Method Description

Antinuclear Antibodies:

The method used to detect antinuclear antibodies (ANA) is enzyme-linked immunosorbent assay (ELISA). A HEp-2 lysate supplemented with specific purified antigens (double-stranded [ds] DNA, histone, SS-A [Ro], SS-B [La], Smith, sm/RNP, Scl-70, Jo-1, and centromere B antigen) are coated onto microtiter plate wells. A dilution of patient serum is added to the well and incubated. After washing to remove unbound serum protein, an enzyme conjugated antihuman IgG antibody is added to detect human IgG bound to the microtiter plate well. After incubation and washing to remove unbound conjugate, a substrate to the enzyme is added to the well. After incubation, the enzyme substrate reaction is stopped. The complete assay is measured on a spectrophotometer plate reader. The optical density measured is proportional to the antibody present in the patient serum. Testing is performed on the Agility instrument by Dynex.(Package insert: ELISA kits. Bio-Rad Laboratories; 07/2014)

Cyclic Citrullinated Peptide:

Cyclic citrullinated peptide (CCP) antibodies in serum are detected by binding to the wells of a commercial microtiter plate coated with synthetic CCP. During the first incubation, serum antibodies bind to adsorbed, solid phase CCP. The wells are then washed to remove unbound serum constituents, and horse radish peroxidase-labeled goat anti-human IgG antibody is added. After further incubation and washing to remove unbound conjugate, substrate (3,3',5,5'-tetramethylbenzidine) is added and allowed to incubate. The reaction between enzyme and substrate is stopped and color in the wells is measured in a microtiter plate reader. The concentration of CCP antibodies is determined by comparison to a 5 point standard curve (15.6-250 U). Testing is performed on the Agility instrument by Dynex.(Package insert: Quanta Lite CCP3 IgG ELISA. INOVA Diagnostics; 02/2020)

dsDNA:

The test kit contains 12 micotiter strips each with 8 break-off reagent wells coated with double-stranded DNA (dsDNA). In the first reaction step, diluted patient samples, calibrators and controls are incubated in the wells. Anti-dsDNA antibodies will bind to the antigens coated in the microtiter wells. The wells are washed to remove any unbound proteins and non-specific antibodies. In a second reaction step, rabbit anti-human IgG HRP enzyme conjugate is added to each well. The enzyme conjugate will bind to any wells that have human IgG binding to the dsDNA antigen. The wells are washed to remove any unbound HRP enzyme conjugate. 3,3,5,5, tetramethylbenzidine (TMB) enzyme substrate is added. If the HRP enzyme is present in the well (positive reaction), the HRP enzyme will react with the TMB substrate and produce a blue color. After an additional incubation time to allow the color development, a stop solution is added which turns the blue color yellow and inhibits further color development to allow for stable spectrophotometric reading. The test strips are placed in a microplate reader and the optical density of the color is measured. The amount of antigen specific bound antibody is proportional to the color intensity.(Package insert: Anti-dsDNA-NcX ELISA (IgG). EUROIMMUN; 7/8/2020)

SS-A/Ro, SS-B/La, RNP, Sm, Scl 70, Jo 1, ribosome P, and centromere B 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, one 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; 2019)