cyanine-dye-3 and Autoimmune-Diseases

A DNA microarray scanner was used as a digital fluorescence microscope to simplify the diagnosis of autoimmune bullous diseases. Frozen sections of skin biopsies were taken from 3 patients with bullous pemphigoid and 1 patient each with lichen planus pemphigoides, linear immunoglobulin (Ig) A disease, and dermatitis herpetiformis. After incubation with cyanine-labeled antibodies, the tissues were scanned at 5-mum resolution using an instrument originally designed to study gene expression. The microarray scanner's large field of view, unlike that of fluorescence microscopy, allowed a view of the entire specimen, considerably easing the orientation of tissue. All images were diagnostic and included a linear pattern along the basement membrane zone (BMZ) using anti-IgG and anti-C3 in all cases of bullous pemphigoid, a linear pattern of IgG along the BMZ in lichen planus pemphigoides, and a linear pattern of IgA along the BMZ in linear IgA dermatosis. IgA deposition along dermal papillary tips was seen in dermatitis herpetiformis, but a granular pattern was indiscernible at the 5-mum resolution. The advantages of the microarray scanner over standard fluorescence microscopy include speed, technical ease, large field of view, potential for visualizing multiple antibodies simultaneously in a tissue, and convenience of digital image archiving.

Topics: Autoimmune Diseases; Basement Membrane; Biopsy; Carbocyanines; Complement C3; Dermatitis Herpetiformis; Equipment Design; Fluorescent Antibody Technique; Fluorescent Dyes; Humans; Immunoglobulin A; Immunoglobulin G; Lichen Planus; Microscopy, Fluorescence; Oligonucleotide Array Sequence Analysis; Pemphigoid, Bullous; Predictive Value of Tests; Skin; Skin Diseases, Vesiculobullous

The concurrent detection and quantification of analytes that vary widely in concentration present a principal problem in multiplexed assay systems. Combining competitive and sandwich immunoassays permits coverage of a wide concentration range, and both highly abundant molecules and analytes present in low concentration can be quantified within the same assay.. The use of different fluorescence readout channels allows the parallel use of a competitive system and a sandwich configuration. The 2 generated assay signals are combined and used to calculate the amount of analyte. The measurement range can be adjusted by varying the competitor concentration, and an extension of the assay system's dynamic range is possible.. We implemented the method in a planar protein microarray-based autoimmune assay to detect autoantibodies against 13 autoantigens and to measure the concentration of a highly abundant protein, total human IgG, in one assay. Our results for autoantibody detection and IgG quantification agreed with results obtained with commercially available assays. The use of 2 readout channels in the protein microarray-based system reduced spot-to-spot variation and intraassay variation.. By combining a direct immunoassay with a competitive system, analytes present in widely varying concentrations can be quantified within a single multiplex assay. Introducing a second readout channel for analyte quantification is an effective tool for spot-to-spot normalization and helps to lower intraassay variation.

Topics: Autoanalysis; Autoantibodies; Autoantigens; Autoimmune Diseases; Carbocyanines; Enzyme-Linked Immunosorbent Assay; Fluorescence; Fluorescent Dyes; Humans; Immunoglobulin G; Protein Array Analysis; Reproducibility of Results