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Protein microarray tecnology for unraveling the antibody specificity repertoire against microbiol proteomes.
No full textProtein microarrays: from serodiagnosis to whole proteome scale analysis of the immune response against pathogenic microorganisms.
No full textProtein arrays for serodiagnosis of disease.
No full textProtein microarrays offer the possibility to circumvent most of the current limitations in the serodiagnosis of allergy, autoimmune, and infectious disease by allowing the simultaneous, multiparametric determination of specific subclasses of antibodies directed against many pathogenic antigens. Microarray immunoassays have been developed with these characteristics. A first-generation assay, for the serodiagnosis of infectious disease, allows the determination of IgG and IgM antibodies to various viral and bacterial antigens. In addition, a second-generation assay, designed for the serodiagnosis of allergic disease, permits the determination of IgE antibodies to various allergens implicated in allergic disease. Slides printed with antibody dilution curves and antigen are first incubated with serum samples and then subsequently with secondary antibodies. For detection of human IgG and IgM, fluorescently labeled secondary antibodies are employed. However, because of low-level concentrations of circulating IgE antibodies, a more sensitive protocol is required for human IgE detection. Here, fluorescence is delivered via the coupling of the secondary antibody to tyramide signal-amplification reagentry. Human IgG, IgM, or IgE bound to the printed antigens can then be revealed by confocal scanning microscopy and quantified with internal calibration curves. Generation of analytical and clinical data have demonstrated that the microarray test format provides equivalent performance to enzyme-linked immunosorbent assay (ELISA) tests and offers a significant advantage in convenience and cost when compared to traditional test formats
Detection of allergen-specific IgE on microarrays by use of signal amplification techniques.
Full text linkAntigen Microarrays for Serodiagnosis of Infectious Diseases
No full textBackground: 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
Device and method for detecting fluorescence comprising a light emitting diode as excitation source
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