates.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 ObjectType-Article-2 ObjectType-Undefined-1 ObjectType-Feature-3 Former Vical employee.
ISSN:	0022-1759 1872-7905
DOI:	10.1016/j.jim.2007.02.001