Background: Progress in robotic printing technology has allowed the development of high-density nucleic acid and protein arrays that have increased the throughput of a variety of assays. We generated protein microarrays by printing microbial antigens to simultaneously determine in human sera antibodies directed against Toxoplasma gondii, rubella virus, cytomegalovirus (CMV), and herpes simplex virus (HSV) types 1 and 2 (ToRCH antigens). Methods: The antigens were printed on activated glass slides with high-speed robotics. The slides were incubated first with serum samples and subsequently with fluorescently labeled secondary antibodies. Human IgG and IgM bound to the printed antigens were detected by confocal scanning microscopy and quantified with internal calibration curves. Both microarrays and commercial ELISAs were utilized to detect serum antibodies against the ToRCH antigens in a panel of characterized human sera. Results: The detection limit (mean 2 SD) of the microarray assay was 0.5 pg of IgG or IgM bound to the slides. Within-slide, between-slide, and between-batch precision profiles showed CVs of 1.7–18% for all antigens. Overall, >80% concordance was obtained between microarray assays and ELISAs in the classification of sera; for T. gondii, CMV, and HSV1, concordance exceeded 90%. Conclusions: The microarray is a suitable assay format for the serodiagnosis of infectious diseases and can be easily optimized for clinical use. The ToRCH assay performs equivalently to ELISA and may have potentially important advantages in throughput, convenience, and cost.
Antigen Microarrays for Serodiagnosis of Infectious Diseases
CRISANTI, Andrea
2002
Abstract
Background: Progress in robotic printing technology has allowed the development of high-density nucleic acid and protein arrays that have increased the throughput of a variety of assays. We generated protein microarrays by printing microbial antigens to simultaneously determine in human sera antibodies directed against Toxoplasma gondii, rubella virus, cytomegalovirus (CMV), and herpes simplex virus (HSV) types 1 and 2 (ToRCH antigens). Methods: The antigens were printed on activated glass slides with high-speed robotics. The slides were incubated first with serum samples and subsequently with fluorescently labeled secondary antibodies. Human IgG and IgM bound to the printed antigens were detected by confocal scanning microscopy and quantified with internal calibration curves. Both microarrays and commercial ELISAs were utilized to detect serum antibodies against the ToRCH antigens in a panel of characterized human sera. Results: The detection limit (mean 2 SD) of the microarray assay was 0.5 pg of IgG or IgM bound to the slides. Within-slide, between-slide, and between-batch precision profiles showed CVs of 1.7–18% for all antigens. Overall, >80% concordance was obtained between microarray assays and ELISAs in the classification of sera; for T. gondii, CMV, and HSV1, concordance exceeded 90%. Conclusions: The microarray is a suitable assay format for the serodiagnosis of infectious diseases and can be easily optimized for clinical use. The ToRCH assay performs equivalently to ELISA and may have potentially important advantages in throughput, convenience, and cost.
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