The specificity of the anti-dsDNA ELISA. A closer look.
The anti-dsDNA ELISA is probably one of the most popular techniques for determining antibody reactivity towards dsDNA, since this assay system has proven high sensitivity and is easy to perform. An important difference from other ELISA systems is the use of an intermediate layer (e.g., protamine sulphate or poly-L-lysine) which has been found to be necessary in order to obtain sufficient coating of dsDNA to the plates. When a panel of monoclonal antibodies to DNA (n = 56), all reactive in this anti-dsDNA ELISA were tested on plates coated only with protamine sulphate (PS) a large number were positive, although with a lower reactivity than with DNA. Binding to protamine sulphate occurred via two mechanisms: (1) DNA/anti-DNA immune complexes, present in hybridoma culture supernatants and adherent to protamine sulphate and (2) some IgM antibodies appear to possess an intrinsic affinity for PS. The latter mechanism gives rise to a false positive reaction in the anti-dsDNA ELISA. It was found that 18% of the clones that were unreactive in any anti-dsDNA assay other than the anti-dsDNA ELISA were labelled 'anti-dsDNA' incorrectly. We therefore propose that antibody reactivity towards dsDNA in an ELISA system must be confirmed in other anti-dsDNA assays before such antibodies can be termed 'anti-dsDNA'.
Brinkman
K
K
Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Termaat
R
R
Van den Brink
H
H
Berden
J
J
Smeenk
R
R
D000818:Animals; D000974:Antibodies, Antinuclear / Q000032:analysis*; D000936:Antigen-Antibody Complex / Q000276:immunology; D004247:DNA / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D007075:Immunoglobulin M / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D011479:Protamines / Q000276:immunology
D000974:Antibodies, Antinuclear; D000936:Antigen-Antibody Complex; D007075:Immunoglobulin M; D011479:Protamines; D004247:DNA