Volume 5 Number 12 December 1978
Volume 5 Number 12 December 1978
Nucleic Acids Acids Research Research Nucleic
Antibodies elicited by defmed oligodeoxyribonucleotide sequences
H.J.Storl+, H.Simon+ and H.Barthelmes&+ -Academy of Sciences of GDR, Res. Cent. of Mol. Biol. and Med., Central Institute of Microbiology and Experimental Therapy, DDR - 69 Jena, Beutenbergstr. I1, and (Clinic of Dermatology, Fac. Med. (CharitT), Humboldt - University, DDR - 104 Berlin, Schumannstr. 20-21, GDR Received 26 July 1978
ABSTRACT
Antibodies to the oligodeoxyribonucleotides d(pT) ,
d(pT)4,
and d(pA-A.T-T) were elicited in rabbits by inmunization &ith electrostatic complexes of the respective haptens with methylated bovine serum albumin (MBSA). The antisera were assayed by complement fixation using denatured DNA's of various sources as antigens. The specificities of the antibodies were determined by estimating the inhibition of the complement fixation reaction by defined oligodeoxyribonucleotides. The antibodies were shown to be specific for the sequence of the oligodeoxyribonucleotides or parts of it.
d(pT)
INTRODUCTION The interaction of proteins with nucleic acids represents a very important feature of the regulation, reproduction and expression of genetic informations involved in DNA. The function of site-specific regulatory proteins, polymerases, modification and restriction enzymes is based on the remarkably accurate recognition of particular nucleotide sequences. A similar very high level of interaction specificity is developed by the immune system, which is capable of recognizing a tremendous variety of antigenic determinants and to respond with the formation of corresponding specific antibodies. Numerous studies have shown, that antibodies reactive with DNA can be elicited experimentally by immunization with bases, nucleosides, nucleotides, oligonucleotides or polynucleotides, covalently coupled to a protein carrier or with electrostatic complexes between a basic protein and oligo- or polynucleotides (1 - 6). In connection with the general problem of the specific interaction of proteins with nucleotide sequences, antibodies to oligonucleotides are of particular interest. The ¢) Information Retrieval Umited 1 Falconberg Court London Wl V 5FG England
4919
Nucleic Acids Research feasibility of producing antibodies to ribodinucleotides and -trinucleotides has already been shown by various investiga-. tors (7 - 15). Only few data are available on antibodies to oligodeoxyribonucleotides and their reactivity with nucleotide sequences in the macromolecular DNA (4, 16 - 18). Among the wide variety of DNA determinants existing in SLE sera, STOLLAR et al. (19) were able to identify oligodeoxythymidylic acids as antigenic determinants for two lupus erythematosus sera. In this paper we describe the experimental production of antibodies to oligodeoxythymidylic acids of defined length, as well as to the sequence d(pA-A-T-T), their reaction with denatured DNA's using complement fixation reactions, and examination of the specificities of the antibodies by inhibition studies. MATERIALS AND METHODS
Oligodeoxynucleotides were prepared and kindly supplied by Dr. R. Weiss and E. Birch-Hirschfeld, ZIMET Jena. Nucleosides, mononucleotides and polynucleotides were purchased from Reanal, Miles, Calbiochem and PL-Biochemicals. High molecular weight DNA's from various sources (protein content 0,1 - 0, 7%) were obtained through the courtesy of E. Sarfert and coworkers, ZIMET Jena. Details of the DNA isolation methods and analytical data have been described previously (20, 21). Conjugates of periodate oxidized ribonucleotides with proteins were prepared according to (3). Under our experimental conditions about 15 moles mononucleotides and 4 to 6 moles oligoribonucleotides were bound per mole bovine serum albumin as determined by ultraviolet difference spectra. Tetrathymidylic acid was coupled to human serum albumin using the carbodiimide procedure (4). lmmunogens were prepared by electrostatic interaction of the oligodeoxyribonucleotides d(pT)3, d(pT)4, d(pT)6 and d(pA-A-T-T) with methylated bovine serum albumin (MBSA) according to PLESCIA et al. (2). Equal amounts (in weight) of oligonucleotide and carrier protein were used. The formation of insoluble material was considerably diminished compared to DNA .MBSA complexes prepared in the same manner. 4920
Nucleic Acids Research All antisera were prepared in random-bred rabbits following injections of 500/ug complex in complete Freund's adjuvant into the foot pads and 500/ug of the same emulsion intraocularly. Reimmunization was done four weeks later under the same conditions as for the primary immunization. After four weeks the animals were boostered i.v. four times with increasing doses (150 to 600/ug complex). Five days later the animals were bled by heart puncture and the sera stored at -20 0C. From the antisera those were selected, which were not anticomplementary. The reactivity of the antisera with denatured DNA's was investigated by micro-complement fixation reaction (22) in a total volume of 7,0 ml. The samples were incubated at 40C for 18 to 20 hrs. DNA used as antigen in complement fixation reactions was made up from stock solutions (500 to 800/ug DNA per ml) with diatilled water to a concentration of 4/ug DNA/ml. The samples were denatured by heating in a boiling water bath for 15 min. and then quickly chilled in ice. Then the DNA solutions were brought to about 0,15 M NaCl by dilution with an equal volume of 0,3 M NaCl. Inhibition assays with various inhibitors were performed by addition of 1 ml of different dilutions of the tested inhibitors instead of 1 ml buffer to the incubation mixture. In all experiments controls without antiserum as well as without antigen were included. The percentage of inhibition was calculated from the percentage of complement fixatior in presence and absence of inhibitors. Concentration of standard solutions of inhibitors was determined from their UV absorption. RESULTS Complexes of d(pT)3, d(pT)4 and d(pT)6 with MBSA were used for the production of antibodies in rabbits. These oligonucleotides were chosen, because it has been assumed that the determinant size on DNA may be in the range of tetra- to heptanucleotides and oligothymidylic acids of this length may serve as sequential determinants for antibodies found in special lupus erythematosus (LE) sera, which react with single-stranded 4921
Nucleic Acids Research portions' of DNA (19). In addition, antibodies to the sequence d(pA-A-T-T), known to be a part of the recognition sequence for the specific endonuclease Eco R I, were produced. At a dilution of 1 : 50 all sera reacted with denatured DNA. The isolation of DNA from bacteria of the Zymosarcina-group can lead to impure products (21). It has been shown that antibodies to the protein impurities can be elicited after immunization of rabbits with DNA preparations from Sarcina maxima, complexed to MBSA (23). To be sure that the serological reaction of our Sarcina maxima DNA with the anti-oligodeoxynucleotide sera involves nucleic acid determinants, the influence of pronase as well as DNAase on the complement fixation reaction with anti-d(pT)3 was investigated (fig. 1). Pretreatment of DNA with pronase results in an increase of the reaction which suggests that degradation of some residual protein in DNA from Sarcina maxima makes additional binding sites accessible for the antibodies. On the other hand, complement fixation is practically abolished by digestion of the DNA with DNAase. The same sensitivity against DIiAase digestion has been observed with DNA of the other sources, but only a slight effect of pronase occurred.
100
, Complement fixation reac-
tions of anti-d(pT)3 with Sardenatured DNA from cina
maxima
.(o -.o)
denatured DNA from
Sarcina
ma-
~~~x ima
t/(--*)
pretreatment of
c l /(s-) a'n /
pretreatment of
4922
DNA with
pronase
DNA with DNAase
Nucleic Acids Research Since our previous studies with an antiserum to the (A+T)rich DNA from Sarcina maxima had shown quantitative differences in the serologic activities of DNA's of various sources, dependent on their (A+T) content (23), the reaction of the anti-oligodeoxynucleotide antibodies was assayed with several DNA's. Because of anticomplementarity of some of our denatured DNA preparations, the highest antigen concentrations used were 1 to 1,3/ug/ml. The results of complement fixation reactions are given in figs. 2 - 5. With all four sera the DNA with the greatest (A+T) content is most reactive. The order of reactivity of the other DNA preparations roughly coincides with the base composition. An exception is found in the reaction of DNA's from E. coli and A-phages. To get more informations as to the nature of the antigenic determinants of the DNA's reacting with the antibodies, some other antigens were tested. The results are listed in tab. 1. Table 1
percentage complement fixation antigen
HSA-d(pT)4 +poly /d(A-T )/poly /d(A-T)/
+poly (dA)poly (dT)
anti-
anti-
d(pT)3
d(pT)4
d(pT)6
d(pA-A-T-T)
65
45
40
n.t.
0
0
0
60
65
40 n.t. n.t.
55
45 55 70
n.t. n.t.
AMP-BSA A-A-BSA poly rA
anti-
anti-
0
0
n.t. n.t. 0
0
antigen: 1,2 ug
double-strafded poly (dA)-poly (dT) and poly /d(A-T)/.poly /d(A-T)/ were denatured as described under methods and imme-
diately transferred into the complement fixation incubation mixture. serum dilution: 1 : 50 n.t. = not tested :
4 ~~~~~~~~~~~~~~~~~~4923
Nucleic Acids Research
t3)
N)J
AD .
.
,
-11
e-o .0 c 60
Qp ¢0
(2 20
0,2
(46
DNA (pg)
1,0
Q2
0,6
N4
1,0
1,C
(g)
Figures 2 - 5 ComplemenT fixation reactions of anti-oligodeoxyribonucleotide sera (1 : 50) with denatured DNA's of various sources. DNA from: (.--*) Sarcina maxima (71 Mol-% (A+T)) (A-.-A&) T2 - phages (61 Mol-% (A+T)) u) i.Proteus mirabilis (58 Mol-% (A+T)) (o-oJ) calf thymus (58 Mol-s% (A+T)) - phages (51 Mol-% (A+T)) (x X)~) (A--*) Escherichia coli (47 Mol-% (A+T)) Micrococcus radiodurans (33 Mol-% (A+T)) ( -V) (c
-V7)
fig. 2: anti-d(pT ) fig. 4: anti-d(pT ) 4924
Streptomyces chrysomallus (28 Mol-% (A+T)) fi& 3: anti-d pT)4 fig. 5: anti-d(pA -A-T-T)
Nucleic Acids Research The antisera to oligodeoxythymidylic acids react with d(pT)4-HSA and regions in poly (dA).poly (dT) remaining unpaired after heating and quenching, but not with alternating d(A-T)-sequences of poly /d(A-T)/.poly /d(A-T)/. On the other hand, anti-d(pA-A-T-T) reacts with both denatured polydeoxynucleotides, with AMP-BSA and ApA-BSA, but not with poly rA. The data suggest that the antibodies are directed to sequences of nucleotides. The specificity of the antibodies was further cleared up by comparing the capacity of several nucleotides and oligonucleotides to inhibit the reaction with DNA. Surprisingly, some of the oligonucleotides with antisera caused complement fixation in absence of DNA at concentrations higher than 50/ug/ml. Therefore, for the inhibition studies in some cases the usable amounts of inhibitors were limited. For this reason no effective inhibition with the anti-d(pT)4 serum could be done. Figs. 6 and 7 demonstrate inhibition analyses with anti-d(pT)3 and anti-d(pT)6. With both systems the DNA antibody binding was effectively inhibited by oligothymidylic acids and, to a less extent, by thymidine and thymine. Other bases, nucleosides and nucleotides, not containing thymine, had no effect (data not shown). Most effective inhibitors were d(pT)5 in the case of anti-d(pT)6 and d(pT)7 with anti-d(pT)3. These findings suggest, that the antibodies actually recognize sequences of thymidylic acids, but the antibody populations are heterogeneous in regard of recognizing the length of the sequences. Table 2 summarizes data on the maximum inhibition obtainable under the experimental conditions used. Fig. 8 shows the -esults of inhibition reactions obtained with anti-d(pA-A-T-T). The purine containing oligonucleotides
d(pA-A-T-T), d(pA-A-A-T), d(pA-A-T) and d(pA)2 are the most effective inhibitors. A decreased affinity of the antibody population towards oligothymidylic acids is observed. Unrelated mono- or oligonucleotides are not inhibiting at all (not shown). This inhibition pattern shows that the anti-d(pA-A-T-T) is not only specific for the whole sequence, but it contains obviously several antibody populations in which a dominant part is of preferred specificity for adjoined adenines.
4925
Nucleic Acids Research
Fi2ures
6 and 7 Inhibition of DNA - anti-oligodeoxyribonucleotide binding by variou1s inhibitors. Inhibitors: (x- x) d(pT)9(9 V) d(pT)5 (rn-u) d(pT)2 'dpT (rs d(pT) (O- O) dT ) 4 ( 0V 3 d(pT T n) ) A d pTT (A-A dEpT 6 Fig. 6: Inhibition of the reaction of anti-d(pT) (1 : 50) with denatured DNA from Sarcina maxima ( 1/ug). Fig. 7: Inhibition of the reaction of anti-d(pT)6 (1: 50) with denatured DNA from Escherichia coli (1ug).
d,p
4926
3T
Nucleic Acids Research Table 2 Maximum inhibition of DNA - anti-d(pT)3 and DNA - antid(pT)6 binding obtainable with the complement fixation system. amount
maximum inhibition (%)
inhibitor
(n Moles)
anti-d(pT)3
anti-d(PT)6
d(pT)9 d(pT)8 d(pT)7 d(pT)6 d(pT)5
18 20 22 27
33 not tested 50 10 19 35 46 24 47 48
13 28 55 55 80 32 52 11 42 40
d(pT)4
d(pT)3 d(pT)2 dT T
33 41 54 80 207 397
DI SCUSSION
For the preparation of antibodies of restricted specificity towards defined oligonucleotide sequences, oligoribonucleotides have been covalently attached to a carrier protein via periodate oxidation (7 - 15) or oligodeoxyribonucleoti(les have been coupled by means of carbodiimides (4, 17, 18). To circumvent the difficulties arising from the sensitivity of these products to RNAase or DNAase we used complexes of oligodeoxyribonucleotides withi MBSA. These complexes were assumed to be highly resistant to the action of DNAase (2), We prepared antisera to d(pT)3, d(pT )4 and d(pT )6 because it had been shown by STOLLAR et al. (19), that such oli-xorners may function as antigenic determinants of denatured DNA for special LE sera. By means of complement fixation we found quantitative differences in reactivity of DNA samples of various provenance. The amounts DNA required for maximum complement fixation are hi. her than with anti-mononucleotide sera (24), sug ;esting that rather a sequence of nucleotides is recogni7zed by the antibodies than 4927
-t)
Nucleic Acids Research
Figure 8 Inhibition of the reaction of denatured E. coli DNA with anti-d(pA-A-T-T) by various inhibitors: (A - ) dA (A-A4) adenine --V) dpA (x ( (i- )d(pA )2 ) d(pA-A-T-T ) )d(pA-A-T) ine dT thym (pT (+-+) d(pA-A-A-T) (a-) d(pT)3 (u-1) d(pT-A) Serum: 1 : 50 dilution, antigen: 1 ug E. coli DIIA
(@
-*(t-)d )2
single bases. This is supported by the data given in table 1 and by the results of inhibition reactions shown in figs. 6 - 8. The specificity of the antibodies is not strongly restricted to the nucleotide sequence used as hapten in the immunization, particularly with reference to the length of the sequence. In the antibody populations some predomninate that recognize portions of the whole sequence. This becomes particularly clear with anti-d(pA-A-T-T) where the purines represent a dominant structure for recognition (cf. table 1, fig. 8). Anti-d(pA-A-T-T) reacts in complement fixation with AMP - BSA and ApA-BSA, but not with poly rA. Other authors, using antimononucleoside sera, were also unsuccessful to demonstrate di4928
Nucleic Acids Research rect reaction of their antisera with RNA (3). The hapten-protein conjugates used as immunogens by these investigators and as antigens in our work have been prepared via periodate oxidation (3). Thereby the ribose moiety is changed and morpholine derivatives are formed. Obviously, the sugar part of the antigen substantially contributes to the serological reactivity. This assumption is supported by the observation that antibodies reacting with RNA can be obtained after immunization with hapten conjugates containing unchanged pentose ring structures (25, 26). The inhibition studies with anti-d(pA-A-T-T) reveal a preferred reactivity of the antibodies with oligodeoxyribonucleotides containing adjacent adenines. The inhibition pattern of d(pA-A-T-T) in comparison to d(pA)2 implies the possibility that several antibody populations exist, some of which recognizing the whole tetranucleotide sequence, others react predominantly with consecutive adenines. However, no attempts have been made to isolate specific antibodies by affinity chromatography. The sites of electrostatic interactions of the oligonucleotides with the carrier in the immunogens play an important part for the antibody specificity. Probably this is the reason for the preferred inhibition of the reaction of anti-d(pT)3 with DNA by d(pT)7, since two adjoining d(pT)3 sequences at the carrier protein could simulate about this sequence length and by this way could lead to the formation of antibodies with corresponding specificities. The sequence d(pT)9 is longer than the determinant size on DNA, wihich has been assumed to be in the range of tetra- to heptanucleotides (19). Therefore, no further increase of the inhibitory effect is observed with d(pT)9 when compared to d(pT)7. The aspects discussed above have also to be taken into consideration for interpretation of the differences in reactivity observed with DNA's of various sources. Moreover, the reaction of the macromolecular antigen with the antibody molecules is greatly influenced by the accessibility of the nucleoside sequences to the antibody binding sites. After denaturation the DNA's exist as randomly coiled structures and the degree of re4929
Nucleic Acids Research activity will be affected by that portion of nucleotides remaining masked after denaturation. The measured reactivity of the DNA's with the anti-oligonucleotide sera must be attributed to the presence of such nucleotide sequences as found out by inhibition analyses.
ACKNOWLEDGMENTS The authors thank Mrs. U. Amthor and Miss C. Thon for expert technical assistance. REFERENCES
(1) Stollar, B.D. (1973) in The Antigens, Sela, M., Ed., Vol. I, pp. 1 - 85. Academic Press, New York (2) Plescia, O.J., Braun, W. and Palczuk, N.C. (1964) Proc. Natl. Acad. Sci. U.S.A. 52, 279 - 285 (3) Erlanger, B.F. and Beiser, S.M. (1964) Proc. Natl. Acad. Sci. U.S.A. 52, 68 - 74
(4) (5) (6) (7) (8) (9)
(10)
(11) (12) (13)
(14) (15) (16)
Halloran, M.J. and Parker, C.W. (1966) J. Immunol. 96, 379 - 385 Lacour, F., Nahon-Merlin, E. and Michelson, M. (1973) Curr. Top. Microbiol. lmmunol. 62, 1 - 39 Stollar, B.D. (1975) Crit. Rev. Biochem. 3, 45 - 69 Beiser, S.M. and Erlanger, B.F. (1966) Cancer Res. 26, 2012 - 2017 Wallace, S.P., Erlanger, B.F. and Beiser, S.M. (1971) Biochemistry 10, 679 - 683 Bonavida, B., Fuchs, S., Sela, M., Rocddy, P.W. and Sober, H.A. (1972) Eur. J. Biochem. 31, 534 - 540 D'Alisa, R.M. and Erlanger, B.F. (1974) Biochemistry 13, 3575 - 3579 D'Alisa, R.M. and Erlanger, B.F. (1976) J. Immunol. 116, 1629 - 1634 Plescia, O.J., Braun. W., Imperator, S., Cora-Block, E., Jaroskova, L. and Schimbor, C. (1968) in Nucleic Acids in Immunology, Plescia, O.J. and Braun, W., Eds., pp. 5 - 17. Springer, New York Leng, M., Drocourt, J.-L., Lavayre, J., Guigues, M. and Michelson, M. (1974) 1982 C. R. Acad. Sci. Paris, Ser. D, 278, 1979 Lavayre, J. and Leng, M. (1976) C. R. Acad. Sci. Paris, Ser. D, 283, 1819 - 1822 Estrada-Parra, S. and Garcia-Ortigoza, E. (1972) Immunochemistry 9, 839 - 842 Plescia, 0.J., Palczuk, N.C., Braun, W. and Cora-Figiero-
ra, E. (1965) Science 148, 1102 - 1103 (17) Khan, S.A., Humayun, M.Z. and Jacob, T.M Nucl. Acids Res. 4, 2997 - 3006
4930
(1977)
Nucleic Acids Research (18) Khan, S.A. and Jacob, T.M. (1977) Nucl. Acids Res. 4, 3007 - 3015 (19) Stollar, B.D., Levine, L., Lehrer, H.I. and Van Vunakis, R. (1 962) Proc. Natl. Acad. Sci. U.S.A. 48, 874 880 (20) Sarfert, E. and Venner, H. (1965) Hoppe-Seylers Z. physiol. Chem. 340, 157 - 173 (21) Sarfert, E. and Venner, H. (1969) Z. allg. Mikrobiol. 9, 153 - 160 (22) Wasserman, E. and Levine, L. (1961) J. Immunol. 87, 290 - 295 (23) Simon, H., Storl, H.J. and Barthelmes, H. (1976) Acta biol. med. germ. 35, 1553 - 1559 (24) Simon, H., Storl, H.J. and Barthelmes, H. (1975) Acta biol. med. germ. 34, 1261 - 1272 (25) Sela, M., Ungar-Waron, H. and Schechter, Y. (1964) Proc. Natl. Acad. Sci. U.S.A. 52, 285 - 292 (26) Ungar-Waron, H., Hurwitz, E., Jaton, J.-C. and Sela, M. (1967) Biochim. Biophys. Acta 138, 513 - 531 -
4931
Volume 5 Number 12 December 1978
Nucleic Acids Acids Research Research Nucleic
Antibodies elicited by defmed oligodeoxyribonucleotide sequences
H.J.Storl+, H.Simon+ and H.Barthelmes&+ -Academy of Sciences of GDR, Res. Cent. of Mol. Biol. and Med., Central Institute of Microbiology and Experimental Therapy, DDR - 69 Jena, Beutenbergstr. I1, and (Clinic of Dermatology, Fac. Med. (CharitT), Humboldt - University, DDR - 104 Berlin, Schumannstr. 20-21, GDR Received 26 July 1978
ABSTRACT
Antibodies to the oligodeoxyribonucleotides d(pT) ,
d(pT)4,
and d(pA-A.T-T) were elicited in rabbits by inmunization &ith electrostatic complexes of the respective haptens with methylated bovine serum albumin (MBSA). The antisera were assayed by complement fixation using denatured DNA's of various sources as antigens. The specificities of the antibodies were determined by estimating the inhibition of the complement fixation reaction by defined oligodeoxyribonucleotides. The antibodies were shown to be specific for the sequence of the oligodeoxyribonucleotides or parts of it.
d(pT)
INTRODUCTION The interaction of proteins with nucleic acids represents a very important feature of the regulation, reproduction and expression of genetic informations involved in DNA. The function of site-specific regulatory proteins, polymerases, modification and restriction enzymes is based on the remarkably accurate recognition of particular nucleotide sequences. A similar very high level of interaction specificity is developed by the immune system, which is capable of recognizing a tremendous variety of antigenic determinants and to respond with the formation of corresponding specific antibodies. Numerous studies have shown, that antibodies reactive with DNA can be elicited experimentally by immunization with bases, nucleosides, nucleotides, oligonucleotides or polynucleotides, covalently coupled to a protein carrier or with electrostatic complexes between a basic protein and oligo- or polynucleotides (1 - 6). In connection with the general problem of the specific interaction of proteins with nucleotide sequences, antibodies to oligonucleotides are of particular interest. The ¢) Information Retrieval Umited 1 Falconberg Court London Wl V 5FG England
4919
Nucleic Acids Research feasibility of producing antibodies to ribodinucleotides and -trinucleotides has already been shown by various investiga-. tors (7 - 15). Only few data are available on antibodies to oligodeoxyribonucleotides and their reactivity with nucleotide sequences in the macromolecular DNA (4, 16 - 18). Among the wide variety of DNA determinants existing in SLE sera, STOLLAR et al. (19) were able to identify oligodeoxythymidylic acids as antigenic determinants for two lupus erythematosus sera. In this paper we describe the experimental production of antibodies to oligodeoxythymidylic acids of defined length, as well as to the sequence d(pA-A-T-T), their reaction with denatured DNA's using complement fixation reactions, and examination of the specificities of the antibodies by inhibition studies. MATERIALS AND METHODS
Oligodeoxynucleotides were prepared and kindly supplied by Dr. R. Weiss and E. Birch-Hirschfeld, ZIMET Jena. Nucleosides, mononucleotides and polynucleotides were purchased from Reanal, Miles, Calbiochem and PL-Biochemicals. High molecular weight DNA's from various sources (protein content 0,1 - 0, 7%) were obtained through the courtesy of E. Sarfert and coworkers, ZIMET Jena. Details of the DNA isolation methods and analytical data have been described previously (20, 21). Conjugates of periodate oxidized ribonucleotides with proteins were prepared according to (3). Under our experimental conditions about 15 moles mononucleotides and 4 to 6 moles oligoribonucleotides were bound per mole bovine serum albumin as determined by ultraviolet difference spectra. Tetrathymidylic acid was coupled to human serum albumin using the carbodiimide procedure (4). lmmunogens were prepared by electrostatic interaction of the oligodeoxyribonucleotides d(pT)3, d(pT)4, d(pT)6 and d(pA-A-T-T) with methylated bovine serum albumin (MBSA) according to PLESCIA et al. (2). Equal amounts (in weight) of oligonucleotide and carrier protein were used. The formation of insoluble material was considerably diminished compared to DNA .MBSA complexes prepared in the same manner. 4920
Nucleic Acids Research All antisera were prepared in random-bred rabbits following injections of 500/ug complex in complete Freund's adjuvant into the foot pads and 500/ug of the same emulsion intraocularly. Reimmunization was done four weeks later under the same conditions as for the primary immunization. After four weeks the animals were boostered i.v. four times with increasing doses (150 to 600/ug complex). Five days later the animals were bled by heart puncture and the sera stored at -20 0C. From the antisera those were selected, which were not anticomplementary. The reactivity of the antisera with denatured DNA's was investigated by micro-complement fixation reaction (22) in a total volume of 7,0 ml. The samples were incubated at 40C for 18 to 20 hrs. DNA used as antigen in complement fixation reactions was made up from stock solutions (500 to 800/ug DNA per ml) with diatilled water to a concentration of 4/ug DNA/ml. The samples were denatured by heating in a boiling water bath for 15 min. and then quickly chilled in ice. Then the DNA solutions were brought to about 0,15 M NaCl by dilution with an equal volume of 0,3 M NaCl. Inhibition assays with various inhibitors were performed by addition of 1 ml of different dilutions of the tested inhibitors instead of 1 ml buffer to the incubation mixture. In all experiments controls without antiserum as well as without antigen were included. The percentage of inhibition was calculated from the percentage of complement fixatior in presence and absence of inhibitors. Concentration of standard solutions of inhibitors was determined from their UV absorption. RESULTS Complexes of d(pT)3, d(pT)4 and d(pT)6 with MBSA were used for the production of antibodies in rabbits. These oligonucleotides were chosen, because it has been assumed that the determinant size on DNA may be in the range of tetra- to heptanucleotides and oligothymidylic acids of this length may serve as sequential determinants for antibodies found in special lupus erythematosus (LE) sera, which react with single-stranded 4921
Nucleic Acids Research portions' of DNA (19). In addition, antibodies to the sequence d(pA-A-T-T), known to be a part of the recognition sequence for the specific endonuclease Eco R I, were produced. At a dilution of 1 : 50 all sera reacted with denatured DNA. The isolation of DNA from bacteria of the Zymosarcina-group can lead to impure products (21). It has been shown that antibodies to the protein impurities can be elicited after immunization of rabbits with DNA preparations from Sarcina maxima, complexed to MBSA (23). To be sure that the serological reaction of our Sarcina maxima DNA with the anti-oligodeoxynucleotide sera involves nucleic acid determinants, the influence of pronase as well as DNAase on the complement fixation reaction with anti-d(pT)3 was investigated (fig. 1). Pretreatment of DNA with pronase results in an increase of the reaction which suggests that degradation of some residual protein in DNA from Sarcina maxima makes additional binding sites accessible for the antibodies. On the other hand, complement fixation is practically abolished by digestion of the DNA with DNAase. The same sensitivity against DIiAase digestion has been observed with DNA of the other sources, but only a slight effect of pronase occurred.
100
, Complement fixation reac-
tions of anti-d(pT)3 with Sardenatured DNA from cina
maxima
.(o -.o)
denatured DNA from
Sarcina
ma-
~~~x ima
t/(--*)
pretreatment of
c l /(s-) a'n /
pretreatment of
4922
DNA with
pronase
DNA with DNAase
Nucleic Acids Research Since our previous studies with an antiserum to the (A+T)rich DNA from Sarcina maxima had shown quantitative differences in the serologic activities of DNA's of various sources, dependent on their (A+T) content (23), the reaction of the anti-oligodeoxynucleotide antibodies was assayed with several DNA's. Because of anticomplementarity of some of our denatured DNA preparations, the highest antigen concentrations used were 1 to 1,3/ug/ml. The results of complement fixation reactions are given in figs. 2 - 5. With all four sera the DNA with the greatest (A+T) content is most reactive. The order of reactivity of the other DNA preparations roughly coincides with the base composition. An exception is found in the reaction of DNA's from E. coli and A-phages. To get more informations as to the nature of the antigenic determinants of the DNA's reacting with the antibodies, some other antigens were tested. The results are listed in tab. 1. Table 1
percentage complement fixation antigen
HSA-d(pT)4 +poly /d(A-T )/poly /d(A-T)/
+poly (dA)poly (dT)
anti-
anti-
d(pT)3
d(pT)4
d(pT)6
d(pA-A-T-T)
65
45
40
n.t.
0
0
0
60
65
40 n.t. n.t.
55
45 55 70
n.t. n.t.
AMP-BSA A-A-BSA poly rA
anti-
anti-
0
0
n.t. n.t. 0
0
antigen: 1,2 ug
double-strafded poly (dA)-poly (dT) and poly /d(A-T)/.poly /d(A-T)/ were denatured as described under methods and imme-
diately transferred into the complement fixation incubation mixture. serum dilution: 1 : 50 n.t. = not tested :
4 ~~~~~~~~~~~~~~~~~~4923
Nucleic Acids Research
t3)
N)J
AD .
.
,
-11
e-o .0 c 60
Qp ¢0
(2 20
0,2
(46
DNA (pg)
1,0
Q2
0,6
N4
1,0
1,C
(g)
Figures 2 - 5 ComplemenT fixation reactions of anti-oligodeoxyribonucleotide sera (1 : 50) with denatured DNA's of various sources. DNA from: (.--*) Sarcina maxima (71 Mol-% (A+T)) (A-.-A&) T2 - phages (61 Mol-% (A+T)) u) i.Proteus mirabilis (58 Mol-% (A+T)) (o-oJ) calf thymus (58 Mol-s% (A+T)) - phages (51 Mol-% (A+T)) (x X)~) (A--*) Escherichia coli (47 Mol-% (A+T)) Micrococcus radiodurans (33 Mol-% (A+T)) ( -V) (c
-V7)
fig. 2: anti-d(pT ) fig. 4: anti-d(pT ) 4924
Streptomyces chrysomallus (28 Mol-% (A+T)) fi& 3: anti-d pT)4 fig. 5: anti-d(pA -A-T-T)
Nucleic Acids Research The antisera to oligodeoxythymidylic acids react with d(pT)4-HSA and regions in poly (dA).poly (dT) remaining unpaired after heating and quenching, but not with alternating d(A-T)-sequences of poly /d(A-T)/.poly /d(A-T)/. On the other hand, anti-d(pA-A-T-T) reacts with both denatured polydeoxynucleotides, with AMP-BSA and ApA-BSA, but not with poly rA. The data suggest that the antibodies are directed to sequences of nucleotides. The specificity of the antibodies was further cleared up by comparing the capacity of several nucleotides and oligonucleotides to inhibit the reaction with DNA. Surprisingly, some of the oligonucleotides with antisera caused complement fixation in absence of DNA at concentrations higher than 50/ug/ml. Therefore, for the inhibition studies in some cases the usable amounts of inhibitors were limited. For this reason no effective inhibition with the anti-d(pT)4 serum could be done. Figs. 6 and 7 demonstrate inhibition analyses with anti-d(pT)3 and anti-d(pT)6. With both systems the DNA antibody binding was effectively inhibited by oligothymidylic acids and, to a less extent, by thymidine and thymine. Other bases, nucleosides and nucleotides, not containing thymine, had no effect (data not shown). Most effective inhibitors were d(pT)5 in the case of anti-d(pT)6 and d(pT)7 with anti-d(pT)3. These findings suggest, that the antibodies actually recognize sequences of thymidylic acids, but the antibody populations are heterogeneous in regard of recognizing the length of the sequences. Table 2 summarizes data on the maximum inhibition obtainable under the experimental conditions used. Fig. 8 shows the -esults of inhibition reactions obtained with anti-d(pA-A-T-T). The purine containing oligonucleotides
d(pA-A-T-T), d(pA-A-A-T), d(pA-A-T) and d(pA)2 are the most effective inhibitors. A decreased affinity of the antibody population towards oligothymidylic acids is observed. Unrelated mono- or oligonucleotides are not inhibiting at all (not shown). This inhibition pattern shows that the anti-d(pA-A-T-T) is not only specific for the whole sequence, but it contains obviously several antibody populations in which a dominant part is of preferred specificity for adjoined adenines.
4925
Nucleic Acids Research
Fi2ures
6 and 7 Inhibition of DNA - anti-oligodeoxyribonucleotide binding by variou1s inhibitors. Inhibitors: (x- x) d(pT)9(9 V) d(pT)5 (rn-u) d(pT)2 'dpT (rs d(pT) (O- O) dT ) 4 ( 0V 3 d(pT T n) ) A d pTT (A-A dEpT 6 Fig. 6: Inhibition of the reaction of anti-d(pT) (1 : 50) with denatured DNA from Sarcina maxima ( 1/ug). Fig. 7: Inhibition of the reaction of anti-d(pT)6 (1: 50) with denatured DNA from Escherichia coli (1ug).
d,p
4926
3T
Nucleic Acids Research Table 2 Maximum inhibition of DNA - anti-d(pT)3 and DNA - antid(pT)6 binding obtainable with the complement fixation system. amount
maximum inhibition (%)
inhibitor
(n Moles)
anti-d(pT)3
anti-d(PT)6
d(pT)9 d(pT)8 d(pT)7 d(pT)6 d(pT)5
18 20 22 27
33 not tested 50 10 19 35 46 24 47 48
13 28 55 55 80 32 52 11 42 40
d(pT)4
d(pT)3 d(pT)2 dT T
33 41 54 80 207 397
DI SCUSSION
For the preparation of antibodies of restricted specificity towards defined oligonucleotide sequences, oligoribonucleotides have been covalently attached to a carrier protein via periodate oxidation (7 - 15) or oligodeoxyribonucleoti(les have been coupled by means of carbodiimides (4, 17, 18). To circumvent the difficulties arising from the sensitivity of these products to RNAase or DNAase we used complexes of oligodeoxyribonucleotides withi MBSA. These complexes were assumed to be highly resistant to the action of DNAase (2), We prepared antisera to d(pT)3, d(pT )4 and d(pT )6 because it had been shown by STOLLAR et al. (19), that such oli-xorners may function as antigenic determinants of denatured DNA for special LE sera. By means of complement fixation we found quantitative differences in reactivity of DNA samples of various provenance. The amounts DNA required for maximum complement fixation are hi. her than with anti-mononucleotide sera (24), sug ;esting that rather a sequence of nucleotides is recogni7zed by the antibodies than 4927
-t)
Nucleic Acids Research
Figure 8 Inhibition of the reaction of denatured E. coli DNA with anti-d(pA-A-T-T) by various inhibitors: (A - ) dA (A-A4) adenine --V) dpA (x ( (i- )d(pA )2 ) d(pA-A-T-T ) )d(pA-A-T) ine dT thym (pT (+-+) d(pA-A-A-T) (a-) d(pT)3 (u-1) d(pT-A) Serum: 1 : 50 dilution, antigen: 1 ug E. coli DIIA
(@
-*(t-)d )2
single bases. This is supported by the data given in table 1 and by the results of inhibition reactions shown in figs. 6 - 8. The specificity of the antibodies is not strongly restricted to the nucleotide sequence used as hapten in the immunization, particularly with reference to the length of the sequence. In the antibody populations some predomninate that recognize portions of the whole sequence. This becomes particularly clear with anti-d(pA-A-T-T) where the purines represent a dominant structure for recognition (cf. table 1, fig. 8). Anti-d(pA-A-T-T) reacts in complement fixation with AMP - BSA and ApA-BSA, but not with poly rA. Other authors, using antimononucleoside sera, were also unsuccessful to demonstrate di4928
Nucleic Acids Research rect reaction of their antisera with RNA (3). The hapten-protein conjugates used as immunogens by these investigators and as antigens in our work have been prepared via periodate oxidation (3). Thereby the ribose moiety is changed and morpholine derivatives are formed. Obviously, the sugar part of the antigen substantially contributes to the serological reactivity. This assumption is supported by the observation that antibodies reacting with RNA can be obtained after immunization with hapten conjugates containing unchanged pentose ring structures (25, 26). The inhibition studies with anti-d(pA-A-T-T) reveal a preferred reactivity of the antibodies with oligodeoxyribonucleotides containing adjacent adenines. The inhibition pattern of d(pA-A-T-T) in comparison to d(pA)2 implies the possibility that several antibody populations exist, some of which recognizing the whole tetranucleotide sequence, others react predominantly with consecutive adenines. However, no attempts have been made to isolate specific antibodies by affinity chromatography. The sites of electrostatic interactions of the oligonucleotides with the carrier in the immunogens play an important part for the antibody specificity. Probably this is the reason for the preferred inhibition of the reaction of anti-d(pT)3 with DNA by d(pT)7, since two adjoining d(pT)3 sequences at the carrier protein could simulate about this sequence length and by this way could lead to the formation of antibodies with corresponding specificities. The sequence d(pT)9 is longer than the determinant size on DNA, wihich has been assumed to be in the range of tetra- to heptanucleotides (19). Therefore, no further increase of the inhibitory effect is observed with d(pT)9 when compared to d(pT)7. The aspects discussed above have also to be taken into consideration for interpretation of the differences in reactivity observed with DNA's of various sources. Moreover, the reaction of the macromolecular antigen with the antibody molecules is greatly influenced by the accessibility of the nucleoside sequences to the antibody binding sites. After denaturation the DNA's exist as randomly coiled structures and the degree of re4929
Nucleic Acids Research activity will be affected by that portion of nucleotides remaining masked after denaturation. The measured reactivity of the DNA's with the anti-oligonucleotide sera must be attributed to the presence of such nucleotide sequences as found out by inhibition analyses.
ACKNOWLEDGMENTS The authors thank Mrs. U. Amthor and Miss C. Thon for expert technical assistance. REFERENCES
(1) Stollar, B.D. (1973) in The Antigens, Sela, M., Ed., Vol. I, pp. 1 - 85. Academic Press, New York (2) Plescia, O.J., Braun, W. and Palczuk, N.C. (1964) Proc. Natl. Acad. Sci. U.S.A. 52, 279 - 285 (3) Erlanger, B.F. and Beiser, S.M. (1964) Proc. Natl. Acad. Sci. U.S.A. 52, 68 - 74
(4) (5) (6) (7) (8) (9)
(10)
(11) (12) (13)
(14) (15) (16)
Halloran, M.J. and Parker, C.W. (1966) J. Immunol. 96, 379 - 385 Lacour, F., Nahon-Merlin, E. and Michelson, M. (1973) Curr. Top. Microbiol. lmmunol. 62, 1 - 39 Stollar, B.D. (1975) Crit. Rev. Biochem. 3, 45 - 69 Beiser, S.M. and Erlanger, B.F. (1966) Cancer Res. 26, 2012 - 2017 Wallace, S.P., Erlanger, B.F. and Beiser, S.M. (1971) Biochemistry 10, 679 - 683 Bonavida, B., Fuchs, S., Sela, M., Rocddy, P.W. and Sober, H.A. (1972) Eur. J. Biochem. 31, 534 - 540 D'Alisa, R.M. and Erlanger, B.F. (1974) Biochemistry 13, 3575 - 3579 D'Alisa, R.M. and Erlanger, B.F. (1976) J. Immunol. 116, 1629 - 1634 Plescia, O.J., Braun. W., Imperator, S., Cora-Block, E., Jaroskova, L. and Schimbor, C. (1968) in Nucleic Acids in Immunology, Plescia, O.J. and Braun, W., Eds., pp. 5 - 17. Springer, New York Leng, M., Drocourt, J.-L., Lavayre, J., Guigues, M. and Michelson, M. (1974) 1982 C. R. Acad. Sci. Paris, Ser. D, 278, 1979 Lavayre, J. and Leng, M. (1976) C. R. Acad. Sci. Paris, Ser. D, 283, 1819 - 1822 Estrada-Parra, S. and Garcia-Ortigoza, E. (1972) Immunochemistry 9, 839 - 842 Plescia, 0.J., Palczuk, N.C., Braun, W. and Cora-Figiero-
ra, E. (1965) Science 148, 1102 - 1103 (17) Khan, S.A., Humayun, M.Z. and Jacob, T.M Nucl. Acids Res. 4, 2997 - 3006
4930
(1977)
Nucleic Acids Research (18) Khan, S.A. and Jacob, T.M. (1977) Nucl. Acids Res. 4, 3007 - 3015 (19) Stollar, B.D., Levine, L., Lehrer, H.I. and Van Vunakis, R. (1 962) Proc. Natl. Acad. Sci. U.S.A. 48, 874 880 (20) Sarfert, E. and Venner, H. (1965) Hoppe-Seylers Z. physiol. Chem. 340, 157 - 173 (21) Sarfert, E. and Venner, H. (1969) Z. allg. Mikrobiol. 9, 153 - 160 (22) Wasserman, E. and Levine, L. (1961) J. Immunol. 87, 290 - 295 (23) Simon, H., Storl, H.J. and Barthelmes, H. (1976) Acta biol. med. germ. 35, 1553 - 1559 (24) Simon, H., Storl, H.J. and Barthelmes, H. (1975) Acta biol. med. germ. 34, 1261 - 1272 (25) Sela, M., Ungar-Waron, H. and Schechter, Y. (1964) Proc. Natl. Acad. Sci. U.S.A. 52, 285 - 292 (26) Ungar-Waron, H., Hurwitz, E., Jaton, J.-C. and Sela, M. (1967) Biochim. Biophys. Acta 138, 513 - 531 -
4931
