Binding of antinucleoside antibody to DNA

lmmunochemistry, 1974, Vol. 11, pp. 321 324. Pergamon Press. Printed in Great Britain

B I N D I N G OF ANTINUCLEOSIDE A N T I B O D Y TO D N A D. S E N I T Z E R , * B. F. E R L A N G E R and S. M. BEISER~" The Department of Microbiology, Columbia University, 630 West 168th Street, New York, N.Y. 10032, U.S.A. (Received 29 October 1973)

Abstract--Reactions between radiolabeled heat-denatured E. coli DNA and antibodies specific for each of the four naturally occurring mononucleosides were detected by the modification of the Farr procedure using antigamma globulin to precipitate the antigen-antibody complex. Nonimmune serum binds less than 5 per cent of the radiolabeled DNA. The homologous hapten is at least 103 times more inhibitory than the heterologous haptens. Formaldehyde-denatured DNAs are also effective inhibitors. M. lysodeikticus DNA:~ (72 per cent G-C), denatured in the presence of 1% formaldehyde inhibited anti-cytidine better than did CI. perfringens DNA (30 per cent G-C) similarly denatured. The reverse was obtained with antithymidine. Thus DNAs of varying base composition can be differentiated from each other.

INTRODUCTION

MATERIALS AND M E T H O D S

Antibodies that react with deoxyribonucleic acid (DNA) were first found in sera of patients with lupus erythematosus (Ceppelini et al., 1957; Robbins et al., 1957; Seligmann, 1957). They have since been raised experimentally in animals by various techniques (cf. Levine et al., 1960; Butler et al., 1962; Plescia et al., 1964; Erlanger and Beiser, 1964). It has, therefore, become feasible to apply highly sensitive and specific immunochemical techniques to the study of nucleic acids and related substances (For reviews on this subject, see Plescia and Braun, 1968; Erlanger et al., 1972). The purpose of this investigation was to develop a sensitive radioimmunoassay for nucleosides, and to use this assay to distinguish among D N A molecules of different base composition. In order to accomplish this, antibodies were prepared to each of the naturally occurring purines and pyrimidines present in D N A (Erlanger and Beiser, 1964). The capacity of each antibody to bind denatured, tritiated D N A was quantitated and inhibition by hapten was studied. Inhibition of binding was found to be highly specific for the homologous haptens, heterologous haptens being more than a thousand-fold less active. Inhibition of binding by various other denatured DNA's, was also studied and a correlation was found with base composition.

D N A Preparations DNA preparations were obtained from Sigma Chemical Corporation and were further purified as described by Marmur (1961). Radiolabelled E. coil D N A Escherichia coil C600 was grown in a minimal medium consisting of Tris, 12 g; KC1, 2 g; NH~C1, 2g; MgC12 •6H20 , 0.5 g; Na2HPOa, 0.185 g; Na2SOa, 0.35 g; glucose, 5 g; casein hydrolysate, 0"1%, thiamine, 0.05 g; H20, 1000 rnl (pH adjusted to 7 with concn HC1). A sterile aqu. soln. of tritiated thymidine (Schwarz/Mann; S.A. 14.3 c/raM) was added at the beginning of logarithmic growth. Growth was terminated when the absorbance at 450 nm of the culture had increased three-fold. Radiolabelled DNA was isolated and purified by a modification of the method of Marmur (1961). In this modification, 250 units of T1 ribonuclease (RNase) (Worthington) and 50 #g of pancreatic RNase (Sigma) per ml DNA were incubated at 37°C for 30 min. D N A conch, was determined by a modification of the Dische diphenylamine reaction (Burton, 1956) using deoxyadenosine as standard. The DNA used was greater than 95 per cent precipitable in cold 5% trichloroacetic acid. Denaturation of the radiolabelled DNA was accomplished by heating solutions containing 2 #g DNA/ml of SSC 10.15 M NaCI, 0-015 Na Citrate p-H 7'0) at 100°C for 10 min and then rapidly cooling in an ice bath. Th~ DNA preparations that were used as inhibitors were denatured as described above, but in the presence of 1% formaldehyde (HCHO), and then dialyzed at 4°C against SSC until free formaldehyde was removed, as determined by its reaction with chromotropic acid as described by Feigel (1946). Denaturation of DNA preparations was monitored by plotting the hyperchromic shift at 260 nm in a Gilford spectrophotometer model 2000.

* Present address: Department of Microbiology, Medical College of Ohio at Toledo, P.O. Box 6190, Toledo, OH 43614, U.S.A. "~This publication is dedicated to the memory of S. M. Beiser. :~ Abbreviations used: DNA, deoxyribonucleic acid; RNI ramunoc heraistry ase, ribonuclease; SSC, standard saline-citrate; HCHO, formaldehyde; 3H-DNA, tritiated DNA; TBS, Tris-buffered Nucleosides were conjugated to BSA by the method of saline; G-C, guanine plus cytosine; Anti-T, anti-thymidine; Erlanger and Beiser (1964). Rabbits were immunized (ErlanAnti-C, anti-cytidine. ger and Beiser, 1964) and the antisera fractionated with 321

322

D. SENITZER, B. F. ERLANGER and S. M. BEISER

Na2SO4, to isolate v-globulin. The assay was performed with a dilution of nucleoside antisera that bound approx. 50 per cent of the added denatured, aH-DNA. Dilutions of antibody, DNA and haptens were made in TBS (Tris-buffered saline: 0.01 M Tris-HC1, 0.15 M NaC1, pH 7.4). Hapten or unlabelled DNA (HCHO denatured and dialyzed) was incubated with 50 #1 of an appropriate dilution of antibody for one hour at 4°C, then 3H-DNA (0.1 #g/50/zl) was added and the mixture kept at 4°C overnight. A quantity of sheep anti-rabbit gamma globulin was added which was 20 per cent more than required to precipitate all the rabbit antibody. After a further incubation at 4°C overnight, the precipitate was collected by centrifugation, washed twice with cold TBS, dissolved in Soluene (Packard Instruments) and counted in a 10 ml Omnifluor (New England Nuclear)using a Picker Ansitron scintillation counter. Binding was determined~ after correcting for quenching, by comparing counts precipitable by cold 5~ TCA with those found in the immune precipitate. Non-specific precipitation was never more than 5 per cent.

specifically and quantitatively inhibited by the homologous hapten in the presence of a mixture of nucleosides (as would be found in DNA) experiments were performed using a mixture of four haptens. The concentration of one hapten was increased to 100 times the concentration of the other three. Table 1 indicates

RESULTS AND DISCUSSION

no significant change in the number of moles of homologous hapten required to produce 50 per cent inhibition. Unlabelled D N A preparations from various sources were then examined for their ability to compete for specific antibody. Because the immunochemical reactivity of D N A is dependent upon its mol. wt (Murakami et al., 1961; Healy et al., 1963) and upon its degree of denaturation (Kohn et al., 1966), these parameters must be taken into account when relating reactivity with base composition. D N A was heat denatured in the presence of 1% formaldehyde to ensure strand separation of D N A containing a high guanine--cytosine

Table 1. Inhibition of anti-T by hapten mixtures Haptens Thymidine Adenosine Guanosine Cytidine

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The capacity of the nucleoside antibody preparations to bind denatured aH-DNA from E. coli C600 was measured by the double antibody technique. A dilution of antibody specific for each of four nucleosides was chosen such that from 30-50 per cent of the added D N A was bound in the absence of inhibitor. The specificity of the preparations were examined by hapten inhibition (Fig. 1). In each case the homologous hapten was at least 103 times more inhibitory than the heterologous haptens. In order to demonstrate that the binding could be

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Fig. 1. Hapten-inhibition of the binding between 50/A of antinucleoside sera and 0.1 #g heat denatured aH-DNA. Antisera dilutions were chosen at which 30-50 per cent of the aH-DNA would be bound. Inhibitor (50 #l) was added to the antinucleoside sera and incubated at 4°C for 1 hr before the aH-DNA was added. The tubes were incubated overnight at 4°C betbre sheep anti-rabbit gamma globulin was added at an amount sufficient to precipitate all added rabbit gamma globulin. The experiment was incubated overnight at 4°C. Precipitates were washed and radioactivity determined. O, adenosine; O, cytidine; &, guanosine; A, thymidine; x, uridine; I~, guanosine monophosphate.

Binding of Antinucleoside Antibody to DNA (G-C) content. Figure 2 depicts remelting curves which indicate little or no secondary structure remaining in any of the denatured DNA preparations. Figure 3 d~epictsthe results of inhibition experiments using two different DNA preparations. Anti-T binding was reduced by as much as 56 per cent by Clostridial DNA; Micrococcal DNA produced only 29 per cent inhibition. In Fig. 4, employing anti-C, the reverse order of inhibition was obtained, i.e. the high G-C DNA produced better than 70 per cent inhibition while the low G - C DNA produced 25 per cent inhibition. The difference in G - C content of the two DNA preparations used in preceding experiments is approximately 40 per cent. In order to determine whether smaller differences could be detected, the same experiment was performed using DNA from M. lysodeikticus,

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Cl. perfringens and calf thymus (45 per cent G ~ ) . Inhibition by anti-C was in accord with the G - C content of the DNAs (Fig. 5): M. lysodeikticus DNA produced 51 per cent inhibition, calf thymus, 37 per cent and Cl. perfringens, 14 per cent. The DNA preparations used in the above experiments had the following $20,w values: E. coil - 13.4, M. Iysodeikticus - 18"0 and C. perfringens - 19"8. We can conclude then that the observed differences in reactivity cannot be ascribed either to different states of denaturation or to molecular size. We have thus been able to demonstrate the specificity of our antisera using a relatively simple double antibody technique. We expect this technique to be of

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324

D. SENITZER, B. F. ERLANGER and S. M. BEISER

value in quantifying nucleoside concentrations in complex mixtures such as sera. We have also demonstrated that D N A s of varying base composition can be differentiated from each other. Such a capability may be of value in the study of the composition of D N A in sera containing circulating nucleic acid-antibody complexes (of. Andres et al.).

Acknowledgement--This work was supported in part by NIH Grant AI-06860-08. REFERENCES

Andres G. A., Aceinni L., Beiser S. M., Christian C. L., Cinotti G. A., Erlanger B. F., Hsu K. C. and Seegal B. C. (1970) J. clin. Invest. 49, 2106. Burton K. (1956) Biochera. J. 62, 315. Butler V. P., Beiser S. M., Erlanger B. F., Tanenbaum S. W., Cohn S. and Bendich A. (1962) Proc. natn. Acad. Sci. U.S.A. 48, 1597.

Ceppellini R., Polli E. and Celuda F. (1957) Proc. Soc. exp. Biol. 96, 572. Erlanger B. F. and Beiser S. M. (1964) Proc. hath. Acad. Sci. U.S.A. 49, 662. Erlanger B. F., Senitzer D., Miller O. J. and Beiser S. M. (1972) Acta endocr., suppl. 168, 206. Feigel F. (1946) Qualitative Analysis by Spot Tests, p. 395. ~rth-Holland, Amsterdam. Healy J. W., Stollar D., Simon M. I. and Levine L. (1963) Archs Biochem. 103, 461. Kohn K. W., Spears C. L. and Doty P. (1966) J. molec. Biol. 19, 266. Levine L., Murakami W. T., van Vunakis H. and Grossman L. (1960) Proc. hath. Acad. Sci. U.S.A. 46, 1038. Marmur J. (1961) J. molec. Biol. 3, 208. Murakami W. T., van Vunakis H., Grossman L. and Levine L. (1961) Hrology 14, 190. Piescia O. J. and Braun W. (1968) Nucleic Acids in Immunology. Springer, New York, N.Y. Plescia O. J., Braun W. and Palczuk N. C. (1964) Proc. hath. Acad. Sci. U.S.A. 52, 279. Robbins W. C., Holman H. R., Deicher H. and Kunkel H. G. (1957) Proc. Soc. exp. Biol. 96, 575. Seligmann M. (1957) C.R. Acad. Sci. 245, 243.