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Preparation of 125I-labeled Native DNA for Use in Radioimmunoassays for Anti-Native-DNA Antibodies.

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Preparation of 12Wabeled Native DNA for Use in
Radioimmunoassays for Anti-Native-DNA Antibodies
Harold Keiser
A method is described for the in vitro labeling of calf thymus native DNA
(nDNA) with 1251. The 121-calf thymus nDNA produced by this method is
suitable for use in the Millipore filter assay for antibodies to nDNA in sera
of patients with systemic lupus erythematosusand gives results equivalent
to those obtained with JH-HeLa cell nDNA labeled in vivo.
Serum levels of antibodies to native D N A
(nDNA) are of great value in monitoring disease activity in patients with systemic lupus erythematosus (SLE) (1,2). T h e most sensitive
procedures for measuring these antibodies, the
Farr (ammonium sulphate precipitation) technique (3) and the recently described Millipore
filter assay (4), are both radioimmunoassays
which require the use of labeled nDNA. )H- and
IT-labeled nDNA can be prepared from viral,
bacterial and human cell lines in culture (4),
but the methods of isolation and purification are
technically complex and feasible only for specialized laboratories. Commercially available
labeled nDNA is of variable quality and its cost
for the Millipore filter assay, which uses 1 pg of
nDNA per test, is prohibitive.
By an adaptation of the procedure outlined
by Commerford (5) and Rosenberg et a1 ( 6 ) ,an
lZ5llabel can be incorporated into calf thymus
nDNA from commercial sources. This simple
From the Department of Medicine, Albert Einstein College of iMedicine, Bronx, New York.
Supported by Grants from the New York Chapter of the
Arthritis Foundation, The Lupus Erythematosus Foundation and NIH Grant #5 R01 AM 07343.
HAROLD KEISER, MD: Postdoctoral Fellow, The Arthritis
Foundation, and Assistant Professor of Medicine, Albert
Einstein College of Medicine.
Reprint requests should be addressed to: Harold Keiser,
M D , Department of Medicine, Albert Einstein College of
Medicine, 1300 Morris Park Avenue, Bronx, NY 10461.
Submitted for publication Feb. 7, 1973; accepted April 2,
1973.
468
procedure yields large quantities of labeled
nDNA suitable for use in radioimmunoassays
for anti-nDNA antibodies.
MATERIAL AND METHODS
Calf thymus DNA (type I, Sigma Chemical Co., St.
Louis, Mo.), 2 mg/ml in SSC solution (0.15 M sodium
chloride, 0.015 M sodium citrate), was passed through a
Millipore filter (type HAWP, Millipore Corp., Bedford,
Mass.) under gentle suction. Filtered nDNA solution (0.3
ml) was added to 0.2 ml of 1.25 x 10 -4 M potassium iodide
equilibrated with 2 mCi of lZ5l(obtained from New England Nuclear Corp., Boston, Mass., as NalZ51in 0.1 ml of
0.1 M NaOH) and 0.3 ml of 0.5 M sodium acetate buffer,
pH 5.0. After mixing, 0.3 ml of 5 x
M thallium trichloride (K & K Laboratories, Plainview, N.Y.) in 0.1 M
sodium acetate buffer, p H 5.0, were added and the reaction
mixture was heated at 56°C for 1 hour. IZSI-nDNAwas
separated from the other reactants by passage through a
Sephadex G-50 column. The yield was 400 pg of l Z 5 l nDNA with a specific activity of 250,000 cpm/ pg.
For use in the Millipore filter assay, the IZ5I-nDNAwas
diluted with nonradioactive nDNA previously passed
through a Millipore filter and 0.15 M tris-HC1 buffer, p H
7.5, to a specific activity of 5-10.000 cpm/pg and a concentration of 10-20 pg/ml as determined by the optical density
at 260 m p (1.0 O.D. units = 50 pg/rnl). The diluted I*5InDNA solution was passed through a Millipore filter and
0.1 rnl aliquots were transferred to 10 x 75 mm plastic disposable test tubes and stored frozen at -20” c .
The Millipore filter assay for anti-nDNA activity was
performed as previously described (4), except that the radioactivity of the washed filters was measured in an automated
gamma counter. In calculating anti-nDNA activity, the specific activity of the Iz5I-nDNA(i.e., cpm precipitated by cold
5% TCA per pg nDNA) must be corrected for radioactive
decay due to the 60 day half-life of 1251.
Arthritis and Rheumatism,Vol. 16, No. 4 (July-August 1973)
PREPARATION OF '*%DNA
2
4
6
8
10
12
M
16
18
20
22 24
'251-calf thymus nDNA (units)
Fig 1. Comparison of anti-nDNA
levels as determined by the Millipore
filter assay testing the same serum
samples with %-HeLa cell nDNA and
1251-calf thymus nDNA. Normal sera
are designated by (A). sera of hospitalized patients without connective
tissue disease by (o), sera of patients
with connective tissue disease other
than SLE by ( 0 ) . sera of patients with
inactive SLE by (O), and sera of
patients with active SLE by (x).
have not been widely available o r generally utilized. This situation should change as the recently described Millipore filter assay (4)comes
into wider use. This assay is rapid, simple to
RESULTS
perform and capable of quantitating high levels
Over a 2-month period, 100 sera were tested
of activity on undiluted sera, and it includes
for anti-nDNA activity by the Millipore filter steps to control for denaturation of the nDNA
assay using both 3H-thymidine-labeled HeLa antigen. However, the Millipore filter assay has
cell nDNA, as previously described (4), and
the major drawback of requiring relatively large
1251-labeledcalf thymus nDNA. With a single
amounts of radioactive nDNA. U p to now raexception, the results using the different nDNA
dioactive nDNA for anti-nDNA assays has
preparations were equivalent (P < .05): high
been produced by in vivo incorporation of an
levels of anti-nDNA activity (> 5 units) were
isotope followed by isolation and purification of
found only in sera of patients with active SLE, the nDNA (4), procedures which are techniwhile normal controls, hospitalized patients, cally specialized and beyond the capability of
patients with inactive SLE and those with other most laboratories. Polynucleotides have been laconnective tissue diseases generally had much beled in vztro with 3H-actinomycin D (7) and by
lower levels (Figure 1). A serum from a 12-year methylation with '%-dimethyl sulfate (8).
old boy with active SLE had very high activity However, the specific activity of nDNA labeled
(114 units) with 3H-HeLa nDNA but only with '%-dimethyl sulfate is too low for it to be
moderately high activity (16 units) with lZ5I-calf useful in a radioimmunoassay and 3Hthymus nDNA. Further investigation will be actinomycin D itself binds to Millipore filters
required to explain this discrepancy.
(9) so that nDNA labeled with it is not suitable
for use in the Millipore filter assay which relies
DISCUSSION
on the filterability of nDNA in the absence of
In spite of its value as an indicator of disease anti-nDNA antibody.
An lZ5I label can be introduced into nDNA by
activity, anti-nDNA antibody determinations
The patient material and criteria for SLE and disease activity were those described previously (4).
Arthritis and Rheumatism,Vol. 16, No. 4 (July-August 1973)
469
KEISER
iodination in the presence of thallium trichloride which results in the incorporation of iodine as 5-iodocytosine through a stable covalent
bond (5). T h e procedure allows preparation of
radioactive nDNA of high specific activity
without significant alteration of its molecular
weight, melting profile, ability to renature or to
transform (5). Studies with iodinated denatured
DNA have shown that it retains its ability to react with specific antisera (6) and the results described above indicate that '251-calf thymus
n D N A is s u i t a b l e f o r u s e i n a r a d i o immunoassay for anti-nDNA antibodies in the
sera of patients with SLE.
There are several potential difficulties in the
use of Iz5I-nDNA prepared by the method described here. T h e addition of iodine to nDNA
and breaks in the nDNA chain caused by radioactive decay are potential sources of altered antigenic specificity. T h e commercial preparation
of calf thymus nDNA used has been found to
contain a small amount of protein impurity
which might incorporate significant amounts of
label at low concentrations of iodide (5). Millipore filtration and the relatively high concentration of iodide used in the method described
here probably minimize this potential problem.
T h e commercial calf thymus nDNA is also far
from uniform in size, in contrast to the nDNA
antigen preparations used in previous studies
(4). Additional preparative steps such as phenol
extraction to remove the protein impurity or
sedimentation velocity centrifugation to isolate
nDNA of more uniform size are feasible. However, for the purposes of a clinical assay, it does
not seem necessary to introduce further complexity to the preparative procedure since I25Icalf thymus nDNA prepared without these ad-
470
ditional steps gave results in the Millipore filter
assay which were equivalent to those obtained
with more carefully standardized 'H-HeLa cell
nDNA.
ACKNOWLEDGMENTS
The advice and encouragement of Drs. Milton Adesnik,
Peter Barland and Donald Marcus and the statistical assistance of Mrs. Joseph DeVito are gratefully acknowledged.
REFERENCES
1. Koffler D, Carr R, Agnello V, et al: Antibodies to
polynucleotides in human sera: Antigenic specificity and relation to disease. .J Exp Med
134:294-3 12,1971
2. Schur PH, Sandson .J: Immunologic factors and
clinical activity in systemic lupus erythematosus.
N Eng ,J Med 278: 533-538,1968
3. Pincus T, Schur PH, Rose JA, et al: Measurement of serum DNA-binding activity in systemic
lupus erythematosus. N Eng J Med 281:701705, 1969
4. Ginsberg B, Keiser H: A Millipore filter assay for
antibodies to native DNA in sera of patients with
systemic lupus erythematosus. Arth Rheum 16:
199-207,1973
5. Commerford SL: Iodination of nucleic acids in
uitro. Biochem 10:1993-1999, 1971
6. Rosenberg B.J, Erlanger BF, Beiser SM: Radioimmunochemical studies on nucleoside-specific
antibodies using iodinated DNA. J Immunol
108:271-274, 1972
7. Carr RI, Koffler D, Agnello V, Kunkel HG:
Studies on DNA antibodies using DNA labeled
with actinomycin-D ('H) or dimethyl ('H) sulphate. Clin Exp Immunol4:527-536,1969
8. Pochon F, Michelson AM: Polynucleotides. IX.
Methylation of nucleic acids, homopolynucleotides
and complexes. Biochim Biophys Acta 149:99106, 1967
9. Ginsberg, B: Personal communication
Arthritis and Rheumatism, Vol. 16, No. 4 (July-August 1973)
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preparation, antibodies, dna, native, labeled, anti, radioimmunoassay, use, 125i
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