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A comparison of methods for labelling DNA for use in the radioimmuno assay of DNA-antibodies
Journal of Immunological Methods 5 (1974) 1-8 © North-Holland,Publishing Company
A COMPARISON OF METHODS FOR LABELLING DNA FOR USE IN T H E R A D I O I M M U N O A S S A Y
OF DNA-ANTIBODiES
S.A. LEON, B. SHAPIRO, G. KOLLMANN and A. GREEN Radiation Research Laboratory, Department of Nuclear Medicine, Division of Radiology, Albert Einstein Medical Center, Philadelphia, Pennsylvania 19141, U.S.A.
Received 2 January 1974
Accepted 10 January 1974
The radioimmunoassay for DNA-antibodies in systemic lupus erythematosus is assessed with DNA labelled with 1251 via chemical iodination on one hand (12Sl-DNA), and with DNA containing [ 12 s I] iododeoxyuridine via biological incorporation on the other ([ 12 sIUdR] DNA). The results show that chemical iodination labels protein impurities in the DNA and reduces the difference in binding of normal vs lupus sera. 125IUdR-DNA is a superior product since no label is introduced in proteins. The binding of 125I-DNAby normal sera ranges from 5 to 20% and from 40 to 70% lupus sera. The same sera yield values of 0-3% for normal and 92-98% for lupus with 12sIUdR-DNA. The latter allows a sharper discrimination between normal and slightly elevated values, as in patients under treatment.
1. INTRODUCTION Antibodies to DNA may be found in the circulation of patients with systemic lupus erythematosus (SLE) (Seligmann and Arana, 1968). Circulating DNA has also been observed and evidence that D N A - a n t i b o d y complexes form in the serum and accumulate in the renal glomeruli has been presented (Stollar, 1968). The appearance and exacerbation of the nephritis attendant in this disease has been shown to be related to the formation and deposition of these complexes in the kidneys (Stollar, 1968). A simple and precise method for measuring very small amounts of DNAantibody has been developed using 14C-labelled DNA (Pincus et al., 1969). This method promises to be the most definitive test for SLE presently available. It was reported to be positive in 75% of lupus patients studied, whereas all other tests were positive in only 2 5 - 6 4 % of the cases. Moreover, the anti-DNA-antibody test showed greater specificity for SLE than the other diagnostic tests used. Since some laboratories do not have liquid scintillation counting facilities, and since liquid scintillation counting is more laborious than gamma counting, the anti-DNA-antibody test for SLE using 14C-labelled DNA has been restricted in its use. The purpose of the present work was to prepare and test 125I-labelled DNA,
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S.A. LEON et al.
which could be used in the anti-DNA-antibody measurement and which would simplify the procedure and make it available to laboratories with gamma counting facilities only. Two methods of labelling DNA with 1251 were employed: (a) direct iodination of salmon sperm DNA by a modification of the method of Commerford (1971) and (b) incorporation of 125I-labelled 5-iododeoxyuridine ( I U d R ) i n t o DNA by growing E. coli, strain 15 TAU.
2. MATERIALS AND METHODS Highly polymerized salmon sperm DNA was purchased from Mann Research Laboratories. 125I-UdR was the product of Amersham/Searle and Na125I was obtained from New England Nuclear Corporation. Thallium trichloride (TICI 3 -H2 O) was purchased from K&K Laboratories. Human albumin, B grade, and human gamma-globulin, fraction II were obtained from Calbiochem.
2.1. Chemical iodination of DNA Ten microliters of DNA solution containing 40/ag were added to 0.2 ml of ammonium acetate buffer (0.1 M ammonium acetate adjusted to pH 5.0 with glacial acetic acid). 25/J1 of 2.5 × 10-4 M KI containing 50/aCiof carrier-free Na125I and 25/al of 1.5 × 10 -3 M T1C13 were added. The mixture was incubated at 60°C for 15 min, then chilled in the ice and the excess T1C13 was neutralized with 25/A of 0.01 M Na2SO 3. The reaction mixture was dialyzed against one liter of cold ammonium acetate buffer (3 times) and then with two changes of borate buffer (0.16 M NaC1 and 0.2 M H3BO 3, adjusted to pH 8.0 with solid NaOH). Under these conditions, the specific activity of the DNA is 8000-10,000 cpm//lg, and the only iodinated residue is cytosine (Commerford, 1971).
2. 2. Preparation of 12 5iUdR_labelled DNA E. coli, strain 15 TAU was grown on minimal medium (Davis and Mingioli, 1950), supplemented with 25/lg/ml thymine, 50 #g/ml arginine and 20/2g/ml uracil, as described for BU-labelled DNA by Hanawalt (1967). When the culture reached O.D. 450 nm of 0.4, the cells were harvested, washed and transferred to fresh medium containing only 1/2g/ml thymine and 200/2Ci of 125IUdR (approx. 20/~g). The culture was grown to an O.D. of 0.7-0.8; the cells were harvested, washed and suspended in NET buffer (Hanawalt, 1967). They were lysed with 1% sodium dodecyl sarcosinate (Geigy) at 60°C for 5 - 7 min, and the DNA was extracted and purified according to Marmur (196l). Partially purified DNA was obtained by isopropanol precipitation after the first chloroform extraction (see table 6). In order to obtain extensively purified DNA, the material was extracted until no protein was visible at the interface, then treated with RNAse and pronase
Methods of labelling DNA used in DNA-Ab radioimmunoassay
3
(Pincus et al., 1969). After the final lsopropanol precipitation, the yield from a 100 ml culture was 96/lg DNA with a specific activity of 9400 cpm//lg. 14C-labelled DNA was prepared from KB cells as described (Pincus et al., 1969). 2.3. Chemical iodination of albumin and ,/-globulin Human albumin and `/-globulin were iodinated exactly as described for DNA, except that 100/ag of each were taken and 10/~Ci of Na 1251 were used. 2.4. Measurement of antibody binding to DNA Essentially the technique of Pincus et al. (1969) was used. Fifty microliters of 125i.labelled DNA (0.1-0.5/ag in borate buffer) were mixed with 50 ~1 of serum (normal or lupus) diluted 1 : 10 in the same buffer. The mixture was incubated at 37°C for 30 min and in the refrigerator overnight. Then 100/ll of cold saturated (NH4)2SO 4 were added, mixed and left to stand for 1 hr in the cold. After centrifugation, 100/al of the supernatant fraction (S) were removed and counted in a Baird Atomic Spectrometer. The remaining supematant containing the precipitate (P) was also counted and the binding was calculated according to the formula: P-S - - X 100 = Percent DNA bound. P+S 2.5. Analytical ultracentrifugation The molecular weight of DNA samples after various treatments was determined by the band sedimentation method (Studier, 1965). In all cases, 10/al of the solution containing • 1 #g DNA was placed in the sample well, whereas 1 M NaC1 0.01 M Tris-chloride pH 7.6 was used as the bulk solution. All experiments were carried out at 25,980 rpm and 20°C, with initial slow acceleration at 5000 rpm for 3 - 5 min. The films were scanned with a Gilford Spectrophotometer, model 2400, equipped with a linear transport attachment. The position of the band was measured in the scans and the molecular weights were calculated (Studier, 1965).
3. RESULTS At first, labelling of DNA with 125I was carried out by direct iodination, using different concentrations of KI. The amounts of 125I were also varied to maintain constant specific activity. Although the binding of 125I-DNA in normal serum was not affected at different KI concentration, the binding in lupus serum appeared to decrease with increasing iodination of DNA (table 1).
S.A. LEON et al. Table 1 Effect of iodination on the binding of 12 s I-DNA by normal and lupus serum. KI concentration
2.5 X 10-s M 2.5 X 10-4 M 2.5 X l0 -3 M
Specific activity * (cpm/**g)
3510 8600 4330
Percent binding Normal
Lupus
15 31 26
59 42 23
* The amount of Na 12 si was adjusted in order to obtain a product with similar specific activity, although the extent of iodination was different (compare with table 2). The effect of self-irradiation of 1251-DNA was measured by both molecular weight determinations and by effect on binding in normal and lupus serum. Table 2 shows that molecular weight is markedly reduced by self-irradiation. The effect on binding in lupus serum was negligible but the effect in normal serum was to increase the binding. This may represent a nonspecific binding to smaller DNA fragments. Because of the effects of extensive iodination and internal irradiation on DNA, subsequent experiments with directly iodinated DNA utilized a minimum amount of iodine (KI concentration 2.5 × 10-5 M) and only enough radioactivity to permit convenient counting (final specific activity 8 - 1 0 , 0 0 0 cpm//~g). The effect of denaturation was also studied and table 3 shows that heat denaturation reduces DNA binding by both normal and lupus serum. The high binding by normal sera of 1251-DNA prepared by direct iodination was of great concern, since normal serum binding of 14C-DNA was 0 in most cases. lodination with non-radioactive iodine, of 14C-DNA, gave the same binding results as non-iodinated 14C-DNA (table 4) for normal serum. The decrease in binding by lupus serum of the iodinated 14C-DNA is consistent with the data in table 1. lodination in itself, therefore, was not the cause of high binding of DNA in normal serum. One possible cause for the high binding was the iodination of trace amounts Table 2 Effect of self-irradiation on the molecular weight of DNA and on the binding by normal and lupus serum. Specific activity (cpm//lg) No iodination
3545 (1 day) 3545 (10 days) 236,000 (1 day) 236,000 (I0 days)
M.W. (X 10-6)
6.54 3.55 2.85 1.74 0.81
Percent binding Normal
Lupus
7 22 29 46
66 81 76 87
Methods o f labelling DNA used in DNA-A b radio immunoassay
5
Table 3 Binding of double- and single-stranded DNA by normal and lupus serum.
Table 4 Effect of iodination of 14C-labelled DNA on the binding by normal and lupus serum.
12 5I-DNA
DNA
Native Denatured
Percent binding Normal
Lupus
33 14
74 40
Percent binding
14C_ 14C-iodinated
Normal
Lupus
0 0
42 30
of protein in the DNA preparation. This would precipitate with the ammonium sulfate and results in an apparent high antibody level. To test this hypothesis, DNA was prepared by growing E. coli in the presence of 1251UdR, for a short time, on the assumption that the label would be incorporated in DNA, but not in protein. Table 5 shows that the DNA from E. coli, labelled with 125IUdR ' whether purified of protein or not, shows no binding in normal serum and almost 100% in lupus serum. DNA extracted from cold E. coli cells, but labelled by chemical iodination instead of IUdR, gave very high binding in normal serum and about the same binding in lupus serum. The reduced binding in lupus serum may be due to iodinated protein which does not precipitate in ammonium sulfate. Table 5 also shows chemically iodinated salmon sperm DNA for comparison. Further studies of the effect of the presence of labelled protein in the DNA preparation were carried out b y adding 125i.labelled globulin and albumin to DNA labelled with 125IUdR. Table 6 shows that addition of labelled globulin raises the ratio in normal serum from 0 to 13%, whereas addition of labelled albumin does not alter it. The protein effects are consistent with the observations on directly iodinated DNA. Because of the difficulty in removing all protein by purification procedures, we have elected to use only 125iUdR.labelled DNA. Table 7 shows the comparison of 14C-labelled DNA and 125iUdR_labelled DNA in 23 normal and 8 lupus sera. For each serum, the value was an average of
Table 5 Comparison of binding of 12SlUdR.DNA and chemically iodinated 12SI_DNA by normal and lupus serum and effect of protein impurities in the preparation. E. coli (15 TAU) DNA
Percent binding Normal
1251 UdR-DNA, purified (< 0.5% protein) 12si UdR-DNA, not purified (~ 20% protein) 1251-DNA (chemically iodinated), 1.0% protein Salmon sperm 12 SI.DNA (chemically iodinated)
Lupus
0
98
0 65 25
93 62 74
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S.A. LEON et al.
Table 6 Effect of traces of 1251_labelled protein precipitable with ammonium sulfate (globulin)and non-precipitable (albumin) on the ratio of binding by normal and lupus serum. Percent binding
12 s i UdR-DNA (2000 cpm) (no additions) + 1251~'-globulin (700 cpm) + 125I-albumin (900 cpm)
Normal
Lupus
0 13 1
75 48 65
]Fable 7 Comparison of the binding of 14C- and 12sIUdR-labelled DNA by normal and lupus sera. Percent binding *
14C-DNA 12s-I-UdR-DNA
Normal
Lupus
2.7 (range: 0-7) 1.0 (range: 0-3)
70 (range: 60 76) 97 (range: 92-98)
* The determinations represent the average value of 23 normal and 8 lupus sera. 4 determinations. The results indicate that the two preparations are comparable in both reactivity and specificity.
4. DISCUSSION The above results with 14C-labelled DNA confirm the work of Pincus et al. (1969). This reagent used in the measurement of anti-DNA-antibody provides a most sensitive and specific test for SLE. The results with chemically iodinated DNA were not comparable to those with the 14C product, in that normal serum gave high binding values (up to 30% in some runs) and lupus serum sometimes gave relatively low binding values (down to 40% in some cases). The apparent cause of both high normal and low lupus values was the presence of protein impurities in the DNA. Since protein is a much better substrate for iodination than DNA, even a small amount of protein would be labelled disproportionately with 125I. Purification procedures would be arduous and would never remove every trace of protein. It is of interest that increased iodination of DNA (chemically) reduced the binding in lupus serum. This could be due partly to increased iodination of protein impurities that do not precipitate with ammonium sulfate in the test. However, since the same effect occurred with 14C-DNA iodinated with nonradioactive iodine, other factors may be involved, lodination, by the chemical procedure described, yield 5-substituted iodocytosine in DNA (Commerford, 1971), and this may distort
Methods o f labelling DNA used in DNA-Ab radioirnmunoassay
7
the binding site and make it less reactive with antibody. Iodine incorporated via IUdR may also induce such alteration but to a lesser extent, presumably because less iodine is incorporated per molecule. It is also of interest that increased internal irradiation (by higher specific activity and prolonged storage) increased binding in normal serum (Hughes, 1971). The reduced molecular weight of the DNA (Tan et al., 1966; Leon et al., 1973) may be responsible for this, but effect on labelled protein impurities (cross-linking) resulting in more of these proteins precipitating with the ammonium sulfate could also be a factor. Breakdown of DNA in the body and subsequent binding to protein moieties, especially of fragments damaged by ultraviolet irradiation after exposure to sunlight (Tan, 1968), might result in immunogenic complexes of DNA, which could stimulate antibody production (Stollar and Sandberg, 1966; Tan et al., 1966; Stollar, 1970) and could be the etiologic mechanism for lupus. The results with 125IUdR.labelled DNA indicated that this product is equal to the 14C-labelled DNA in reactivity and specificity. Since the test procedure with the gamma-labelled DNA is much simpler, the use of 1251-DNA in the anti-DNAantibody test is the method of choice. In addition, the same system may be used for a very sensitive determination of DNA in nanogram quantities (Wikman-Coffelt, 1972). For this purpose, chemically iodinated DNA of high specific activity is a good reagent and protein contamination and label may not interfere in the test. It has been reported that the levels of DNA in serum increase during active episodes of SLE. In other conditions, such as leukemia, lymphosarcoma and liver disease, low levels of DNA have been detected (Carr, 1969). It might be important to monitor these levels during treatment in order to evaluate the response. Although no DNA was detected in the serum of patients with multiple myeloma, rheumatoid arthritis and other cliseases it is possible that only with a radioimmunoassay would their levels be determined. Work along these lines is in progress in this laboratory.
5. SUMMARY Chemical iodination of DNA with Na1251 will also label trace impurities of protein which take up a disproportionate amount of radioactivity. Biological labelling of DNA by growing cells of a strain of E. coli in the presence of [ 125i] iododeoxyuridine yields a superior reagent, since no label is introduced in material other than DNA. Therefore extensive purification of the product is not necessary. The lower specific activity of 125IUdR-DNA compared to 125I-DNA is not a disadvantage, since the damage by self-irradiation is reduced. In the assay for DNAantibody levels, it is shown that high specific activity and prolonged storage increase the non-specific binding of DNA by components of normal serum. On the other hand, the specific binding by the antibodies in SLE sera is not affected. The binding of 125I-DNA by normal sera ranges from 5 to 20% and from 40 to 70% by
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S.A. LEON et al.
lupuS sera. When t h e same sera are t e s t e d w i t h 1 2 5 I U d R - D N A ,
the values are
0 - 3 % for n o r m a l a n d 9 2 - 9 8 % for lupus sera.
ACKNOWLEDGEMENT We are grateful t o D.I. Martin, M.Sc., for his able assistance.
REFERENCES Carr, R.I., 1969, DNA antibodies and DNA in serum, Ph.D. Thesis, Rockefeller University, New York. Commerford, S.L., 1971, Biochemistry 10, 1993. Davis, B.D. and E.S. Mingioli, 1950, J. Bacteriol. 60, 17. Hanawalt, P.C., 1967, in: Methods in Enzymology, eds. L. Grossman and K. Moldave (Academic Press, New York) p. 7 0 2 - 7 0 8 . Hughes, G.R.V., 1971, Lancet Oct. 16, 861. Leon, S.A., G.J. KoUmann and B. Shapiro, 1973, Int. J. Radiat. Biol. 23,325. Marmur, J., 1961, J. Mol. Biol. 3,208. Pincus, T., P.H. Schur, J.A. Rose, J.L. Decker and N. Talal, 1969, New Engl. J. Med. 281,701. Seligmann, M. and R. Arana, 1968, in: Nucleic acids in immunology, eds. O.J. Plescia and W. Braun (Springer-Verlag, New York) p. 9 8 - 1 1 3 . Stollar, B.D., 1968, in: Nucleic acids in immunology, eds. O.J. Plescia and W. Braun (SpringerVerlag, New York) p. 114-124. Stollar, B.D., 1970, Biochim. Biophys. Acta 209,541. Stollar, B.D. and A.L. Sandberg, 1966, J. Immunol. 96,755. Studier, F.W., 1965, J. Mol. Biol. 11,373. Tan, E.M., 1968, Science 161, 1353. Tan, E.M., P.H. Schur, R.I. Cart and H.G. Kunkel, 1966, J. Clin. Invest. 45, 1732. Wikman-Coffelt, J., 1972, Biochem. Biophys. Res. Commun. 48,502.
A COMPARISON OF METHODS FOR LABELLING DNA FOR USE IN T H E R A D I O I M M U N O A S S A Y
OF DNA-ANTIBODiES
S.A. LEON, B. SHAPIRO, G. KOLLMANN and A. GREEN Radiation Research Laboratory, Department of Nuclear Medicine, Division of Radiology, Albert Einstein Medical Center, Philadelphia, Pennsylvania 19141, U.S.A.
Received 2 January 1974
Accepted 10 January 1974
The radioimmunoassay for DNA-antibodies in systemic lupus erythematosus is assessed with DNA labelled with 1251 via chemical iodination on one hand (12Sl-DNA), and with DNA containing [ 12 s I] iododeoxyuridine via biological incorporation on the other ([ 12 sIUdR] DNA). The results show that chemical iodination labels protein impurities in the DNA and reduces the difference in binding of normal vs lupus sera. 125IUdR-DNA is a superior product since no label is introduced in proteins. The binding of 125I-DNAby normal sera ranges from 5 to 20% and from 40 to 70% lupus sera. The same sera yield values of 0-3% for normal and 92-98% for lupus with 12sIUdR-DNA. The latter allows a sharper discrimination between normal and slightly elevated values, as in patients under treatment.
1. INTRODUCTION Antibodies to DNA may be found in the circulation of patients with systemic lupus erythematosus (SLE) (Seligmann and Arana, 1968). Circulating DNA has also been observed and evidence that D N A - a n t i b o d y complexes form in the serum and accumulate in the renal glomeruli has been presented (Stollar, 1968). The appearance and exacerbation of the nephritis attendant in this disease has been shown to be related to the formation and deposition of these complexes in the kidneys (Stollar, 1968). A simple and precise method for measuring very small amounts of DNAantibody has been developed using 14C-labelled DNA (Pincus et al., 1969). This method promises to be the most definitive test for SLE presently available. It was reported to be positive in 75% of lupus patients studied, whereas all other tests were positive in only 2 5 - 6 4 % of the cases. Moreover, the anti-DNA-antibody test showed greater specificity for SLE than the other diagnostic tests used. Since some laboratories do not have liquid scintillation counting facilities, and since liquid scintillation counting is more laborious than gamma counting, the anti-DNA-antibody test for SLE using 14C-labelled DNA has been restricted in its use. The purpose of the present work was to prepare and test 125I-labelled DNA,
2
S.A. LEON et al.
which could be used in the anti-DNA-antibody measurement and which would simplify the procedure and make it available to laboratories with gamma counting facilities only. Two methods of labelling DNA with 1251 were employed: (a) direct iodination of salmon sperm DNA by a modification of the method of Commerford (1971) and (b) incorporation of 125I-labelled 5-iododeoxyuridine ( I U d R ) i n t o DNA by growing E. coli, strain 15 TAU.
2. MATERIALS AND METHODS Highly polymerized salmon sperm DNA was purchased from Mann Research Laboratories. 125I-UdR was the product of Amersham/Searle and Na125I was obtained from New England Nuclear Corporation. Thallium trichloride (TICI 3 -H2 O) was purchased from K&K Laboratories. Human albumin, B grade, and human gamma-globulin, fraction II were obtained from Calbiochem.
2.1. Chemical iodination of DNA Ten microliters of DNA solution containing 40/ag were added to 0.2 ml of ammonium acetate buffer (0.1 M ammonium acetate adjusted to pH 5.0 with glacial acetic acid). 25/J1 of 2.5 × 10-4 M KI containing 50/aCiof carrier-free Na125I and 25/al of 1.5 × 10 -3 M T1C13 were added. The mixture was incubated at 60°C for 15 min, then chilled in the ice and the excess T1C13 was neutralized with 25/A of 0.01 M Na2SO 3. The reaction mixture was dialyzed against one liter of cold ammonium acetate buffer (3 times) and then with two changes of borate buffer (0.16 M NaC1 and 0.2 M H3BO 3, adjusted to pH 8.0 with solid NaOH). Under these conditions, the specific activity of the DNA is 8000-10,000 cpm//lg, and the only iodinated residue is cytosine (Commerford, 1971).
2. 2. Preparation of 12 5iUdR_labelled DNA E. coli, strain 15 TAU was grown on minimal medium (Davis and Mingioli, 1950), supplemented with 25/lg/ml thymine, 50 #g/ml arginine and 20/2g/ml uracil, as described for BU-labelled DNA by Hanawalt (1967). When the culture reached O.D. 450 nm of 0.4, the cells were harvested, washed and transferred to fresh medium containing only 1/2g/ml thymine and 200/2Ci of 125IUdR (approx. 20/~g). The culture was grown to an O.D. of 0.7-0.8; the cells were harvested, washed and suspended in NET buffer (Hanawalt, 1967). They were lysed with 1% sodium dodecyl sarcosinate (Geigy) at 60°C for 5 - 7 min, and the DNA was extracted and purified according to Marmur (196l). Partially purified DNA was obtained by isopropanol precipitation after the first chloroform extraction (see table 6). In order to obtain extensively purified DNA, the material was extracted until no protein was visible at the interface, then treated with RNAse and pronase
Methods of labelling DNA used in DNA-Ab radioimmunoassay
3
(Pincus et al., 1969). After the final lsopropanol precipitation, the yield from a 100 ml culture was 96/lg DNA with a specific activity of 9400 cpm//lg. 14C-labelled DNA was prepared from KB cells as described (Pincus et al., 1969). 2.3. Chemical iodination of albumin and ,/-globulin Human albumin and `/-globulin were iodinated exactly as described for DNA, except that 100/ag of each were taken and 10/~Ci of Na 1251 were used. 2.4. Measurement of antibody binding to DNA Essentially the technique of Pincus et al. (1969) was used. Fifty microliters of 125i.labelled DNA (0.1-0.5/ag in borate buffer) were mixed with 50 ~1 of serum (normal or lupus) diluted 1 : 10 in the same buffer. The mixture was incubated at 37°C for 30 min and in the refrigerator overnight. Then 100/ll of cold saturated (NH4)2SO 4 were added, mixed and left to stand for 1 hr in the cold. After centrifugation, 100/al of the supernatant fraction (S) were removed and counted in a Baird Atomic Spectrometer. The remaining supematant containing the precipitate (P) was also counted and the binding was calculated according to the formula: P-S - - X 100 = Percent DNA bound. P+S 2.5. Analytical ultracentrifugation The molecular weight of DNA samples after various treatments was determined by the band sedimentation method (Studier, 1965). In all cases, 10/al of the solution containing • 1 #g DNA was placed in the sample well, whereas 1 M NaC1 0.01 M Tris-chloride pH 7.6 was used as the bulk solution. All experiments were carried out at 25,980 rpm and 20°C, with initial slow acceleration at 5000 rpm for 3 - 5 min. The films were scanned with a Gilford Spectrophotometer, model 2400, equipped with a linear transport attachment. The position of the band was measured in the scans and the molecular weights were calculated (Studier, 1965).
3. RESULTS At first, labelling of DNA with 125I was carried out by direct iodination, using different concentrations of KI. The amounts of 125I were also varied to maintain constant specific activity. Although the binding of 125I-DNA in normal serum was not affected at different KI concentration, the binding in lupus serum appeared to decrease with increasing iodination of DNA (table 1).
S.A. LEON et al. Table 1 Effect of iodination on the binding of 12 s I-DNA by normal and lupus serum. KI concentration
2.5 X 10-s M 2.5 X 10-4 M 2.5 X l0 -3 M
Specific activity * (cpm/**g)
3510 8600 4330
Percent binding Normal
Lupus
15 31 26
59 42 23
* The amount of Na 12 si was adjusted in order to obtain a product with similar specific activity, although the extent of iodination was different (compare with table 2). The effect of self-irradiation of 1251-DNA was measured by both molecular weight determinations and by effect on binding in normal and lupus serum. Table 2 shows that molecular weight is markedly reduced by self-irradiation. The effect on binding in lupus serum was negligible but the effect in normal serum was to increase the binding. This may represent a nonspecific binding to smaller DNA fragments. Because of the effects of extensive iodination and internal irradiation on DNA, subsequent experiments with directly iodinated DNA utilized a minimum amount of iodine (KI concentration 2.5 × 10-5 M) and only enough radioactivity to permit convenient counting (final specific activity 8 - 1 0 , 0 0 0 cpm//~g). The effect of denaturation was also studied and table 3 shows that heat denaturation reduces DNA binding by both normal and lupus serum. The high binding by normal sera of 1251-DNA prepared by direct iodination was of great concern, since normal serum binding of 14C-DNA was 0 in most cases. lodination with non-radioactive iodine, of 14C-DNA, gave the same binding results as non-iodinated 14C-DNA (table 4) for normal serum. The decrease in binding by lupus serum of the iodinated 14C-DNA is consistent with the data in table 1. lodination in itself, therefore, was not the cause of high binding of DNA in normal serum. One possible cause for the high binding was the iodination of trace amounts Table 2 Effect of self-irradiation on the molecular weight of DNA and on the binding by normal and lupus serum. Specific activity (cpm//lg) No iodination
3545 (1 day) 3545 (10 days) 236,000 (1 day) 236,000 (I0 days)
M.W. (X 10-6)
6.54 3.55 2.85 1.74 0.81
Percent binding Normal
Lupus
7 22 29 46
66 81 76 87
Methods o f labelling DNA used in DNA-A b radio immunoassay
5
Table 3 Binding of double- and single-stranded DNA by normal and lupus serum.
Table 4 Effect of iodination of 14C-labelled DNA on the binding by normal and lupus serum.
12 5I-DNA
DNA
Native Denatured
Percent binding Normal
Lupus
33 14
74 40
Percent binding
14C_ 14C-iodinated
Normal
Lupus
0 0
42 30
of protein in the DNA preparation. This would precipitate with the ammonium sulfate and results in an apparent high antibody level. To test this hypothesis, DNA was prepared by growing E. coli in the presence of 1251UdR, for a short time, on the assumption that the label would be incorporated in DNA, but not in protein. Table 5 shows that the DNA from E. coli, labelled with 125IUdR ' whether purified of protein or not, shows no binding in normal serum and almost 100% in lupus serum. DNA extracted from cold E. coli cells, but labelled by chemical iodination instead of IUdR, gave very high binding in normal serum and about the same binding in lupus serum. The reduced binding in lupus serum may be due to iodinated protein which does not precipitate in ammonium sulfate. Table 5 also shows chemically iodinated salmon sperm DNA for comparison. Further studies of the effect of the presence of labelled protein in the DNA preparation were carried out b y adding 125i.labelled globulin and albumin to DNA labelled with 125IUdR. Table 6 shows that addition of labelled globulin raises the ratio in normal serum from 0 to 13%, whereas addition of labelled albumin does not alter it. The protein effects are consistent with the observations on directly iodinated DNA. Because of the difficulty in removing all protein by purification procedures, we have elected to use only 125iUdR.labelled DNA. Table 7 shows the comparison of 14C-labelled DNA and 125iUdR_labelled DNA in 23 normal and 8 lupus sera. For each serum, the value was an average of
Table 5 Comparison of binding of 12SlUdR.DNA and chemically iodinated 12SI_DNA by normal and lupus serum and effect of protein impurities in the preparation. E. coli (15 TAU) DNA
Percent binding Normal
1251 UdR-DNA, purified (< 0.5% protein) 12si UdR-DNA, not purified (~ 20% protein) 1251-DNA (chemically iodinated), 1.0% protein Salmon sperm 12 SI.DNA (chemically iodinated)
Lupus
0
98
0 65 25
93 62 74
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S.A. LEON et al.
Table 6 Effect of traces of 1251_labelled protein precipitable with ammonium sulfate (globulin)and non-precipitable (albumin) on the ratio of binding by normal and lupus serum. Percent binding
12 s i UdR-DNA (2000 cpm) (no additions) + 1251~'-globulin (700 cpm) + 125I-albumin (900 cpm)
Normal
Lupus
0 13 1
75 48 65
]Fable 7 Comparison of the binding of 14C- and 12sIUdR-labelled DNA by normal and lupus sera. Percent binding *
14C-DNA 12s-I-UdR-DNA
Normal
Lupus
2.7 (range: 0-7) 1.0 (range: 0-3)
70 (range: 60 76) 97 (range: 92-98)
* The determinations represent the average value of 23 normal and 8 lupus sera. 4 determinations. The results indicate that the two preparations are comparable in both reactivity and specificity.
4. DISCUSSION The above results with 14C-labelled DNA confirm the work of Pincus et al. (1969). This reagent used in the measurement of anti-DNA-antibody provides a most sensitive and specific test for SLE. The results with chemically iodinated DNA were not comparable to those with the 14C product, in that normal serum gave high binding values (up to 30% in some runs) and lupus serum sometimes gave relatively low binding values (down to 40% in some cases). The apparent cause of both high normal and low lupus values was the presence of protein impurities in the DNA. Since protein is a much better substrate for iodination than DNA, even a small amount of protein would be labelled disproportionately with 125I. Purification procedures would be arduous and would never remove every trace of protein. It is of interest that increased iodination of DNA (chemically) reduced the binding in lupus serum. This could be due partly to increased iodination of protein impurities that do not precipitate with ammonium sulfate in the test. However, since the same effect occurred with 14C-DNA iodinated with nonradioactive iodine, other factors may be involved, lodination, by the chemical procedure described, yield 5-substituted iodocytosine in DNA (Commerford, 1971), and this may distort
Methods o f labelling DNA used in DNA-Ab radioirnmunoassay
7
the binding site and make it less reactive with antibody. Iodine incorporated via IUdR may also induce such alteration but to a lesser extent, presumably because less iodine is incorporated per molecule. It is also of interest that increased internal irradiation (by higher specific activity and prolonged storage) increased binding in normal serum (Hughes, 1971). The reduced molecular weight of the DNA (Tan et al., 1966; Leon et al., 1973) may be responsible for this, but effect on labelled protein impurities (cross-linking) resulting in more of these proteins precipitating with the ammonium sulfate could also be a factor. Breakdown of DNA in the body and subsequent binding to protein moieties, especially of fragments damaged by ultraviolet irradiation after exposure to sunlight (Tan, 1968), might result in immunogenic complexes of DNA, which could stimulate antibody production (Stollar and Sandberg, 1966; Tan et al., 1966; Stollar, 1970) and could be the etiologic mechanism for lupus. The results with 125IUdR.labelled DNA indicated that this product is equal to the 14C-labelled DNA in reactivity and specificity. Since the test procedure with the gamma-labelled DNA is much simpler, the use of 1251-DNA in the anti-DNAantibody test is the method of choice. In addition, the same system may be used for a very sensitive determination of DNA in nanogram quantities (Wikman-Coffelt, 1972). For this purpose, chemically iodinated DNA of high specific activity is a good reagent and protein contamination and label may not interfere in the test. It has been reported that the levels of DNA in serum increase during active episodes of SLE. In other conditions, such as leukemia, lymphosarcoma and liver disease, low levels of DNA have been detected (Carr, 1969). It might be important to monitor these levels during treatment in order to evaluate the response. Although no DNA was detected in the serum of patients with multiple myeloma, rheumatoid arthritis and other cliseases it is possible that only with a radioimmunoassay would their levels be determined. Work along these lines is in progress in this laboratory.
5. SUMMARY Chemical iodination of DNA with Na1251 will also label trace impurities of protein which take up a disproportionate amount of radioactivity. Biological labelling of DNA by growing cells of a strain of E. coli in the presence of [ 125i] iododeoxyuridine yields a superior reagent, since no label is introduced in material other than DNA. Therefore extensive purification of the product is not necessary. The lower specific activity of 125IUdR-DNA compared to 125I-DNA is not a disadvantage, since the damage by self-irradiation is reduced. In the assay for DNAantibody levels, it is shown that high specific activity and prolonged storage increase the non-specific binding of DNA by components of normal serum. On the other hand, the specific binding by the antibodies in SLE sera is not affected. The binding of 125I-DNA by normal sera ranges from 5 to 20% and from 40 to 70% by
8
S.A. LEON et al.
lupuS sera. When t h e same sera are t e s t e d w i t h 1 2 5 I U d R - D N A ,
the values are
0 - 3 % for n o r m a l a n d 9 2 - 9 8 % for lupus sera.
ACKNOWLEDGEMENT We are grateful t o D.I. Martin, M.Sc., for his able assistance.
REFERENCES Carr, R.I., 1969, DNA antibodies and DNA in serum, Ph.D. Thesis, Rockefeller University, New York. Commerford, S.L., 1971, Biochemistry 10, 1993. Davis, B.D. and E.S. Mingioli, 1950, J. Bacteriol. 60, 17. Hanawalt, P.C., 1967, in: Methods in Enzymology, eds. L. Grossman and K. Moldave (Academic Press, New York) p. 7 0 2 - 7 0 8 . Hughes, G.R.V., 1971, Lancet Oct. 16, 861. Leon, S.A., G.J. KoUmann and B. Shapiro, 1973, Int. J. Radiat. Biol. 23,325. Marmur, J., 1961, J. Mol. Biol. 3,208. Pincus, T., P.H. Schur, J.A. Rose, J.L. Decker and N. Talal, 1969, New Engl. J. Med. 281,701. Seligmann, M. and R. Arana, 1968, in: Nucleic acids in immunology, eds. O.J. Plescia and W. Braun (Springer-Verlag, New York) p. 9 8 - 1 1 3 . Stollar, B.D., 1968, in: Nucleic acids in immunology, eds. O.J. Plescia and W. Braun (SpringerVerlag, New York) p. 114-124. Stollar, B.D., 1970, Biochim. Biophys. Acta 209,541. Stollar, B.D. and A.L. Sandberg, 1966, J. Immunol. 96,755. Studier, F.W., 1965, J. Mol. Biol. 11,373. Tan, E.M., 1968, Science 161, 1353. Tan, E.M., P.H. Schur, R.I. Cart and H.G. Kunkel, 1966, J. Clin. Invest. 45, 1732. Wikman-Coffelt, J., 1972, Biochem. Biophys. Res. Commun. 48,502.