Pathogenic autoantibodies in systemic lupus erythematosus are derived from both self-reactive and non-self-reactive B cells

Previous studies have shown that both murine and human anti-double-stranded DNA (anti-dsDNA) antibodies can develop from non-DNA-reactive B cells and suggest a crucial role for somatic mutation in dsDNA binding. However, since only a limited number of human anti-dsDNA antibodies have been analyzed previously, we could not exclude other mechanisms for the generation of anti-dsDNA antibodies in patients with systemic lupus erythematosus (SLE). Therefore, we isolated IgM anti-dsDNA antibodies from peripheral blood B cells of a patient with SLE. Three somatically mutated IgM anti-DNA antibodies with pathogenic potential (glomerular binding) were reverted to their germline configuration. Although all three IgM anti-dsDNA antibodies came from the same lupus patient, they displayed different profiles. Reversion to the germline sequence of autoantibodies A9 and B5 resulted in decreased dsDNA binding. In contrast, the germline form of G3-recognized dsDNA as well as the mutated counterpart. These results suggest that mutated IgM anti-dsDNA antibodies may develop from both DNA- and non-DNA-reactive B cells. The implications are that B cell activation occurs in response to self and non-self antigens, while selection after activation may be mediated by self antigen in SLE. Moreover, ineffective tolerance checkpoints may exist before and after antigen activation in SLE.     

Light chain determines the binding property of human anti-dsDNA IgG autoantibodies

We have previously prepared human anti-double-stranded (ds) DNA IgG Fab clones using phage-display technology. Nucleotide sequence analysis of genes of immunoglobulin (Ig) heavy and light chain variable regions in these Fab clones suggested that the DNA-binding activity of the clones depended on light chain usage. To confirm the role of the light chain in antibody binding to DNA, we constructed in the present study's new recombined Fab clones by heavy and light chain shuffling between the original anti-dsDNA Fab clones. Clones constructed by pairing Fdgamma fragments with the light chain from a high DNA-binding clone showed high DNA-binding activities, whereas other constructed clones using light chains from low DNA-binding clones showed low DNA-binding activities. Our results indicate that light chains in anti-dsDNA antibodies can determine the DNA-binding activity of the antibodies. Ig chain shuffling of phage-display antibodies may be useful for investigating the molecular mechanisms for antigen-antibody binding of human autoantibodies.     

SLE血清中抗-DNA抗体分离和性质的研究

本文用国产高分子树脂(T)接枝小牛胸腺DNA,通过亲合层析从系统性红斑狼疮SLE患者血清中纯化出抗-ds DNA抗体和抗-ss DNA抗体。酶联免疫吸附分析(ELISA)的研究表明:SLE抗-DNA抗体和DNA结合的差异性很大,是高度非均一性的。抗-ss DNA抗体不仅组成成分比抗-ds DNA抗体复杂,ss DNA/抗-ssDNA亲合能力也明显高于ds DNA/抗-ds DNA。纯化的抗-DNA抗体以IgG类抗体占主导,同时也有其它类型抗体存在(例如IgM等)。抗-ds DNA抗体有较抗-ss DNA抗体高的IgG含量(两者的IgG/IgM分别是7.0和4.0),说明IgG抗-DNA抗体更倾向于同dsDNA结合。     

Induction of anti-double stranded DNA antibodies in normal mice by immunization with bacterial DNA

Because of recent observations suggesting that bacterial DNA is immunogenic, the induction in normal mice of antibodies to Escherichia coli (EC) dsDNA was investigated. BALB/c and C57BL/6 mice were immunized with dsEC or ds calf thymus (CT) DNA complexed to methylated BSA in adjuvant; antibody responses were measured by ELISA. In both strains, dsEC DNA immunization induced a much higher anti-dsDNA response to dsEC DNA than did dsCT DNA immunization. Neither immunized group showed an appreciable antibody response when tested on dsCT DNA. Anti-dsDNA antibodies were also demonstrated by ELISA using synthetic DNA duplexes as well as a filter binding assay using 3H-labeled dsEC DNA as Ag. These results suggest that bacterial dsDNA is immunogenic and that at least some anti-dsDNA specificities can arise by immunization.     

Detection of masked anti-DNA antibodies in lupus sera by a monoclonal anti-idiotype

We report here the use of a monoclonal anti-idiotype 3I to human anti-DNA antibodies to detect in serum idiotype-positive antigen-binding antibodies lacking DNA-binding activity as measured by conventional antigen binding assays. We studied paired serum samples from 13 patients with systemic lupus obtained at two times in the course of their disease: in each patient, one serum sample has anti-DNA activity and the second serum sample has no anti-dsDNA activity detectable by Millipore filter, ELISA, or Crithidia assay. Reactivity with 3I as detected with a radioimmunoassay (RIA) was present in all 13 sera with anti-dsDNA activity. Six patients showed a decrease in 3I reactivity to normal levels in the second serum sample, in which anti-dsDNA antibodies were not detectable by conventional antigen-binding assays. The other seven patients' second serum sample continued to show elevated 3I reactivity by RIA even though no anti-dsDNA activity was apparent. When the 3I-reactive antibodies from these latter patients' sera were eluted from a 3I affinity column, they revealed DNA-binding activity. Furthermore, dsDNA binding by these sera was apparent when they were displayed on Western blots of isoelectric focusing gels run in 8 M urea and incubated with radiolabeled dsDNA. These results indicate that the 3I anti-idiotype can detect anti-DNA antibodies in some sera of SLE patients that lack anti-DNA activity by ordinary assays. These antibodies may be inhibited in binding dsDNA by excess antigen or autologous anti-idiotype, and their DNA binding activity can be unmasked by procedures promoting immune complex dissociation.     

Stable expression of a recombinant human antinucleosome antibody to investigate relationships between antibody sequence,binding properties,and pathogenicity

When purified under rigorous conditions, some murine anti-double-stranded-DNA (anti-dsDNA) antibodies actually bind chromatin rather than dsDNA. This suggests that they may actually be antinucleosome antibodies that only appear to bind dsDNA when they are incompletely dissociated from nucleosomes. Experiments in murine models suggest that antibody–nucleosome complexes may play a crucial role in the pathogenesis of glomerulonephritis in systemic lupus erythematosus. Some human monoclonal anti-DNA antibodies are pathogenic when administered to mice with severe combined immunodeficiency (SCID). Our objective was to achieve stable expression of sequence-altered variants of one such antibody, B3, in Chinese hamster ovary (CHO) cells. Purified antibodies secreted by these cells were tested to investigate whether B3 is actually an antinucleosome antibody. The pathogenic effects of the antibodies were tested by implanting CHO cells secreting them into SCID mice. Purified B3 does not bind to dsDNA unless supernatant from cultured cells is added, but does bind to nucleosomes. The strength of binding to dsDNA and nucleosomes is dependent on the sequence of the light chain. Mice that received CHO cells secreting wild-type B3 developed more proteinuria and died earlier than control mice that received nonsecreting CHO cells or mice that received B3 with a single light chain mutation. However, none of the mice had histological changes or deposition of human immunoglobulin G in the kidneys. Sequence changes may alter the pathogenicity of B3, but further studies using different techniques are needed to investigate this possibility.     

Primary structures and chain dominance of anti-DNA antibodies

Using several anti-DNA autoantibodies, we analyzed the relative involvement of heavy and light chains in their interactions with DNA. We previously obtained eight hybridomas producing monoclonal anti-DNA autoantibodies by fusing spleen cells from an MRL-lpr/lpr mouse with myeloma cells. The chain dominance was analyzed by UV cross-linking experiments, in which the antibodies were covalently cross-linked with radioisotope-labeled oligonucleotides by short-wavelength UV-light, and the cross-linked H and L chains were analyzed by SDS-PAGE and densitometric scanning. Among these, three were found to be heavy chain dominant antibodies in which heavy chains are dominantly involved in DNA binding. The other five were co-dominant antibodies in which both heavy and light chains are involved in DNA binding. To determine the factor(s) that can explain the chain dominance in DNA binding, we determined the amino acid sequences of the variable regions of both heavy (VH) and light (VL) chains of all eight monoclonal antibodies. By analyzing the data, we were able to draw the following conclusions: (1) The arginine residues are found in the CDR3 regions of both VH and VL of the co-dominant antibodies; whereas, the same residues are found only in the CDR3s of VH, but not in VL, of the heavy chain dominant antibodies. (2) The net charges of the V regions affect the chain dominance. From the results of this study it is suggested that the presence of arginine residue in CDR3 is a critical factor in determining chain-dominance, as well as DNA binding of anti-DNA antibodies in general.     

The critical role of arginine residues in the binding of human monoclonal antibodies to cardiolipin

Previously we reported that the variable heavy chain region (VH) of a human beta2 glycoprotein I-dependent monoclonal antiphospholipid antibody (IS4) was dominant in conferring the ability to bind cardiolipin (CL). In contrast, the identity of the paired variable light chain region (VL) determined the strength of CL binding. In the present study, we examine the importance of specific arginine residues in IS4VH and paired VL in CL binding. The distribution of arginine residues in complementarity determining regions (CDRs) of VH and VL sequences was altered by site-directed mutagenesis or by CDR exchange. Ten different 2a2 germline gene-derived VL sequences were expressed with IS4VH and the VH of an anti-dsDNA antibody, B3. Six variants of IS4VH, containing different patterns of arginine residues in CDR3, were paired with B3VL and IS4VL. The ability of the 32 expressed heavy chain/light chain combinations to bind CL was determined by ELISA. Of four arginine residues in IS4VH CDR3 substituted to serines, two residues at positions 100 and 100 g had a major influence on the strength of CL binding while the two residues at positions 96 and 97 had no effect. In CDR exchange studies, VL containing B3VL CDR1 were associated with elevated CL binding, which was reduced significantly by substitution of a CDR1 arginine residue at position 27a with serine. In contrast, arginine residues in VL CDR2 or VL CDR3 did not enhance CL binding, and in one case may have contributed to inhibition of this binding. Subsets of arginine residues at specific locations in the CDRs of heavy chains and light chains of pathogenic antiphospholipid antibodies are important in determining their ability to bind CL.     

Optical biosensor-based characterization of anti-double-stranded DNA monoclonal antibodies as possible new standards for laboratory tests

The serum determination of circulating anti-double-stranded (ds)DNA autoantibodies is a routine measure for the laboratory diagnosis of systemic lupus erythematosus. Since available assays differ substantially and no feasible calibrator is available, the aim of this study was to evaluate a recently introduced surface plasmon resonance (SPR) biosensor chip for binding studies between dsDNA and anti-dsDNA autoantibodies and to demonstrate its usefulness for the characterization of new monoclonal antibody (mAb) standards and standardization of assays.We characterized two human and one murine monoclonal anti-dsDNA antibodies by measuring the kinetic on- and off-rates using the biosensor and calculating functional affinity (avidity) as the ratio of these. Obtained equilibrium dissociation constants were verified by an independent method and inhibition experiments were performed to determine reactivities to DNA of various length and composition.While all mAbs exhibited comparable avidities, which could be confirmed by gel shift experiments, one of them proved to have slower association and dissociation kinetics. This was the only mAb providing positive results in the Farr RIA. In inhibition experiments with ss- and ds-oligonucleotides 10, 24 and 42 bp in length, the mAbs acted substantially different.The study demonstrates how putative standards for the anti-dsDNA determination can be characterized using SPR biosensor technology. Our results suggest that kinetic rate constants seem to be decisive in explaining the behaviour of mAbs. Different reactivities to various DNA species should be taken into account with respect to varying DNA sources in commonly used laboratory assays.     

Antibodies elicited in response to EBNA-1 may cross-react with dsDNA

Background

Several genetic and environmental factors have been linked to Systemic Lupus Erythematosus (SLE). One environmental trigger that has a strong association with SLE is the Epstein Barr Virus (EBV). Our laboratory previously demonstrated that BALB/c mice expressing the complete EBNA-1 protein can develop antibodies to double stranded DNA (dsDNA). The present study was undertaken to understand why anti-dsDNA antibodies arise during the immune response to EBNA-1.

Methodology/Principal Findings

In this study, we demonstrated that mouse antibodies elicited in response to EBNA-1 cross-react with dsDNA. First, we showed that adsorption of sera reactive with EBNA-1 and dsDNA, on dsDNA cellulose columns, diminished reactivity with EBNA-1. Next, we generated mononclonal antibodies (MAbs) to EBNA-1 and showed, by several methods, that they also reacted with dsDNA. Examination of two cross-reactive MAbs—3D4, generated in this laboratory, and 0211, a commercial MAb—revealed that 3D4 recognizes the carboxyl region of EBNA-1, while 0211 recognizes both the amino and carboxyl regions. In addition, 0211 binds moderately well to the ribonucleoprotein, Sm, which has been reported by others to elicit a cross-reactive response with EBNA-1, while 3D4 binds only weakly to Sm. This suggests that the epitope in the carboxyl region may be more important for cross-reactivity with dsDNA while the epitope in the amino region may be more important for cross-reactivity with Sm.

Conclusions/Significance

In conclusion, our results demonstrate that antibodies to the EBNA-1 protein cross-react with dsDNA. This study is significant because it demonstrates a direct link between the viral antigen and the development of anti-dsDNA antibodies, which are the hallmark of SLE. Furthermore, it illustrates the crucial need to identify the epitopes in EBNA-1 responsible for this cross-reactivity so that therapeutic strategies can be designed to mask these regions from the immune system following EBV exposure.     

Mutational analysis of arsonate binding by a CRIA+ antibody. VH and VL junctional diversity are essential for binding activity

To assess the impact of various heavy and light chain mutations on p-azophenylarsonate binding, murine antibodies have been produced in insect cells (SF9) utilizing a baculovirus expression system. When expressed in this system, an antibody composed of a canonical CRIA+ heavy and light chain can bind antigen and express idiotype indistinguishably from analogous hybridoma-derived antibodies. Antibodies comprised of either light chains mutant at the V-J junction or heavy chains mutant at the V-D junction were found to be incapable of binding arsonate. In addition, substitutions in the first and second complementarity determining regions of the heavy chain were shown to play a role in arsonate binding, most likely related to affinity maturation targeted at the carrier protein. These results confirm the obligatory role that junctional diversity plays in the generation of arsonate-specific antibodies, as well as extend our understanding of the role of other variable region amino acids in arsonate binding.     

Quantitative analyses of C3b capture and immune adherence of IgM antibody/dsDNA immune complexes

We isolated the IgM fraction from the plasma of an SLE patient with high titer anti-dsDNA antibodies and prepared soluble IgM/dsDNA immune complexes (IC) that fixed C and captured sufficient C3b to bind to human E via their C3b/C4b receptor, CR1 (immune adherence, IA). We used specific 125I-labeled mAb to IgM, C3b, and IgG to measure the stoichiometries of these C-opsonized IC. They contained 10 to 60 C3b and 10 to 30 IgM per PM2 dsDNA, had no detectable IgG, and the vast majority of the C3b was bound to the IgM, and not to the dsDNA. These stoichiometries are in contrast to those we observed for comparable E-bound IC prepared with IgG anti-dsDNA antibodies (100 to 200 C3b, and 200 to 500 IgG). Our results help explain the greater lability of the IgM IC with respect to IA as evidenced by their plasma-mediated release from human E (presumably due to factor I), and confirm previous predictions of a lower density of "packing" of IgM on dsDNA, compared to IgG. The detailed stoichiometry of C3b capture by the IgM IC (typically 1.5 to 3 C3b per IgM) suggests that individual IgM molecules with multiple C3b facilitate IC binding to clusters of CR1. Finally, comparison of the IgM/dsDNA IC with other IgM IC which have been investigated with respect to C activation, and review of the proposed mechanism by which IgM activates C, suggests that the nature of the Ag plays a fundamental role in determining whether or not an IgM IC can activate C and participate in IA.     

Effect of dsDNA binding to SmD-derived peptides on clinical accuracy in the diagnosis of systemic lupus erythematosus

Systemic lupus erythematosus is characterized by antibodies to a variety of intracellular self-antigens, such as dsDNA and Sm, and these serve as hallmarks in the diagnosis of systemic autoimmune diseases. Several studies have shown that SmD1 and SmD3 synthetic peptides represent highly functional antigens for autoantibody detection and thus for diagnostic applications. The present study analysed the technical and clinical accuracy of an anti-SmD1 (amino acids 83–119) and an anti-SmD3 (amino acids 108–122) ELISA for the detection of anti-Sm antibodies. Depending on the cut-off value of the SmD1 ELISA, we found a high degree of concordance between the two tests. At an optimized cut-off value of 100 units for SmD1 we found the same clinical sensitivity (12.5%) and specificity (100%) in a group of systemic lupus erythematosus patients (n = 48) and in controls (n = 99). The concordance at this cut-off value was 100% (P < 0.0001; χ² = 127.61). Using a second panel of sera (n = 65) preselected based on positive anti-Sm results, we confirmed the high degree of concordance between the two assays. Using dsDNA-coated ELISA plates and biotinylated peptides we confirmed the high dsDNA binding properties for SmD1, which were significantly higher than the SmD3-derived peptide. However, no cross-linking of anti-dsDNA antibodies to SmD1 was observed after adding increasing amounts of dsDNA to anti-dsDNA positive, anti-SmD1 negative serum. We therefore conclude that the reported difference in the sensitivity is related to the different cut-off levels and not to the detection of anti-dsDNA antibodies bridged via dsDNA to the SmD1 peptide. Moreover, we found that a subpopulation of anti-Sm antibodies cross-reacted with SmD1 and SmD3. Taken together, the data indicate that both SmD peptide ELISAs represent accurate assays and may be used as important standards for the detection of anti-Sm antibodies.     

Development and selection of edited B cells in B6.56R mice

Tolerance to dsDNA is broken in mice with a high-affinity anti-DNA H chain transgene, 56R, on the C57BL/6 background (B6.56R). B6.56R produce more anti-dsDNA Abs than BALBc.56R. To investigate how anti-DNA Abs are regulated on the B6 background, phenotypic and genetic studies were performed. B6.56R have reduced numbers of B cells and phenotypically altered B cell subsets, including relative increases in the proportions of IgM-negative bone marrow B cells, cells with a marginal zone phenotype, and cells with a transitional T3 phenotype. The peripheral B cell repertoire in B6.56R is restricted: most B cells express the 56R H chain and use a similar, limited subset of editor L chains. DNA binding is more common in B6.56R because the repertoire is shifted toward L chains that are more permissive for DNA binding. H chain editing is also observed and is increased in spontaneous as compared with LPS hybridomas. A subset of spontaneous hybridomas appears to lack H chain expression.     

Specific recognition of a dsDNA sequence motif by an immunoglobulin VH homodimer

Anti-DNA antibodies have the potential to be applied in vast fields of fundamental as well as medical research. They are found in autoimmune diseases, such as systemic lupus erythemotosus. In most cases, anti-dsDNA antibodies do not present sequence specificity and are of low affinity. The dominant role of VH domains in DNA recognition induced us to search for binders based on VH dimers (VHD), previously reported to bind different protein antigens. We screened a phage displayed homo-VHD library against a 19-bp dsDNA sequence. A sequence-specific binder was selected, which recognizes the terminal located CTGC motif with a Kd of 250 nM. Association of the two identical VH domains of the molecule was shown to be essential for binding.     

NMR evidence for mechanical coupling of phosphate B(I)-B(II) transitions with deoxyribose conformational exchange in DNA

2a2 is the most commonly rearranged gene in the human V(lambda )locus. It has been postulated that certain immunoglobulin genes (including 2a2) are rearranged preferentially because their germline sequences encode structures capable of binding to a range of antigens. Somatic mutation could then increase the specificity and affinity of binding to a particular antigen.We studied the properties of five IgG molecules in which the same heavy chain was paired with different light chains derived from 2a2. The pattern of somatic mutations in 2a2 was shown to be crucial in conferring the ability to bind DNA, but two different patterns of mutation each conferred this ability.Computer-generated models of the three-dimensional structures of these antibodies illustrate the ability of 2a2 to form a DNA binding site in different ways. Somatic mutations at the periphery of the DNA binding site were particularly important. In two different light chains, mutations to arginine at different sites in the complementarity determining regions (CDRs) enhanced binding to DNA. In a third light chain, however, mutation to arginine at a different site blocked binding to DNA.     

Human and murine anti-DNA antibodies induce the production of anti-idiotypic antibodies with autoantigen-binding properties (epibodies) through immune-network interactions

To examine the potential role of immune-network interactions in the production of lupus autoantibodies, normal NZW rabbit antibody responses were analyzed after immunization with one of the following Ig preparations: human lupus serum anti-dsDNA antibodies, human lupus serum anti-ssDNA antibodies, a mixture of human lupus serum anti-dsDNA and anti-ssDNA antibodies, the MRL-lpr/lpr anti-dsDNA mAb H241, and the MRL-lpr/lpr anti-ssDNA mAb H130. Four of five rabbits produced Ig typical of lupus autoantibodies: individual rabbit Ig cross-reacted with multiple autoantigens including nucleic acids, cardiolipin, SmRNP, glomerular extract, laminin, and exogenous Ag. Rabbit anti-Id against human anti-dsDNA antibodies were highly specific for dsDNA. Notably, in each serum the autoantibody activity was confined to the anti-Id Ig fraction. A similar spontaneously occurring Id-anti-Id interaction was also found between anti-ssDNA and anti-dsDNA antibodies isolated from an individual lupus patient. These results indicate that lupus autoantibodies which share Ag binding properties with pathogenic Ig, including both cross-reactive and anti-dsDNA antibodies, can induce the production of Ig with similar autoantigen binding properties through immune-network interactions. This phenomenon, if unregulated, could lead to the amplification of pathogenic autoantibody production in individuals with systemic lupus.     

Detection of antibodies to DNA: a technical assessment.

Antibodies to DNA can be found in the circulation of the majority of patients with Systemic Lupus Erythematosus (SLE). They are quite specific for this disease, which makes their detection an important diagnostic aid to the clinician. Fluctuations in the level of anti-dsDNA in an individual patient generally parallel the clinical state of that patient. Furthermore, the presence of anti-dsDNA may precede the diagnosis of SLE by more than a year. Four methods relevant for the measurement of anti-dsDNA antibodies are discussed in this paper: the ELISA, the indirect immunofluorescence test on Crithidia luciliae, the PEG assay, and the Farr assay. Each of these methods detects a part of the spectrum of anti-dsDNA antibodies present in the circulation of an individual patient. The ELISA is the most sensitive method, whereas the Farr assay is the most specific for SLE. However, with the latter method only antibodies of a relative high avidity for DNA are detected. Mild forms of SLE, where patients only have anti-dsDNA of a low avidity in their circulation, may easily be missed by this technique. Clinically, high avidity anti-dsDNA is related with the more frequent occurrence of nephritis, whereas low avidity anti-dsDNA antibodies are more often found in patients with central nervous system involvement.     

Detection of antibodies to DNA: a technical assessment

Antibodies to DNA can be found in the circulation of the majority of patients with Systemic Lupus Erythematosus (SLE). They are quite specific for this disease, which makes their detection an important diagnostic aid to the clinician. Fluctuations in the level of anti-dsDNA in an individual patient generally parallel the clinical state of that patient. Furthermore, the presence of anti-dsDNA may precede the diagnosis of SLE by more than a year. Four methods relevant for the measurement of anti-dsDNA antibodies are discussed in this paper: the ELISA, the indirect immunofluorescence test onCrithidia luciliae, the PEG assay, and the Farr assay. Each of these methods detects a part of the spectrum of anti-dsDNA antibodies present in the circulation of an individual patient. The ELISA is the most sensitive method, whereas the Farr assay is the most specific for SLE. However, with the latter method only antibodies of a relative high avidity for DNA are detected. Mild forms of SLE, where patients only have anti-dsDNA of a low avidity in their circulation, may easily be missed by this technique. Clinically, high avidity anti-dsDNA is related with the more frequent occurrence of nephritis, whereas low avidity anti-dsDNA antibodies are more often found in patients with central nervous system involvement.     

How human IgGs against DNA recognize oligonucleotides and DNA

In the literature, there are no available data on how anti‐DNA antibodies recognize DNA. In the present work, to study the molecular mechanism of DNA recognition by antibodies, we have used anti‐DNA IgGs from blood sera of patients with multiple sclerosis. A stepwise increase in ligand complexity approach was used to estimate the relative contributions of virtually every nucleotide unit of different single‐ (ss) and double‐stranded (ds) oligonucleotides to their affinity for IgG fraction having high affinity to DNA‐cellulose. DNA‐binding site disposed on the heavy chain demonstrates higher affinity to different dNMPs (K_d = 0.63μM‐3.8μM) than the site located on the light chain (28μM‐170μM). The heavy and light chains interact independently forming relatively strong contacts with 2 to 4 nucleotides of short homo‐ and hetero‐d(pN)_2‐9. Then the increase in the affinity of different d(pN)_n became minimal, and at n ≥ 8 to 9, all dependencies reached plateaus: approximately 3.2nM to 20nM and approximately 200nM to 460nM for the heavy and light chains, respectively. A similar situation was observed for different ribooligonucleotides, in which their affinity is 6‐fold to 100‐fold lower than that for d(pN)_n. Transition from ss to ds d(pN)_n leads to a moderate increase in affinity of ligands to DNA‐binding site of heavy chains, while light chains demonstrate the same affinity for ss and ds d(pN)_n. Long supercoiled DNA interacts with both heavy and light chains with affinity of approximately 10‐fold higher than that for short oligonucleotides. The thermodynamic models were constructed to describe the interactions of IgGs light and heavy chains with DNA.