Plasma DNA Assay Methods

At the outset, we modified our methodology to augment the sensitivity of the assay. Previously, plasma samples were heat inactivated at 56° С for 60 minutes to destroy complement activity. We found that we could increase the sensitivity of plasma DNA detection by adding sodium chloride to the plasma before heat inactivation. A 500-p.l aliquot of test plasma was added to 25 m of sodium chloride to approximate a 1 M sodium chloride solution. This saline/plasma solution was then incubated for 30 minutes at 60°C.

Serum samples containing antibodies to native DNA were ob­tained from patients with SLE. Using the Crithidia luciliae fluores­cence assay as a screening method, we found that serum samples from patients with titers of anti-double-stranded DNA (anti- dsDNA) below 1/320 did not precipitate well by counterimmunoe- lectrophoresis (CIE). To ensure that the antisera from patients with SLE provided precipitating antibody specific for dsDNA, these serum samples were tested with commercial DNA (Worthington calf thymus type I, Sigma) that had been purified by passage through a hydroxyapatite column (BioRad Laboratories, Richmond, Calif). By running the SLE patient serum samples against serial dilutions of the purified dsDNA, we were able to look for serum samples that gave well-defined precipitin lines. After testing several different antisera, we found two that gave consistent and similar results. Twenty-six patients were tested with both antisera; all but one patient (negative with one serum, weakly reactive with the other) had the same results with both serum samples. Since there was essentially no difference between the two serum samples, the remaining patients were tested with the serum we had in greater quantity, This particular serum was kindly provided by Paul Plotz, MD, National Arthritis Institute, Bethesda, Md. A 1:6 dilution of this serum afforded detected of pure dsDNA at a level of 0.75 \Lg/ ml in normal plasma.
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Counterimmunoelectrophoresis was performed as described previously® except that the serum was electrophoresed for seven minutes before adding the patients plasma. The plate was then run for an additional 53 minutes to give a total test time of 60 minutes. The plates were interpreted immediately after the electrophoresis was completed and recorded as positive or negative for a precipitin line. Positive results were semiquantitated by titering. The total tikne from thawing the plasma sample to reading the result was approximately two hours.

The specificity of the precipitin line for DNA was tested by three methods. First, DNase digestion was performed on plasma to which purified dsDNA had been added as well as on plasma from 42 patients testing positive by CIE. The second method involved blocking of the anti-DNA with DNA. Three hundred microliters of pure dsDNA (concentration of 29.4 fxg/ml of DNA) was added to 100 u.1 of the anti-DNA test serum and incubated for one hour at 37°C. The mixture was then assayed by CIE against plasma containing 6.3 ^JLg/ml of dsDNA. The third method combined the first two methods. The test serum containing anti-DNA was incubated for two hours at 37°C with excess DNA. Then DNase was added and incubated for another two hours at 37°C. As expected, the anti-DNA formed precipitin lines when assayed with plasma DNA. All three methods confirmed that the assay measured DNA and not some other interfering substance.

In an earlier report from this laboratory, nonspecific precipitin lines were noted when heparin was used as the anticoagulant in the blood collection tubes. A subsequent study noted that the concen­tration of heparin in collection tubes were at least 14 U/ml of plasma in contrast to plasma levels of heparin following intravenous bolus or continuous infusion which rarely rises above 2.0 U/ml. At that time a heparin concentration of 2.5 U/ml did not cause any precipitin lines, suggesting that heparin given in therapeutic doses to patients would not interfere with the plasma DNA determinations. For further confirmation, we ran plasma heparin concentrations ranging from 1.25 to 10 U/ml against several anti-DNA serum samples in CIE. No precipitin lines formed indicating that heparin given as therapy does not result in false-positive DNA lines.