Measurement of low avidity anti-dsDNA by the Crithidia luciliae test and the PEG assay.

With the immunofluorescence technique (IFT) using Crithidia luciliae as a substrate, 14,417 sera sent to our laboratory for routine anti-dsDNA determination, were screened for the presence of antibodies to dsDNA. The 1,260 sera that were found IFT positive were then assayed with the Farr radioimmunoassay, in which 3H-labelled PM2-DNA is used as antigen. Only 470 sera (37%) were found to be Farr positive. This discrepancy is, at least partially, caused by the fact that the Farr assay does not detect anti-DNA of low avidity, whereas the Crithidia-IFT does. Sixty-eight percent of the IFT-positive/Farr negative sera were found positive with the PEG assay, a radioimmunoassay that also employs double stranded PM2-DNA as antigen, and that also detects anti-dsDNA of low avidity. The IFT performed on IFT positive/Farr negative sera was found to be rather irreproducible. It was shown that this was due to local increases of the salt concentration resulting from the way the assay was performed. The problem could be overcome by careful control of the assay conditions, i.e. never letting Crithidia slides dry up after washing with PBS. In the PEG assay, these sera sometimes showed a DNA binding that decreased with time. It could be shown that this is caused by a parallel increase in pH during the incubation as a result of CO2 evaporation from the serum.

Specificity in systemic lupus erythematosus of antibodies to double-stranded DNA measured with the polyethylene glycol precipitation assay.

Recently, a new radioimmunoassay--the polyethylene glycol (PEG) assay--was introduced to measure antibodies to double-stranded (ds) DNA. In this method, polyethylene glycol precipitation of formed 3H-DNA/antiDNA complexes is used instead of the ammonium sulfate precipitation used in the Farr assay. In contrast to the Farr assay, with which only high-avidity antibodies to dsDNA are detected, the PEG assay also reportedly measures anti-dsDNA of relatively low avidity. We studied whether this gain in antibody measurement results in loss of specificity for systemic lupus erythematosus. When the PEG assay was applied to a selected panel of 440 sera from patients with various well-defined autoimmune diseases and to a group of 197 normal human control sera, matched sex and age to the patients, the method was found to be fairly specific for systemic lupus erythematosus, although the sera from some patients with myasthenia gravis and some with autoimmune liver disease were also found positive. Screening of 352 additional serum specimens, sent to our laboratory for diagnostic reasons, revealed that, with the PEG assay, an extra population of relatively low-avidity antibodies to dsDNA--missed by the Farr assay--was detected. Upon clinical evaluation, we found that the patients in whom such antibodies were detected generally fulfilled a number of the preliminary criteria of the American Rheumatism Association for systemic lupus erythematosus, but that this diagnosis often was not made. We claim that the presence of low-avidity antiDNA characterizes a milder form of the disease in which patients often show only a single clinical feature of the disease. We conclude that results of the PEG assay add valuable diagnostic and clinical information to results obtained by the Farr assay.

Avidity of antibodies to dsDNA: comparison of IFT on Crithidia luciliae, Farr assay, and PEG assay.

The Farr assay is thought to detect only antibodies to DNA of relative high avidity. This is due to the high salt concentration of the employed ammonium sulfate precipitation, which dissociates DNA-anti-DNA complexes of low avidity. A recently introduced method to detect anti-DNA, the PEG assay, circumvents these dissociating reaction conditions by using polyethylene glycol instead of ammonium sulfate to precipitate the complexes; we therefore thought to measure antibodies to DNA of low avidity as well. We tested this assumption in several ways. It was found that the PEG assay detects a population of antibodies to DNA that are missed by the Farr assay. Complexes made with these antibodies were salt labile and could readily be dissociated by means of excess DNA, whereas Farr-positive antibodies formed stable complexes with DNA. Avidity studies using the method described by Celada et al. indicated that the anti-DNA detected by the PEG assay but missed by the Farr assay was of relatively low avidity. An inverse correlation between avidity and slope of the binding curves in the PEG assay was observed. These results confirm the notion that the PEG assay detects antibodies to DNA of low avidity. The fact that the Farr assay does not measure these antibodies confers possible diagnostic importance upon the PEG assay.