Human Anti-dsDNA ELISA (IgG)
Antibodies against DNA are distinguished into two different types: antibodies against double stranded, native DNA (dsDNA) and antibodies against single-stranded, denatured DNA. The dsDNA-speicific antibodies (anti-dsDNA) are considered a specific marker for systemic lupus erythematosus (SLE), due to the high clinical associations [1-3]. The presence of these autoantibodies could be virtually diagnostic for SLE [1, 3]. In fact, anti-dsDNA antibodies may be present in patients even before they develop clinical features of SLE [2, 4]. Therefore, monitoring the condition of anti-dsDNA antibodies is essential for maintaining the healthy condition as well as identifying the progression of SLE. Moreover, the identification of anti-dsDNA antibodies in other pathological conditions and in healthy subjects is very rare (less than 0.5%) .
PRINCIPLE OF THE ASSAY
The micro-plate is pre-coated with double-stranded DNA (dsDNA) antigens. Standards and samples are added into the wells and any dsDNAspeicific antibodies presented in the sample is bound by the immobilized antigens. After washing, any unbound antibodies will be removed. Then Goat anti-human IgG-HRP conjugate is added. The conjugate binds to the captured dsDNA-specific antibodies. Then substrate is catalyzed by the HRP to produce a blue color that changes to yellow after adding the stopping buffer. The density of the yellow coloration is directly proportional to the amount of captured dsDNA-specific antibodies in the plate. The light absorbance (OD value) under 450nm wavelength of the wells is determined using a microplate reader. The antibody concentration of the unknown sample can be estimated with the provided calibrators in the kit, which are calibrated against International Standard WHO Wo/80. The kit offers semiquantitative and quantitative interpretation of the data, which is in the section of INTERPRETATION.
The linearity of Anti-dsDNA ELISA (IgG) was determined by assaying 8 serial dilutions of 5 serum samples. The linear regreassion was calculated, calculated, R2 amounting to >0.97 within the concentration range of 5 IU/mL to 600 IU/mL.
The reproducibility of the test was investigated by determine the intra- and inter-assay coefficients of variation using 3 sera. The Intra-assay CVs are based on 20 determinations and the inter-assay CVs on 4 determinations performed in 6 different plates.
Intra-assay variation, n=20
Inter-assay variation, n=4 x 6
Mean value (IU/mL)
Mean value (IU/mL)