Studies on Measurement of Anti-DNA Antibodies in SLE Sera using Millipore Filter Radioimmunoassay.

Abstract

The measurement of anti-native DNA (n-DNA) and denatured DNA (d-DNA) antibodies in sera from patients with SLE and other rheumatic diseases ussng millipore filter redioimmunoassay was studied methodologically and clinically. The ¹²⁵I-n-DNA and d-DNA produced by the in vitro labelling of calf thymus n-DNA and heat denatured DNA with ¹²⁵I. Radioactive DNA was separated from other reactants by passage throgh a Sephadex G-50 column. The specific activities of ¹²⁵I-n-DNA and d-DNA were 0.3μCi/mg and 0.94μCi/mg, respectively. Test sera were heated at 56°C for 30 minutes. Fifty microliters of heated serum were added to labelled DNA in 200μ liter of 0.15 M Tris buffer, PH7.5, and 150μ liter of distilled water.The assay mixture was incubated at 37°C for 15 minutes afeer which the reaction was stopped by the addition of 4ml of SSC solution (mixture of 0.15M Sodium Chloride and 0.015M Solium Citrate). The diluted assay mixture was passed through a prewetted millipore filter (type-HAWP) under gentle suction. The radioactivity of the filter membrane was measured in a scintillation counter, Sera were obtained from 32 healthy controls, 22 patients with rheumatic diseases other than SLE and 33 patients with SLE. As the results, the millipore filter assay using preheated sera was capable of detecting specific heat stable DNA binding activity in SLE sera. Complex formation between radioactive DNA and anti-DNA antibodies in SLE serum was biphasic, with a rapid initial phase complete in 10 minutes. Optimum. density of salts in Tris buffer was determined to be 0.15M. When unlabelled n-DNA and d-DNA were added to 50μl of serum from the SLE patients with high titer of anti-n-DNA and-d-DNA antibodies, unlabelled n-DNA inhibited the binding of radioactive n-DNA, and unlabelled d-DNA inhibited both the binding of radioactive n-DNA and d-DNA. The high levels of anti-n-DNA antibodies were found in sera from patients with SLE and reflected the degree of disease activity. On the other hand, anti-d-DNA antibodies appeared in sera from not only SLE but other rheumatic diseases. This simply and rapidly perfomed assay is easily quantitied and can be useful in the diagnosis and management of patients wit SLE.