Automated detection of anti-double-stranded DNA antibody in systemic lupuserythematosus serum by flow immunoassay

We describe a novel automated now immunoassay system for quantification of and-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 b y an ion-exchange column on the basis of the difference in isoelectric poin t. 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 sign al and concentration of anti-dsDNA monoclonal antibody in the range of 0-30 0 mu g/mL. This simple technique permits the assay of anti-dsDNA autoimmune antibodies within 25 min. The ion-exchange column was simply regenerated b y occasional elution with eluent (20 mM N-methylpiperazine, pH 5.5) supplem ented with 0.5 M NaCl, to remove unreacted ALP-DNA.