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. 2000 Nov;101(3):371–377. doi: 10.1046/j.1365-2567.2000.00119.x

Specificity and binding kinetics of murine lupus anti-DNA monoclonal antibodies implicate different stimuli for their production

E R Eivazova *, J M McDonnell †, B J Sutton †, N A Staines *
PMCID: PMC2327084  PMID: 11106941

Abstract

The origin and relative biological importance of the many different DNA-reactive antibodies that appear in systemic lupus erythematosus are not well understood. A detailed analysis of their fine specificity and binding characteristics with DNA is a necessary step in understanding their biology. We have examined here two monoclonal antibodies (mAb) IV-228 and V-88 that are, respectively, characteristic of antibodies, which bind exclusively to single-stranded (ss) DNA and to both double-stranded (ds) DNA and ssDNA. By surface plasmon resonance (SPR) on BIAcore, we characterized the kinetics of binding of each antibody to synthetic ss and ds oligonucleotides. Antibody V-88 and IV-228 showed different patterns of reactivity for both ss and ds oligonucleotides, characterized by distinctly different kinetic parameters. Analysis of their binding kinetics indicates the importance of base composition in defining DNA epitopes, and shows that some epitopes, such as that recognized by mAb V-88, are expressed on dsDNA and ssDNA, whereas others, as recognized by IV-228, are not. The base preferences of V-88 for ds GC-rich structures over AT-rich, and of IV-228 for ss T-rich structures, also reveal distinct differences between these antibodies. We conclude that the different binding properties of the antibodies will relate to their biological activities. The base preferences of the antibodies suggest that they might be induced by different immunological stimuli, such as those that could be provided by the various DNA fragments and structures released during programmed cell death.

Introduction

Anti-DNA autoantibodies are a major component of systemic lupus erythematosus (SLE) and play an important role in the pathology of lupus nephritis. The appearance of these antibodies in humans and in murine models of lupus correlates with the progression of the disease, and by comparison with all the other lupus autoantibodies, those against double-stranded (ds) DNA are thought to be the most pathogenic and involved in the development of renal pathology.1–4 However, due to the systemic character and complexity of the disease, it still remains unclear what exactly are the primary stimuli that drive such autoantibody responses and the mechanisms that regulate the whole pathological process in lupus. Numerous studies on the production of lupus autoantibodies in mice and humans imply the involvement of factors such as genetic background, antibody idiotypes and the antigenicity of bacterial DNA.5–10 We have demonstrated one way in which antibody production might be stimulated: in MRL/Mp-lpr/lpr (MRL) and (NZB × NZW)F1 (BWF1) mice, titres of anti-DNA antibodies correlate with the spontaneous appearance of anti-idiotype antibodies, which were defined by their specificity for synthetic peptides representing sequences of the VH region of anti-DNA monoclonal antibody (mAb) V-88.11 Although both anti-single-stranded (ss) DNA and anti-dsDNA antibodies can be detected in the sera of diseased individuals, it is only the anti-dsDNA antibodies that show a significant correlation with anti-idiopeptide antibody levels. Furthermore, some autoantibodies possess dual specificity for both DNA and anti-DNA antibody idiotopes.12 It is thus possible that the production of anti-dsDNA antibodies is driven by antigenic idiotopes on DNA-binding antibodies.

In the present study, we focused on two DNA binding mAb: V-88 and IV-228, derived from lupus-prone BWF1 and MRL mice, respectively. mAb V-88 is a well characterized and modelled antibody,13 which reacts with both ssDNA and dsDNA, and mAb IV-228 was chosen as a representative anti-DNA antibody with only ssDNA specificity.4,14 It was also demonstrated earlier that these antibodies can bind to renal immune deposits in kidneys of lupus mice.15

To characterize these monoclonal DNA-binding antibodies further, we conducted a study of their specificities and binding kinetics with defined ss and ds oligonucleotides and native DNA, using surface plasmon resonance (SPR) -based biosensor technology (BIAcore). This method provides a powerful and simple approach for direct measurements of the binding between analyte and ligand and its visualization in realtime.16–19

The analysis reveals distinctive differences in the specificities, affinities and binding kinetics of these anti-dsDNA and anti-ssDNA mAbs with ss and ds oligonucleotides. We infer from this that antibodies with specificity for dsDNA are induced by stimuli different from those that induce antibodies with specificity for ssDNA. These autoimmune antibodies could be generated as a result of immune reactions against various DNA fragments which are cleaved and released during the degradation of genomic DNA in cells undergoing apoptosis. This supports the concept that, in such complex systemic autoimmune diseases, there are multiple components and stimuli, which contribute to the development of the disease pathology.

Materials and methods

Monoclonal antibodies with specificity for DNA

Antibody V-88 [immunoglobulin G1κ (IgG1κ)] was derived from an adult female BWF1 mouse.14 It binds both ssDNA and dsDNA and is encoded by a VH 7183 family member and a Vl gene closely related to K5.1.4 Antibody IV-228 (IgG2a) was derived from an adult MRL mouse. It has specificity for ssDNA and does not bind to dsDNA. V-88 and IV-228 immunoglobulins were purified from ascites fluid using ImmunoPure® Immobilized Protein G according to the manufacturer's instructions (Pierce Chemical Company, Rockford, IL, USA).

Preparation of ds and ssDNA

The dsDNA was prepared from calf thymus native DNA (Sigma-Aldrich, Poole, Dorset, UK) first by treatment with S1 nuclease (Sigma) to remove ssDNA and then by phenol–chloroform extraction to remove protein.20 The ssDNA was prepared by heating native DNA in a boiling water bath for 15 min and cooling rapidly. The ratio of optical densities at 260 nm and 280 nm of such preparations (OD260 : OD280) was typically > 1·8, indicating the absence of protein and RNA.

Synthetic 25-mer ss oligonucleotides: (G)25, (C)25, (A)25 and (T)25 were used biotinylated at their 5′ end for SPR and enzyme-linked immunosorbent assay (ELISA) immobilization, and some were made non-biotinylated (Molecular Biology Unit, The Randall Institute) to enable production of ds oligonucleotide fragments by annealing. Double-stranded oligonucleotides (G–C)25 and (A–T)25 were produced using equimolar amounts of ss 25-mer oligonucleotides (with one biotinylated strand) in 0·1 m HEPES + 0·1 m NaCl + 0·1 m ethylenediaminetetraacetic acid (EDTA) by heating in a water bath at 95° for 5 min and leaving the solution to cool at room temperature. To ensure the purity of ds oligonucleotide preparations, they were treated with S1 nuclease as described above.

Anti-DNA antibody ELISA

ELISA for anti-dsDNA and anti-ssDNA antibodies was performed on polystyrene microplates (Nunc, Life Technologies Ltd, Paisley, UK), precoated with poly l-lysine,14 washed with phosphate-buffered saline (PBS), and coated with 50 µl of either dsDNA or ssDNA (10 µg/ml) diluted with PBS and incubated for 1 hr at 37°. The plates were washed in PBS and blocked with 2% w/v skimmed milk powder in PBS + 0·05% Tween-20 (Sigma).

Monoclonal antibodies were diluted with 1% skimmed milk powder in PBS + 0·05% Tween-20 and incubated for 1 hr at 37° with immobilized antigen. Following further washing, a horseradish peroxidase-labelled goat anti-mouse IgG reagent (Jackson Laboratories, Bar Harbor, ME) was added for 1 hr and the reaction was developed by adding O-phenylenediamine (Sigma) in citrate phosphate buffer (0·01 m, pH 6) containing 1 µl/ml H2O2 and incubating plates in the dark at room temperature. The colour reaction was stopped with 0·5 m citric acid and absorbance values were measured at 450 nm using a Titertek Multiskan Plus spectrophotometer (ICN Flow, Irvine, UK).

A biotin–streptavidin ELISA for determination of antibody reactivity with oligonucleotides was developed and optimized for effective microplate coating and resolution. Microplate wells were treated with 50 µl of streptavidin (Sigma) at 3 µg/ml in PBS, and allowed to dry overnight at 37°, then washed in PBS and blocked with 1% skimmed milk powder in PBS + 0·05% Tween-20 for 1 hr at 37°. Then, 5 µg/ml of biotinylated oligonucleotide solution was applied for 2 hr followed by washing with PBS + 0·1% Tween-20 and incubation with primary antibody for 1 hr at 37° and the assay was concluded as described above.

Surface plasmon resonance

The kinetics of antibody binding to immobilized oligonucleotides were determined by the technique of SPR with a BIAcore instrument (Biacore AB, Stevenage, UK). This system has the technical advantage of using unlabelled reactants and allowing real-time monitoring of their interactions, determination of the on- and off-rates and intrinsic affinity of their interaction.16 For these reasons it is highly suited to the analysis of interactions between immunoglobulins from the same species and antibody–DNA binding analysis.

Preliminary experiments were conducted to test various parameters and establish the optimal conditions for the experiments with respect to immobilization density, concentrations, flow rates (of 5–25 µl/min) and association/dissociation times. Specific binding surfaces were prepared by coupling biotinylated ss and ds oligonucleotides (produced as described above), diluted in HBS buffer, pH 7·4 (HEPES 10 mm, NaCl 150 mm, EDTA 3·4 mm) to the streptavidin sensor chip surface (Sensor Chip SA, Pharmacia Biosensor, Stevenage, UK). A range of immobilization densities of ss and ds oligonucleotides on the sensor chip was also initially tested in the experiments but the assays reported here typically used immobilization levels of 500–1000 resonance units (RU). This level was optimized empirically by first testing lower amounts of immobilized ligand. All measurements were performed at a continuous flow rate of 5 µl/min in sterile biospecific inter-action analysis (BIA)-certified HBS buffer (above) containing 0·005% v/v surfactant P-20. Increasing the flow rate had no effect on interaction characteristics. Generally, analytes were injected for 300 seconds, followed by HBS for approximately 400 seconds to observe the dissociation of bound analyte. Regeneration of ligand-bound surfaces after binding of each analyte was achieved with three 60-second pulses of GnHCl 0·8 m at a flow rate of 10 µl/min. The washing agent did not dissociate the streptavidin–biotin complex, and had no effect on the subsequent activity of the chip. Non-specific binding of the analyte to the sensor surface was assessed by performing sample injections onto a streptavidin sensor surface which had no DNA coupled to it. Under these conditions, there was negligible non-specific binding for all of the analytes tested. Binding kinetics were evaluated using the BIA evaluation analysis package (version 2·1, Pharmacia Biosensor) and in-house curve fitting routines using a matlab (MathWorks) platform. Bulk refractive index effects were subtracted from the specific binding prior to kinetic analysis. Fits were performed using simple biomolecular interaction models or more complex biphasic models. These gave satisfactory descriptions of the kinetic characteristics and permitted a fair comparison of kinetics and affinities for the different interactions measured, as shown by the sensorgrams and residual plots. For the purposes of comparing the different binding interactions, kon and koff are each described by a single rate constant, corresponding to a best estimate of the net observed rate constant.21 Simulations of sensorgrams, using kinetic parameters derived from the kinetic analysis were obtained using BIAsimulation software (version 1·1, Pharmacia Biosensor).

Results

Binding of mAbs V-88 and IV-228 with oligonucleotides by ELISA

Both mAbs were tested in ELISA for the ability to react with ss and ds oligonucleotides. They both bound to (G)25, (C)25 and (T)25 but neither of them reacted with (A)25 (Fig. 1a,b). The reactivity of mAb IV-228 for (T)25 was significantly higher than for (G)25 and (C)25, whereas V-88 displayed comparable reactivity with all three ss oligonucleotides. The lack of binding to A25 is not a technical artefact because antibodies in human and murine lupus sera are able to bind A25 in this assay (not shown).

Figure 1.

Figure 1

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Binding of mAb V-88 (a) and IV-228 (b) to 25-mer ss oligonucleotides by ELISA. The mAb were added to biotinylated ss oligonucleotide, immobilized on streptavidin-coated plates, in doubling dilutions. The starting concentration for V-88 was 160 µg/ml, and IV-228 was used from 16 µg/ml, since a much lower amount of IV-228 antibody was required to develop the binding reaction.

Binding of V-88 to ds oligonucleotides showed a very distinct preference for (G–C)25 over (A–T)25 ds oligonucleotides (Fig. 2). There was much higher reactivity with (G–C)25 than with (A–T)25 and binding to native dsDNA was similar to (G–C)25 oligonucleotide binding. This indicates that mAb V-88 preferably recognizes G–C-containing oligonucleotides. There was no binding reaction of antibody IV-228 to dsDNA or ds oligonucleotides.

Figure 2.

Figure 2

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Binding of mAb V-88 to ds (G–C)25-(A–T)25 oligonucleotides and to native dsDNA by ELISA. The starting concentration of V-88, added in double dilutions to biotinylated oligonucleotides on streptavidin-coated plates, and to dsDNA on poly l-lysine coated-plates, was 160 µg/ml. There was no binding reaction of IV-228 to dsDNA or ds oligonucleotides.

Kinetic measurements of mAb V-88 with ss and ds oligonucleotides

The binding kinetics of mAb V-88 with ss and ds oligonucleotides were studied by SPR. The association and dissociation phases of the response curves showed that antibody bound to ss (T)25, (G)25 and (C)25 with similar kinetics. However, V-88 did not bind with ss (A)25. Binding of V-88 to ss oligonucleotides showed a preference for (T)25 with a dissociation constant five times higher than those with (C)25 and (G)25 (Table 1).

Table 1.

Affinity and kinetic data for binding of mAb V-88 to ds and ss oligonucleotides

mAb/ligand kon (m−1 s−1) koff (s−1) Kd (m)
mAbV-88/(G–C)25 2·9 ± 0·52 × 104 5·3 ± 0·73 × 10−4 1·8 ± 0·85 × 10−8
mAbV-88/(A–T)25 1·7 ± 0·43 × 104 2·6 ± 0·79 × 10−3 1·5 ± 0·74 × 10−7
mAbV-88/(T)25 4·3 ± 1·46 × 104 3·1 ± 1·41 × 10−4 7·2 ± 0·57 × 10−7
mAbV-88/(C)25 1·1 ± 0·23 × 104 1·3 ± 0·59 × 10−3 1·2 ± 0·82 × 10−7
mAbV-88/(G)25 1·1 ± 0·24 × 104 1·2 ± 0·51 × 10−3 1·1 ± 0·76 × 10−7
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Comparison of V-88 binding curves with ds oligonucleotides showed that mAb V-88 has a distinct preference for (G–C)25 over (A–T)25. For these experiments the range of V-88 concentrations was the same as for the ss oligonucleotide studies. Sensorgrams clearly showed that the antibody dissociated from (G–C)25 with a much slower rate than from (A–T)25 (Fig. 3a,b). The calculated apparent dissociation rate constants for (G–C)25 (koff = 5·3 × 10−4/second and for (A–T)25 (koff = 2·6 × 10−3/second) give a Kd value for (G–C)25 about 10 times smaller than for (A–T)25. The dissociation constants and the rate constants are summarized in Table 1.

Figure 3.

Figure 3

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Analysis of binding kinetics of mAb V-88 to immobilized ds(G-C)25 (a) and (A-T)25 (b) oligonucleotides by SPR. The mAb V-88 was injected onto the sensor surface in six concentrations starting from 160 μg/ml in doubling dilutions. The immobilization level of ds oligonucleotides on the sensor chip surface was 800 RU. The association and dissociation phases of the interaction were fitted to a biphasic model on binding. The residual plots (lower panel of each figure), obtained by substracting the calculated fit from experimental data, are shown for association and dissociation phases of each binding reaction and represent the highest concentration in each case. The residual values are close to zero and are randomly distributed along the curve.

In control experiments for non-specific binding, when mAbV-88 was injected over the streptavidin-coated chip with no immobilized ligand bound, there was no binding observed. The specificity of the reactions between V-88 and oligonucleotides observed in BIAcore are in good agreement with the ELISA results above.

Kinetic measurements of mAb IV-228 with ss and ds oligonucleotides

Binding assays between mAb IV-228 and oligonucleotides were performed according to the protocol described earlier. Initially, mAb IV-228 was used at the same concentrations as for V-88 antibody but this gave very high responses. The mAb then was tested over a range of concentrations and response curves comparable to those obtained with V-88 were obtained with IV-228 in the range of 1·6–0·1 µg/ml. Consistent with the ELISA results described above, IV-228 antibody reacted well with ss (T)25, (G)25 and (C)25 with a strong preference for (T)25 but did not react with (A)25 (Table 2). The IV-228 reaction with (T)25 was characterized by a very low dissociation rate, much slower than from (C)25 and (G)25 which showed comparable rates of association and dissociation. For (T)25, the calculated apparent dissociation constant Kd = 1·5 × 10−10 was 300 times smaller than the Kd values for (G)25 and (C)25, which were comparable with each other (Table 2). At this range of concentration, mAb IV-228 did not react with either of the ds oligonucleotides, which reveals its specificity for unpaired bases. However, at a higher antibody concentration (160 µg/ml) some binding with ds (G–C)25 up to 100 RU was recorded, but the dissociation rate was 50 times greater than from ss oligonucleotides (data not shown). There was no reaction detected between mAb IV-228 and ds (A–T)25 oligonucleotide.

Table 2.

Affinity and kinetic data for binding of mAb IV-228 to ds and ss oligonucleotides

mAb/ligand kon (m−1 s−1) koff (s−1) Kd (m)
mAb IV-228/(T)25 4·5 ± 1·86 × 105 6·7 ± 0·76 × 10−5 1·5 ± 0·95 × 10−10
mAb IV-228/(G)25 4·3 ± 1·61 × 104 2·1 ± 0·49 × 10−3 4·8 ± 0·58 × 10−8
mAb IV-228/(C)25 4·1 ± 1·85 × 104 1·4 ± 0·75 × 10−3 3·4 ± 0·32 × 10−8
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Discussion

Here, we have characterized for the first time not only the binding specificities, but also the binding kinetics of two anti-DNA mAbs, derived from MRL and BWF1 lupus-prone mice, using 25-mer synthetic ss and ds oligonucleotides by means of SPR. Both V-88 and IV-228 mAb demonstrated specific interaction with (G)25, (C)25 and (T)25 as evidenced by their different kinetic patterns of reactivity. Neither of the analysed mAb reacted with (A)25. Kinetic and affinity data were comparable for both V-88 and IV-228 binding to (G)25 and (C)25, but there was much stronger binding of IV-228 to (T)25 by two orders of magnitude (Table 1). Both mAbs were therefore characterized by obvious base preferences in their binding to ss oligonucleotides, demonstrating that nucleotide base determinants are clearly involved in DNA–antibody interactions.

Antibody IV-228 showed a very strong reactivity against ss oligonucleotides at low concentrations, at which it did not bind to ds oligonucleotides. The highest affinity was seen for the binding of IV-228 with ss oligonucleotide (T)25 but the interactions with (G)25 and (C)25 were also strong. Although at 100-fold higher concentrations, IV-228 displayed some small reaction with ds oligonucleotide (G–C)25, its dissociation rate was very high and there was no reaction with ds (A–T)25 at all.

Antibody V-88 bound well with both ss and dsDNA but showed a clear preference for (G–C)25 over (A–T)25. Its dissociation constant for (G–C)25 was approximately 10 times lower than for (A–T)25, due principally to a much slower dissociation rate from (G–C)25 (Fig. 3a,b).

The results therefore indicate that anti-DNA antibody binding is base dependent and that the sugar phosphate backbone of the DNA does not contribute directly to the epitopes recognized by these antibodies. This is consistent with previous data that these mAb do not bind phospholipids.22 It is also evident that there are conformationally distant epitopes in ss and dsDNA that are recognized differently by the mAbs. For example, IV-228 binds to ssDNA of a certain base composition with high affinity, but binds only very poorly to the same oligonucleotides arranged in a ds structure. Furthermore, these results demonstrate that anti-DNA antibodies display individual patterns of specificity, and these two examples fit with the general observation that while most dsDNA-binding mAbs have a similar affinity for ssDNA, most ssDNA-reactive mAbs have negligible affinity for dsDNA.23 It implies that no two anti-DNA mAbs will react with exactly the same nucleic acid epitopes, and that particular DNA bases are critical for binding. The antibodies studied here are two examples of the diversity of autoantibodies characteristic of systemic lupus disease.

It is likely that the production of DNA-reactive antibodies of diverse specificity is driven by correspondingly diverse antigenic stimuli. The various intracellular components released during apoptosis could provide these immunogenic stimuli.24 One hallmark of apoptosis is DNA fragmentation arising from degradation of nuclear DNA.25–27 However, DNA is heterogeneous in structure, and various nuclear fragments are released sequentially by the apoptotic process that starts with DNA cleavage by caspase-activated DNase.26 This proceeds to excision of high molecular weight DNA fragments from 50 to 300 kb representing DNA loops, rich in G–C sequences.27,28 Fragments rich in A–T, derived from the bases of the DNA loops attached to the nuclear scaffold,29 are released later. The early availability of G–C-rich structures could stimulate anti-dsDNA antibodies with G–C-binding preference, exemplified by V-88 studied here. Later in apoptosis, continuing DNA degradation releases other nuclear structures that would drive production of DNA-binding autoantibodies of different fine specificity,30,31 such as mAb IV-228 that binds specifically to ssDNA. A model of these events in the apoptotic degradation of DNA is described by Fig. 4.

Figure 4.

Figure 4

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Model of apoptotic degradation of DNA initiated by CAD cleavage. The sequence of events starts with cleavage of condensed chromatin by activated CAD nuclease, followed by excision of 30–100 kilobase (kb) loops (with high GC content) and, after subsequent internucleosomal cleavage, formation of oligonucleosomal ladders of different sizes. Other fragments of loops, rich in AT, remain attached to the nuclear matrix and are released later. As the nuclear envelope collapses, various fragments of different sizes and composition are released and represent a source of potential autoantigens to drive autoimmune responses. It is known30 that different cell organelles or their fragments are released in apoptotic blebs, but smaller DNA fragments may escape this mode of elimination and are able to stimulate B cells for autoantibody production.

The diversity of the expressed B-cell repertoire expands during the development of lupus disease32 and our model here predicts the stimulation of B cell responses by nucleic acid structures of diverse conformation and base composition, that are released at different stages of apoptotic fragmentation of DNA. This would contribute to a corresponding diversity in the specificity of expressed B-cell repertoire, reflecting the diversity of the DNA epitopes recognized by the autoantibodies.22

Acknowledgments

This work was supported by grants from Lupus UK and The Arthritis Research Campaign. The authors wish to thank Dr Yegor Vassetzky (CNRS, Montpellier) for helpful discussions.

Glossary

Abbreviations

dsDNA

double-stranded DNA

mAb

monoclonal antibody

ka

association rate constant

kd

dissociation rate constant

Ka

association constant

Kd

dissociation constant

SPR

surface plasmon resonance

ssDNA

single-stranded DNA

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