Automated detection of anti-double-stranded DNA antibody in systemic lupus erythematosus serum by flow immunoassay.
We describe a novel automated flow immunoassay system for quantification of anti-double-stranded (ds) DNA autoimmune antibodies in the serum of patients suffering from systemic lupus erythematosus. dsDNA (360 bp) was covalently coupled with alkaline phosphatase (ALP) to form a novel analytical reagent (ALP-DNA). After immunoreaction, antibody-antigen complexes between ALP-DNA and anti-dsDNA monoclonal antibody were separated from unreacted ALP-DNA by an ion-exchange column on the basis of the difference in isoelectric point. Antibody-antigen complexes were subsequently quantified by luminescence following addition of 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane. The assay yielded a linear relationship between signal and concentration of anti-dsDNA monoclonal antibody in the range of 0-300 micrograms/mL. This simple technique permits the assay of anti-dsDNA autoimmune antibodies within 25 min. The ion-exchange column was simply regenerated by occasional elution with eluent (20 mM N-methylpiperazine, pH 5.5) supplemented with 0.5 M NaCl, to remove unreacted ALP-DNA.
Lim
T
T
Department of Biotechnology, Tokyo University of Agriculture and Technology, Japan.
Komoda
Y
Y
Nakamura
N
N
Matsunaga
T
T
D000974:Antibodies, Antinuclear / Q000032:analysis*; D001322:Autoanalysis; D004247:DNA / Q000032:analysis / Q000276:immunology*; D017931:DNA Primers; D006801:Humans; D007118:Immunoassay; D007202:Indicators and Reagents; D008180:Lupus Erythematosus, Systemic / Q000097:blood*
D000974:Antibodies, Antinuclear; D017931:DNA Primers; D007202:Indicators and Reagents; D004247:DNA