Influence of molecular weight of DNA on the determination of anti-DNA antibodies in systemic lupus erythematosus (SLE) sera by radioimmunoassay

Abstract

Using a radioimmunoassay (RIA) based on the Farr technique with radioactively labeled 3-H-DNA for quantitative measurements of anti-DNA antibodies in sera of patients with systemic lupus erythematosus (SLE), the influence of molecular weight of DNA (ranging from 0.1 times 10-6 to 22.0 times 10-6 daltons) on binding and precipitation in this system has been investigated. Comparing our results with mathematical models it follows that one antibody molecule is fixed on the average to a statistical DNA segment of 2 times 10-6 to 4 times 10-6 daltons. Furthermore binding capacity of the DNA was found to be independent of the molecular weight, as demonstrated in a double label experiment using 14-C and 3-H-labeled DNA of different size. However, the amount of radioactivity precipitated was found to depend on the molecular weight of the labeled DNA following a non-linear function. It was calculated that a minimal ratio of fixed antibody molecules per a certain size of DNA was necessary for precipitation. The mathematical treatment of the observed non-linear precipitation dependence will be discussed using various statistical models. Our results indicate that the quantitative measurements of anti-DNA antibodies with the Farr technique e.g. for diagnosis and control of SLE in clinical immunology is highly dependent on the molecular weight of the labeled DNA used in the assay system and reliable results are only obtained with DNA of a sufficiently high molecular weight.

Reevaluation of the ammonium sulfate assay for dna antibody

“…We do not agree with the suggestion of Geisert et al (20) that only DNA of "sufficiently" high molecular weight will allow reliable results by the NH,SO, assay. Since normal sera are just as sensitive to DNA size as SLE sera, high molecular weight DNA limits the potential range of the assay by increasing the background level of binding.…”

“…Effect of DNA Molecular Weight on Anti-DNA Level. Previous studies have demonstrated that the observed anti-DNA level increases as the molecular weight of the DNA increases (10,20,21). The difficulty in completely explaining this phenomenon by application of the Poisson distribution (see Discussion) prompted us to extend the observations to a greater number of SLE sera as well as to normal sera.…”

“…Similar discrepancies were found by others (20,21). By taking into account the molecular weight distribution for each size DNA and assuming that the minimum antibody/antigen ratio required for precipitation increases along with the molecular weight of the DNA, Geisert et al (20) were able to obtain a reasonable approximation of the actual results. However, our data (Figure 4) show that all SLE sera do not respond in exactly the same way to DNA molecular weight, suggesting that the mathematical model of Geisert et al (20) may not apply to all SLE sera.…”

Anti‐dsdna and complement profiles as prognostic guides in systemic lupus erythematosus

“…Such variations give rise to enormous fluctuations of DNA binding values (6,12). Because in longitudinal studies a proper quantitation of anti-dsDNA is essential, such fluctuations have to be controlled by using DNA with a homogeneous molecular weight or alternatively by correcting variations via the introduction of a panel of standard sera (21).…”

Detection of low avidity anti‐dna antibodies in systemic lupus erythematosus

“…There is some evidence that suggests that the high salt concentration used in the assay may cause dissociation of all but those complexes that contain the highest avidity antibodies (1 8). In addition, it is now recognized that this method is most applicable in the study of the binding of antibodies to high molecular weight DNA (19,20). Recent evidence suggests that low molecular weight DNA may in fact play a significant role in the pathogenesis of SLE (21,22).…”