|Values are valid only on day of printing.|
Antinuclear Antibodies (ANA):
ANA are measured by a commercial enzyme-linked immunosorbent assay (ELISA). Nuclear antigens from HEp2 cells are adsorbed to the wells of a microtiter plate. Diluted sera, calibrator, or controls are added to the wells and incubated. After washing to remove unbound serum proteins, an enzyme-conjugated antihuman IgG antibody is added to detect IgG antibodies bound to the microtiter wells. After incubation and washing to remove unbound conjugate, substrate is added to the wells. After incubation, the enzyme-substrate reaction is stopped. The absorbance in each microtiter well is measured by an automated plate reader. The absorbances are proportional to the levels of antibodies to nuclear antigens present in the test sera, calibrator, or controls.(Package insert: Autoimmune EIA: ANA Screening Test. Bio-Rad Laboratories, Hercules, CA 11/2011)
Cyclic Citrullinated Peptide (CCP):
CCPantibodies are measured by a commercial ELISA. A mixture of synthetic citrullinated peptides is adsorbed to the wells of a microtiter plate. Diluted sera, standards, or controls are added to the wells and incubated. After washing to remove unbound serum proteins, horseradish peroxidase-conjugated, monoclonal antihuman IgG antibody is added to detect IgG antibodies that have bound to the microtiter wells. After incubation and washing to remove unbound conjugate, tetramethylbenzidine (TMB) substrate is added to the wells. After incubation, the enzyme-substrate reaction is stopped. The absorbance in each microtiter well is measured by use of an automated plate reader. The absorbances are proportional to the levels of CCP antibodies, and the concentrations of antibodies in the test sera, and controls are determined by comparison to a multipoint standard curve.(Package insert: Quanta Lite CCP3 IgG ELISA. INOVA Diagnostics, San Diego, CA 11/2010)
Double-Stranded DNA (dsDNA):
dsDNA antibodies are measured by a commercial ELISA. Microwells are precoated with calf thymus double-stranded DNA (dsDNA) antigen. The calibrators, controls, and diluted patient samples are added to the wells and autoantibodies recognizing the dsDNA antigen bind during the first incubation. After washing the wells to remove all unbound proteins, purified peroxidase-labeled goat antihuman IgG conjugate is added. The conjugate binds to the captured human autoantibody and the excess unbound conjugate is removed by a further wash step. The bound conjugate is visualized with 3,3',5,5' TMB substrate, which gives a blue reaction produce, the intensity of which is proportional to a concentration of autoantibody in the sample. Sulfuric acid is added to each well to stop the reaction. This produces a yellow end-point color, which is read at 450 nm.(Package insert: QUANTA Lite dsDNA SC ELISA, INOVA Diagnostics Inc, San Diego, CA 07/2012).
Confirmatory testing for borderline dsDNA by ELISA testing is performed by immunofluorescence assay (IFA). Autoantibodies in a test sample directed against dsDNA bind to antigens in the substrate placed on the slide, which, in this case, is Crithidia luciliae. Washing removes excess serum from the substrate. Fluorescein-conjugated (FITC) antiserum added to the substrate attaches to the bound autoantibody. After a second washing step to remove excess conjugate, the substrate is coverslipped and viewed for fluorescent patterns with a fluorescent microscope. Observation of specific fluorescent patterns on the substrate indicates the presence of autoantibodies in the test sample.(Package insert: Bio-Rad Kallestad Crithidia luciliae Substrate. Bio-Rad Laboratories, Hercules, CA 07/2009)
SS-A/Ro, SS-B/La, RNP, Sm, Scl 70, Jo 1, ribosome P, and centromere antibodies are measured by a commercial multiplex flow immunoassay system. Recombinant or purified antigens are coupled covalently to polystyrene microspheres that are impregnated with fluorescent dyes to create unique fluorescent signatures, 1 microsphere type for each antigen. Diluted sera, calibrators, and controls are added to a mixture containing the antigen-coupled microspheres. Antibodies to each antigen bind to their homologous antigen-coupled microspheres. The microspheres are washed to remove extraneous serum proteins. Phycoerythrin (PE)-conjugated antihuman IgG antibody is then added to detect IgG antibodies bound to the microspheres. The microspheres are washed to remove unbound conjugate, and bound conjugate is detected by laser photometry. A primary laser determines the fluorescent signature of each microsphere, and a secondary laser reveals the level of PE fluorescence associated with the microsphere surface. Results are calculated for each antigen-coated microsphere type by comparing the median fluorescence response to a series of multipoint calibration curves.(Package insert: BioPlex 2200 ANA Screen. Bio-Rad Laboratories, Hercules, CA 03/2012)
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