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 μg/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.