ss-DNA Ab ELISA Kit

Sample

Serum, plasma

Species Reactivity

Human

Detection Method

iELISA

Intended Use

Enzyme Immunoassay for quantitative determination of IgG Autoantibodies to ssDNA in human serum or plasma.

Contents of Kit

1. Divisible microplate: Consisting of 12 modules of 8 wells each. Ready to use. 96 (8×12) wells
2. Calibrators A-F (0, 12.5, 25, 50, 100, 200 U/ml), containing ssDNA antibodies in a serum/buffer matrix (PBS, BSA, detergent, NaN₃ 0.09%),yellow. Ready to use. 1.5 ml × 6
3. Control positive and (1) negative (2), containing ssDNA antibodies in a serum/buffer matrix (PBS, BSA, detergent, NaN₃ 0.09%, yellow. Ready to use. 1.5 ml × 2
4. Sample buffer P, containing PBS, BSA, detergent, preservative NaN₃ 0.09%, yellow, 5× conc. 20 ml
5. Enzyme Conjugate, containing anti-human IgG antibodies, HRP labelled; PBS, BSA, detergent, preservative ProClin 300 0.05%, light red. Ready to use. 15 ml
6. TMB Substrate; containing 3,3',5,5'-Tetramethylbenzidin, colorless. Ready to use. 15 ml
7. Stop solution (contains acid). Ready to use. 15 ml
8. Wash solution, containing Tris, detergent, preservative NaN₃ 0.09%; 50× conc. 20 ml

Storage

Store the kit at 2-8°C in the dark. Do not expose test reagents to heat, sun or strong light during storage and usage. Store microplate sealed and desiccated in the clip bag provided. Unopened reagents are stable until expiration of the kit. See labels for individual batch. Diluted Wash solution and Sample Buffer are stable for at least 30 days when stored at 2-8°C. We recommend consumption on the same day.

Performance Characteristics

Calibration
This assay system is calibrated in relative arbitrary units, since no international reference preparation is available for this assay.
Expected values
In a normal range study with samples from healthy blood donors the following ranges have been established with this ELISA assay: Cut-off 20 U/ml.

Detection Limit

Functional sensitivity was determined to be: 1 U/ml.

General Description

Autoimmune diseases are characterized by the occurence of antibodies against own antigenic structures - so-called autoantibodies. Presence of autoantibodies to native Desoxyribonucleic acids (n-DNA, dsDNA, double-stranded DNA) is typical for the clinical picture of Systemic Lupus erytrematodes (SLE). Antibodies against dsDNA belong to the group of Anti Nuclear Antibodies (ANA), which are directed against various structures of the nucleus of the cell. They appear in a variety of rheumatoid diseases. Besides the ANA antibodies another group of autoantibodies is of interest, which are directed against the so- called Extractable Nuclear Antigens (ENA). The ARA criteria of the American Rheumatism Association provide an extensive diagnostic scheme for the diagnosis of Systemic Lupus erythematodes (SLE). In case that at least 4 of the eleven ARA criteria are fulfilled, SLE is highly predictive. Antibodies to dsDNA are found during the active phases of SLE, where the serum concentration exhibits positive correlation to the severity of the disease. An ongoing therapy may be monitored by the aid of autoantibody determination. Diagnostic sensitivity of the anti-dsDNA determination in cases of SLE is approximately 91 % combined with a diagnostic specificity of nearly 96 percent.
Antibodies against DNA can be differentiated into two groups:
1. antibodies, that bind only to native double-stranded DNA (dsDNA) and
2. antibodies recognizing single-stranded DNA (ssDNA) too.
Measurement of anti nuclear antibodies (ANA, or anti nuclear factor (ANF)) by indirect immunofluorescence test (IFT) is widely accepted as screening method in suspected SLE. Since in some stages of the diseases or during therapy IFT sometimes gives false results, a more specific test system is needed. Negative IFT for anti nuclear antibodies does not exclude the presence of anti-dsDNA antibodies, since the antigenic structures may masked by other structures. Furthermore the ANA titers determined by IF test show only week correlation to the severity of the disease.
Most antibodies against dsDNA are directed against the phosphate units of DNA. Thus, these autoantibodies also bind to DNA single strains. For quantitation of anti-dsDNA it has to be proven, that the antigen preparation exhibits no contamination with single stranded DNA.
Autoantibodies against single-stranded DNA are mainly directed against its basic compound, which in the native DNA is masked inside the helical structure. In serum of SLE patients antissDNA antibodies are found with a frequency of up to 87 percent during acute phases and 43 percent during inactive phases. SLE like diseases are caused by some drugs. For differential diagnosis of drug-induced LE the determination of anti-ssDNA is a valuable diagnostic tool. In drug- induced LE anti-ssDNA is elevated in more the 50 percent of alle cases. Furthermore elevated anti-ssDNA serum concentrations have been reported in Mononucleosis, Hepatitis and various forms of Leukemia.