Method Description

Double-stranded DNA antibodies, IgG:

The test kit contains 12 microtiter 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-horseradish peroxidase (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)

Crithidia luciliae immunofluorescence test screen:

Patient serum is diluted 1:10 and allowed bind to the kinetoplast of the non-pathogenic hemoflagellate Crithidia luciliae adhered to the slide. The Crithidia luciliae contains a dense, pure concentration of circular dsDNA that provides a unique substrate free from contaminating nuclear antigens like histones or single-stranded DNA. Following binding, unbound diluted patient serum is washed off the slide and fluorescent-conjugated antiserum added to the substrate on the slide and incubated. A second washing step is performed to remove excess conjugate. The substrate is cover slipped and reviewed for fluorescent patterns with a fluorescent microscope. Observation of specific fluorescent patterns on the substrate indicate the presence of autoantibodies in the test sample.(Package insert: Immuno Concepts IgG ANTI-nDNA Fluorescent Test System. Immuno Concepts; Rev 4.0, 2023)

Crithidia luciliae immunofluorescence test titer:

Patient serum with a positive screen at the 1:10 dilution is titered up to 1:640 and allowed to bind to the kinetoplast of the non-pathogenic hemoflagellate Crithidia luciliae adhered to the slide. The Crithidia luciliae contains a dense, pure concentration of circular dsDNA that provides a unique substrate free from contaminating nuclear antigens like histones or single-stranded DNA. Following binding, unbound diluted patient serum is washed off the slide and fluorescent-conjugated antiserum added to the substrate on the slide and incubated. A second washing step is performed to remove excess conjugate. The substrate is cover slipped and reviewed for fluorescent patterns with a fluorescent microscope. Observation of specific fluorescent patterns on the substrate indicate the presence of autoantibodies in the test sample. Positive results are reported with an end point titer up to 1:640. Results higher than 1:640 are reported as greater than 1:640.(Package Insert: Immuno Concepts IgG ANTI-nDNA Fluorescent Test System. Immuno Concepts; Rev 4.0, 2023)