Immunology Letters, 34 (1992) 31-.36 0165 -- 2478 / 92 / S 5.00 ~) 1992 Elsevier Science Publishers B.V. All rights reserved IMLET 01840

Novel monoclonal antibodies to double stranded D N A that require Ca 2+ or Mg 2+ for their binding Koichi Imaoka ° and Yoshiyuki Kanai Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan (Received 18 May 1992: revision received and accepted 29 June 1992)

1. Summary Murine monoclonal antibody (MoAb) 2B5 (IgG2aK) was obtained by its binding on a solid phase to double-stranded (ds) DNA from one of the mutant CBA/KI(CBA/KIJms-lpr~g/Iprcg) mice which were recently found, in our institute, to develop lymphadenopathy associated with the production of anti-double-stranded (ds) antibodies. MoAb 2B5 was highly specific for dsDNA, as shown by enzyme-linked immunosorbent assay (ELISA). The dsDNA binding of 2B5 was decreased dose-dependently by the chelating agent EDTA, being lost completely with 2.5-5.0 mM EDTA, whereas dsDNA on the solid phase remained intact after incubation with EDTA. Addition of Ca z~ or Mg 2+ to antibody in culture supernatant that had lost dsDNA binding activity by dialysis against Ca 2 + and Mg 2 ÷ -free buffer restored its binding with dsDNA to the original level, indicating that MoAb 2B5 requires Ca 2+ or Mg 2. for its binding with dsDNA. It is unknown whether MoAb 2B5 recognizes new conformational epitopes created in the presence of Ca 2 ÷ or Mg z+, but this MoAb should be useful in studies Key words: Anti-dsDNA monoclonal antibody; CBA/KIJmsIpffg/lpr cs mouse; EDTA; Ca2+/Mg 2÷ Correspondence to: Yoshiyuki Kanai, Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108, Japan.

on the modes of interaction of DNA with antibodies and DNA-binding proteins. Introduction There are several reports on Ca2+-dependent antibodies [1~]. For instance, Maurer et al. [1] and Liberti et al. [2] described Ca2+-dependent antibodies obtained by immunization of sheep with the synthetic random polymer poly(L-Glu6°L-Ala3°-L-Tyrl0)n. Moreover, Favre and Volloton [4] reported the effects of Ca 2+ on the reaction between angiotensin II and anti-angiotensin II antibody. These reports all deal with experimentally induced antibodies against protein antigens. But there are as yet no reports on experimentally induced or naturally occurring divalent cation-dependent antibodies against DNA. During studies on MoAbs obtained from one of the mutant CBA/KI mice (CBA/KIJms-lprCg/ lpffg) recently found to develop general lymphadenopathy associated with the production of anti- dsDNA antibody [7,8], we found that two MoAbs, named 2B5 and lD6, require Ca 2+ or Mg 2+ to bind to d s D N ~ on a solid phase. The characteristics of these two MoAbs seemed to be essentially the same in terms of both their-binding specificities to dsDNA and the divalent cation requirements for this binding. Therefore, we describe here details of only 2B5 with special emphasis on its requirement of Ca 2+ or Mg 2+ for binding to dsDNA.

*Present address: Department of Microbiology, Institute of Public Health, Minato-ku, Tokyo 108, Japan.

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3.

3.1.

Materials and Methods

MoAbs

Murine hybridomas were obtained as described previously [9]. MoAbs 2B5 and 1D6 were products of newly obtained hybridomas from a 9month-old female CBA/KlJms-lprCg/lpr cg mouse, which showed general lymphadenopathy associated with production of anti-dsDNA antibodies [7,8]. The isotype of these MoAbs was IgG2aK. MoAb 2C10 (lgG2bK), which is specific for dsDNA [10], was used as a control. These hybridomas were cultured in Dulbecco's Modified Eagle's Medium ( D M E M ) containing 4.3 g glucose/l and supplemented with 10% fetal bovine serum (FCS), penicillin, streptomycin, non-essential amino acids, 10 mM Hepes buffer and L-glutamine [9]. D M E M is reported to contain approximately 2.0 mM Ca 2- and 0.8 mM Mg 2+ by the supplier (Gibco).

3.2.

Characterizations of MoAbs 2B5 and ID6

The characteristics of the MoAbs for antigen binding were examined by both direct binding ELISA and competitive ELISA as described [9]. In both assays, the culture supernatants of hybridomas were diluted with the culture medium described above in place of so-called dilutor, which is composed of 5% FCS, 5 mM EDTA and Trisbuffered saline (TBS: 25 mM Tris, 140 mM NaCI, pH 7.4) [9]. As a second antibody, alkaline phosphatase-conjugated goat anti-mouse lgG antibody (Sigma) diluted 1:1000 with dilutor was used in assays.

3.3.

Addition of chelating agents

For determination of the effects of chelating agents on the dsDNA bindings of MoAbs 2B5 and 1D6, culture supernatants of hybridomas were supplemented with EDTA solution (pH 7.4) to give the concentrations shown in Fig. 3, and then their dsDNA binding activities were measured by direct binding ELISA, as described [9].

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3.4. Addition o/divalent cations Hybridoma supernatant antibody was precipitated by half saturation with ammonium sulfate (pH 7.4) and dissolved in a small volume of TBS. After extensive dialysis against TBS, the sample was dissolved and made up to its original volume in TBS. Then various amounts of CaCl2, MgCl2 or both were added and dsDNA binding activities were measured by direct binding ELISA as described above. In this experiment Ca 2' and Mg 2" were chosen because the culture medium contained substantial amounts of Ca 2' and Mg 2 ' as described above and because Ca 2 + and Mg 2 ' are major cations in vivo [! l].

3.5.

Effect of EDTA on the immune complex

Hybridoma supernatants were added to dsDNA-coated microtiter plates and incubated for 1 h at room temperature. The plates were then washed three times with TBS containing 0.05% Tween-20 and once with TBS alone, and incubated with the various concentrations of EDTA in TBS shown in Fig. 5 for 1 h at room temperature. The antibody remaining on the plates was then measured by ELISA as described above. 4.

Results and Discussion

Anti-dsDNA antibodies are produced spontaneously in both patients with systemic lupus erythematosus (SLE) and murine models of SLE [12,13], and many monoclonal anti-DNA antibodies have been obtained from murine models of SLE [13]. Recently we found mutant mice, named CBA/KlJms-lprC~/lpr cg, that develop general lymphadenopathy and produce anti-dsDNA antibodies [7,8]. In this study we obtained two MoAbs, 2B5 and I D6, from one of these mutant mice by bindings of the MoAbs to dsDNA coated on a solid phase and examined the characteristics of these MoAbs by ELISA. Direct binding ELISA showed that both 2B5 and l D6 preferred dsDNA to single-stranded (ss) DNA, and did not bind to Z-DNA, poly(ADP-ribose) or poly(I) (Fig. l). Competitive ELISA confirmed that MoAbs 2B5 and 1D6 are both specific for the dsDNA config-

1.0 x

0.5

1 2 Reciprocal

5 of

10 20 Dilution

Fig. I. Binding preference of MoAb 2B5 shown by direct binding ELISA. The polynucleotides used were prepared as described [9]. Means and SD of values in triplicate experiments are shown. O, dsDNA; O, ssDNA; A , Z-DNA; A, poly(ADPfi~sc); D, poly(dT); l , poly(I).

uration, dsDNA being more inhibitory than ssDNA (Fig. 2). The results in these two assays I00

and those in subsequent experiments on cation-requirements were essentially the same for the two MoAbs, so here we present only the data on 2B5. Anti-DNA antibodies have been classified into three groups on the basis of their antigen binding characteristics: (1) those reacting mainly with dsDNA; (2) those reacting with both dsDNA and ssDNA; and (3) those reacting mainly with ssDNA [14]. Stollar and Papalian demonstrated that on folding back, the secondary structure of ssDNA becomes similar to that of dsDNA, so groups 1 and 2 cannot be clearly differentiated [15]. However, we concluded that 2B5 prefers the dsDNA configuration from the results of both direct and competitive ELISAs (Figs. 1 and 2). We found that the dsDNA binding activities of MoAbs 2B5 and ID6 were lost completely after dialysis against TBS or on addition of EDTA to the reaction mixtures. As we thought that these losses of activity might be due to depletion of cations, we tested the effects of adding various amounts of EDTA to the culture supernatants. We found that the dsDNA binding activities of these MoAbs were decreased dose-dependently by EDTA and were completely lost in the presence of 2.5-5 mM EDTA (Fig. 3), whereas that of MoAb 2C10, which was derived from an auto-

-

I00

-

50 ~ 50

0 1

2 5 10 20 Competitor, ~g/ml

Fig. 2. Binding specificity of MoAb 2B5 shown by competitive ELISA. The polynucleotides used were prepared as described [9]. Means of triplicate experiments are shown. O, dsDNA; O, ssDNA; A, Z-DNA; /k, poly(ADP-ribose); El, poly(dT); R , poly(I).

0

L

0.I

~

I

I EDTA.2Na(mM)

~

t

I0

Fig. 3. Effect of EDTA on MoAb 2B5 binding to dsDNA. See text for details. Means and SD of triplicate experiments are shown.

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i .0 [-

0£

0

0.0 5

t 0.5 Cation(raM)

J 5

Fig. 4. Effects o f Ca 2 ~ and M g 2 ' on M o A b 2B5 binding to d s D N A . See text for details. Means and SD o f triplicate experiments are shown. O, Ca 2 " ; C), Mg2+; ~., Ca 2. plus Mg 2+ .

immune MRL/1 mouse and is specific for dsDNA [10], was not affected by EDTA (data not shown). EDTA had similar effects on ssDNA binding by these MoAbs (data not shown). Pretreatment of dsDNA-coated microtiter plates with the same solution did not alter the binding of the MoAbs to the plates, indicating that EDTA did not cause cleavage of dsDNA from the solid phase. As the culture medium used in these experiments contained 2.0 mM Ca 2" and 0.8 mM Mg 2' , these results suggested that Ca 2- and/or Mg 2 ~ was required for the binding of 2B5 and I D6 to dsDNA. In fact, addition of Ca 2' or Mg 2+ to dialyzed MoAbs 2B5 and 1D6 that had lost their dsDNAbinding activities restored these activities dose-dependently: Ca 2+ was more effective than Mg 2 ' in restoring activity and no synergism of the effects of the two cations was observed (Fig. 4). These results indicate that 2B5 and 1D6 MoAbs require Ca 2~ or Mg 2+ for their binding to dsDNA. To confirm this finding, we examined the effect of EDTA on the pre-formed immune complex between 2B5 and dsDNA on the solid phase. As shown in Fig. 5, the dsDNA binding of MoAb 2B5 was decreased by incubation of the complex with EDTA solution. Similar results were obtained with MoAb ID6 (data not shown). In contrast, the dsDNA binding of 2C10, used as a control, was not decreased by incubation of the com34

plex with EDTA, Incidentally, we found that after dissociation of MoAbs with EDTA, the platcs could be used again like newly prepared dsDNAcoated plates. The epitopes of dsDNA recognized by 2B5 or I D6 in the presence of Ca 2- or Mg 2 " were reversible, like those recognized by 2C10, which disappear in the presence of 0.3--0.5 M NaCI and reappear in normal saline [16]. There are several reports on antibodies that require Ca: ' for their binding to target antigens [16]. Liberti et al. showed that Ca 2~ -induced conformational changes in antigens by bridging the carboxyl groups of glutamic acids, resulting in the creation of new antigenic determinants [2]. All the antibodies reported were directed to protein antigens and required only Ca 2 ' , not Mg 2 ", for increase in their binding activities against these protein antigens. Thus 2B5 and I D6 are the first known MoAbs that require Ca 2- or Mg 2+ for binding to dsDNA. Nucleic acids contain four different potential sites for binding of metal ions: negatively charged phosphate oxygen atoms, ribose hydroxyls, base ring nitrogens, and exocyclic base keto groups [11]. Phosphate oxygens and sugar hydroxyls are

100

0

K'4~

t-#

0.1"

,

L

=

1 10 20 EDTA.2Na (raM)

Fig. 5. Effect of EDTA on the pre-formed immune complex of MoAb 2B5 ( A ) or MoAb 2C10 ( 0 ) with dsDNA. Means and SD for triplicate experiments are shown.

preferential binding sites for alkaline earth cations including Ca 2+ or Mg 2+ [11]. Moreover, Mg 2+ is known to increase the stability of the double-helical structure of DNA [17]. From the above considerations we conclude that 2B5 and 1D6 probably recognize new conformational epitopes created in the presence of Ca 2 ÷ or Mg 2.. These MoAbs will be useful for studying the modes of interaction of antibodies and DNAobinding proteins with DNA.

References [1] Maurer, P.H., Clark, L.G. andd Liberti, P.A. (1970) J. Immunol. 105, 567. [2] Liberti, P.A., Callahan, H.J. and Maurer, P.H. (1974) Adv. Exp. Med. Biol. 48, 161. [3] Callahan, H.J. and Maurer, P.H. (1975) lmmunol. Commun. 4, 537. [41 Favre, L. and Volloton, M.B. (1973) Immunochemistry 10, 43. [5] Frankel, M.E., Callahan, H.J. and Liberti, P.A. (1977) Fed. Proc. 36, 1286.

[6] Maurer, P.H. and Callahan, H.J. (1980) Methods Enzymol. 70, 49. [7] Kimura, M., Mohri, H., Shimada, K., Wakabayashi, T., Kanai, Y. et al. (1990) Clin. Exp. Immunol. 79, 123. [8] Matsuzawa, A., Moriyama, T., Kaneko, T., Tanaka, M. Kimura, M., et al. (1990) J. Exp. Med. 171,519. [9] Kanai, Y., Akatsuka, T., Kubota, T., Goto, S. and Stollar, B.D. (1985) Clin. Exp. Immunol. 59, 139. [10] Kubota, T., Akatsuka, T. and Kanai, Y. (1986) Immunol. Lett. 14, 53. Ill] Saenger, W. (1984) in: Principles of Nucleic Acid Structure. Springer Advanced Texts in Chemistry (C. R. Cantor, ed.) p. 201. Springer Verlag, New York, Berlin, Heidelberg, Tokyo. [121 Tan, E.M. (1989) Adv. Immunol. 44, 93. [13] Theofilopoulos, A.N. and Dixon, F.J. (1985) Adv. Immunol. 37, 269. [14] Arana, R. and Seligmann, M. (1967) J. Clin. Invest. 46, 1867. [15] Stollar, B.D. and Papalian, M. 0980) J. Clin. Invest. 66, 210. [16] Kanai, Y. and Kubota, T. (1989) lmmunol. Lett. 22, 293. [17] Eichhorn, G.L. and Shin, Y.A. (1968) J. Am. Chem. Soc., 90. 7323.

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