Hydroxyl radical modification of human serum albumin generated cross reactive antibodies

Hydroxyl radical-mediated in vitro modification of human serum albumin (HSA) showed 59.2% hyperchromicity at λ_max, 30% loss of alpha helical structure and 71.4% loss of tryptophan fluorescence. The reactive oxygen species (ROS)-modified HSA was highly immunogenic in rabbits as compare to native HSA. The antibody binding was inhibited to the extent of 97% with the immunogen as inhibitor, indicating the induction of immunogen specific antibodies. Experimentally induced antibodies against modified HSA exhibited diverse antigen binding characteristics. Native plasmid DNA, ROS-modified plasmid DNA and ROS-chromatin were found to be an effective inhibitor of induced antibody–immunogen interaction. Induced antibodies against native HSA showed negligible binding to the above mentioned nucleic acid antigens. Band shift assay reiterated the recognition towards nucleic acid antigens. Thus, the induced antibodies against ^√OH modified HSA resembled the diverse antigen-binding characteristics of naturally occurring systemic lupus erythematosus (SLE) anti-DNA autoantibodies.     

Physicochemical and immunological studies on 4-hydroxynonenal modified HSA: Implications of protein damage by lipid peroxidation products in the etiopathogenesis of SLE

4-Hydroxynonenal (HNE) is the most abundant and toxic aldehyde generated by the oxidation of plasma membrane polyunsaturated fatty acids. Systemic lupus erythematosus (SLE), a chronic autoimmune disease, is primarily characterized by increased levels of autoantibodies, predominantly against ds-DNA. However, the initial antigenic stimulus for the disease etiopathogenesis has remained elusive. HNE has been extensively used as a biomarker of oxidative stress. It can form adduct with proteins, making them highly immunogenic. Increased levels of such aldehyde–protein adducts have been reported in various pathological states, including autoimmune disorders like SLE and arthritis. In the present study, HNE-mediated structural changes in human serum albumin (HSA) were characterized by UV, fluorescence, CD and FT-IR spectroscopy as well as by polyacrylamide gel electrophoresis. Furthermore, immunogenicity of native and HNE-modified HSA was probed in female rabbits. The HNE-modified HSA was highly immunogenic eliciting high titre immunogen specific antibodies. Binding of SLE anti-DNA antibodies was analyzed by direct binding and competition ELISA. The data show preferential binding of SLE autoantibodies to HNE-modified HSA as compared to native HSA or native DNA. Our results suggest that HNE modification generates neoepitopes on HSA causing enhanced autoantibodies production. The results point towards the possible role of HNE-modified HSA in SLE etiopathogenesis.     

Antibodies Against Free Radical Modified Native DNA Recognize B-Conformation

Hydroxyl radical, a prominent entity of reactive oxygen species, is known to modify cellular DNA and has been implicated in several human diseases. In the present studies, the radical was generated by exposure of hydrogen peroxide to 254 nm light in the presence of native calf thymus DNA. Single strand breaks, decrease in Tm and modification of adenine and thymine were some of the modifications observed in nDNA. Antibodies induced in experimental animals against the modified DNA were immunogen specific. These antibodies also recognize native B-conformation. It was observed that naturally occurring anti-native DNA autoantibodies from SLE sera recognize modified DNA in direct binding and competition ELISA. Gel retardation assay reiterated the formation of immune complexes between induced antibodies and DNA fragments of around 300 bp (B-conformation). The possible significance of these findings in the etiology of SLE has been discussed.     

Hydroxyl Radical Modification of Polyguanylic Acid: Role of Modified Guanine in Circulating SLE Anti‐DNA Autoantibodies

The hydroxyl radical generated by UV irradiation of hydrogen peroxide cause an extensive damage to guanine residues of ribohomopolymer, polyguanylic acid, poly (G) as investigated by spectrophotometric measurements, agarose gel electrophoresis, Sephadex G‐200 gel filtration and DEAE Sephadex A‐25 column chromatography. Native and ROS‐poly (G) were highly immunogenic inducing high titre antibodies in rabbits. The antibodies showed wide range of cross reactivity with various synthetic polynucleotides exhibiting B‐, A‐, and allied conformations. The diverse antigen binding characteristics of the induced antibodies resembles to those of naturally occurring lupus anti‐DNA autoantibodies. Sera from various SLE patients showed preferential binding to ROS‐poly (G) than native poly (G), indicating that oxidatively modified guanine residues are better recognised. The significance of these findings in the induction of SLE anti‐DNA autoantibodies by oxygen free radicals modified guanine residues in DNA has been discussed.     

Hydroxyl radical modification of polyguanylic acid: role of modified guanine in circulating SLE anti-DNA autoantibodies

The hydroxyl radical generated by UV irradiation of hydrogen peroxide cause an extensive damage to guanine residues of ribohomopolymer, polyguanylic acid, poly (G) as investigated by spectrophotometric measurements, agarose gel electrophoresis, Sephadex G-200 gel filtration and DEAE Sephadex A-25 column chromatography. Native and ROS-poly (G) were highly immunogenic inducing high titre antibodies in rabbits. The antibodies showed wide range of cross reactivity with various synthetic polynucleotides exhibiting B-, A-, and allied conformations. The diverse antigen binding characteristics of the induced antibodies resembles to those of naturally occurring lupus anti-DNA autoantibodies. Sera from various SLE patients showed preferential binding to ROS-poly (G) than native poly (G), indicating that oxidatively modified guanine residues are better recognised. The significance of these findings in the induction of SLE anti-DNA autoantibodies by oxygen free radicals modified guanine residues in DNA has been discussed.     

Polynucleotide specificity of anti-reactive oxygen species (ROS) DNA antibodies.

Hydrogen peroxide in the presence of short wavelength UV light was able to induce alterations in native DNA fragments of 300 bp (ROS-DNA), thereby rendering it immunogenic in experimental animals. The specificity of induced antibodies was investigated by direct binding and competition ELISA. Inhibition studies revealed nearly 89% inhibition in the antibody binding by the immunogen and recognition of native B-, A- and allied conformations presented by various synthetic polynucleotides. Gel retardation assay reiterated the formation of immune complexes between induced antibodies and native and ROS-DNA fragments. It was observed that naturally occurring anti-DNA autoantibodies from systemic lupus erythematosus (SLE) sera recognize ROS-DNA. The comparison of the specificities of anti-DNA autoantibodies from 10 SLE patients showed a 20-50-fold preference for ROS-DNA over native DNA. These results demonstrate that anti-DNA antibodies can be induced by ROS-DNA, and that some of the autoimmune DNA binding antibodies found in SLE may result from response to reactive oxygen species.     

Peroxynitrite-induced modification of H2A histone presents epitopes which are strongly bound by human anti-DNA autoantibodies: role of peroxynitrite-modified-H2A in SLE induction and progression

Peroxynitrite is a potent oxidant and nitrating agent and has in vivo existence. It is a powerful proinflammatory substance and may increase vascular permeability in inflamed tissues. Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease of unknown etiology. Since its discovery, numerous self- and non-self, nuclear, and cytoplasmic antigens have been suggested as stimuli for SLE initiation, but the exact trigger is yet to be identified. In this study, an attempt has been made to investigate the binding characteristics of SLE anti-DNA autoantibodies to native DNA and native and peroxynitrite-modified H2A histone to explore the possible role of modified protein antigen(s) in SLE initiation and progression. The nuclear protein (H2A histone) was modified by peroxynitrite synthesized in our laboratory. The peroxynitrite-modified H2A revealed generation of nitrotyrosine, dityrosine, and carbonyls when subjected to investigation by physicochemical methods. Binding characteristics and specificity of SLE anti-DNA antibodies were analyzed by direct binding and inhibition enzyme-linked immunosorbent assay. The data show preferential binding of SLE autoantibodies to peroxynitrite-modified H2A histone in comparison with native H2A histone or native DNA. A band shift assay further substantiated the enhanced recognition of peroxynitirite-modified H2A histone by anti-DNA autoantibodies. The results suggest that peroxynitrite modification of self-antigen(s) can generate neoepitopes capable of inducing SLE characteristic autoantibodies. The preferential binding of peroxynitrite-modified H2A histone by SLE anti-DNA antibodies points out the likely role of oxidatively modified and nitrated H2A histone in the initiation/progression of SLE. Moreover, oxidatively modified and nitrated nuclear protein antigen, rather than nucleic acid antigens, appear to be more suitable as a trigger for SLE.     

Antigen binding characteristics of experimentally-induced antibodies against hydroxyl radical modified native DNA

Hydroxyl radical, generated by UV irradiation of hydrogen peroxide caused damage to native calf thymus DNA leading to strand breaks, base modification and decrease in melting temperature. The ROS-DNA was highly immunogenic in goat. The antibody activity was inhibited to the extent of 89% with the immunogen as inhibitor. Antigenic specificity of anti-ROS-DNA antibodies by competition ELISA showed multiple cross-reactivity, recognizing B-, A- and allied conformations. The immune complex formation with native and ROS-DNA was substantiated by band shift assay. A striking observation, is the enhanced recognition of ROS-thymine and ROS-poly(dT) by the induced antibodies. These investigations suggest that ROS might be the plausible candidate for the presentation of unique epitopes on ROS-DNA, in particular of thymine, inducing antibodies cross-reacting with native DNA and play an important role in the pathogenesis of SLE.     

Studies with Synthetic Peptides of 80 kDa Human Sperm Antigen (80 kDa HSA)

PROBLEM: The 80 kDa human sperm antigen (HSA) is a sperm-specific and conserved antigen, capable of inducing immunological infertility. Partial N-terminal amino acid sequences of 80 kDa HSA (Peptide NT) and its peptides obtained by digestion with endoproteinase Lys-C (peptides 1-4) and endoproteinase Glu-C (peptides 5-6) did not show any sequence homology with reported known proteins deposited in the Gen-Bank. These sequenced peptides were synthesized and conjugated to key hole limpet haemocyanin (KLH) and evaluated for its antifertility effects. The present communication describes the characterization of these peptides and their antibodies. METHOD OF STUDY: Peptides NT, 1, 2, 3 and 4 were synthesized and conjugated to KLH. Antibodies to KLH conjugated peptides were raised in rabbits by active immunization and the antibody titer was determined by enzyme-linked immunosorbent assay (ELISA) using sperm extract coated wells. The binding specificity of the synthetic peptides or purified 80 kDa HSA to their antibodies was assessed in the presence of various doses of respective synthetic peptides or 80 kDa HSA. The binding specificity was further confirmed by Western blot analysis. Antipeptide antibodies were also checked for sperm agglutinating activity, in-vitro. RESULTS: Active immunization of rabbits elicited significant antibody titers against the synthetic peptides, except for peptide 3. Antipeptide antibodies specifically recognized the native protein in an ELISA and induced in-vitro agglutination of human, rat and monkey sperm. In addition, Western blot analysis showed that these antipeptide antibodies specifically bind to the 80 kDa HSA band of the sperm extract. CONCLUSION: Synthetic peptides of 80 kDa HSA are immunogenic and antibodies raised against these peptides recognize the native protein detected by ELISA, Western blot analysis. In addition, they possess sperm agglutinating activity. These findings suggest that they are promising candidates in the development of immunocontraceptives.     

A novel trait of naturally occurring anti-DNA antibodies: dissociation from immune complexes in neutral 0.3-0.5 M NaCl

Monoclonal and polyclonal anti-DNA antibodies from autoimmune mice, and experimentally induced rabbit anti-nucleic acid polyclonal antibodies were tested for stability of binding to nucleic acids in the presence of various concentrations of NaCl by an enzyme-linked immunosorbent assay (ELISA). Murine monoclonal antibodies 2C10 (IgG2b) and 1A2 (IgG2a), which are known to react specifically with double-stranded (ds) DNA, dissociated completely from their complexes with DNA when washed with a neutral 0.5 M NaCl solution. Another monoclonal antibody (MoAb) (IgM,kappa), polyreactive with single-stranded (ss) DNA, cardiolipin, and trinitrophenylhapten (TNP), was also dissociated from its complexes with ss DNA, but not from its complexes with TNP, by 0.3-0.5 M NaCl. Similar differences were observed in the binding stability of serum antibodies from autoimmune mice to DNA and TNP. In contrast, anti-nucleic acid polyclonal antibodies induced in rabbits by immunization with poly(I), poly(dT) or poly(ADP-ribose) were not significantly dissociated from their immune complexes with relevant antigens or DNA by 0.5 M NaCl. The finding that nucleic acid antigens were not detached from a solid phase by washing with 0.5 M NaCl solution indicated that the reduction of binding of anti-DNA antibodies in both MoAbs and naturally occurring antibodies was really due to dissociation of the antibodies from immune complexes. This is the first demonstration that DNA epitopes recognized by naturally occurring antibodies in both SLE and its mouse models are sensitive to neutral NaCl concentrations. This novel trait of naturally occurring antibodies will be very useful in studies on the nature of immune complexes in sera and kidneys of cases of systemic lupus erythematosus (SLE).     

Production of anti-dengue NS1 monoclonal antibodies by DNA immunization

Monoclonal antibodies against dengue NS1 protein were generated following immunization of mice with plasmid DNA encoding the transmembrane form of NS1 from dengue serotype 2 virus. A mammalian expression vector, pDisplay, was engineered to direct cell surface expression of dengue NS1 and tested for transient expression in COS cells. Two mice were immunized intramuscularly with six doses of 100 microg of plasmid at 2-week intervals; one mouse received a booster of live virus prior to the last plasmid injection. Both mice showed antibody responses against dengue antigens in dot enzyme immunoassay. Following fusion, hybridomas were screened with dot enzyme immunoassay against all four dengue serotypes. Specificity to the NS1 protein was confirmed by western blot analysis. Among five anti-dengue NS1 monoclonal antibodies generated, two clones were serotype 2 specific, two clones reacted with all four serotypes and the last also reacted with Japanese encephalitis virus. Reactivity against native or denatured forms of NS1 revealed three clones with reactivity to linear epitopes and two clones recognizing conformational epitopes. Such diverse specificity of anti-dengue NS1 monoclonal antibodies indicates that DNA immunization, especially with the combination of virus boosting, is an efficient way of producing monoclonal antibodies against viral protein. This has opened up a possibility of producing monoclonal antibodies to rare viral proteins that are difficult to isolate or purify.     

Specificity of anti-DNA antibodies induced in normal mice by immunization with bacterial DNA.

To determine the specificity of anti-DNA antibodies induced in normal mice by immunization with bacterial DNA, sera from BALB/c mice immunized with single-stranded DNA from Escherichia coli (EC) were tested for binding to a panel of synthetic DNA and RNA homopolymers as well as duplexes. Results of these studies indicate that sera from EC DNA immunized mice preferentially bind certain DNA and RNA homopolymers as well as DNA duplexes. Furthermore, the specificity of the antibodies from immunized mice resembled those of sera from autoimmune MRL-lpr/lpr mice in terms of the synthetic antigens recognized, although some differences were noted in the magnitude of the response to individual duplexes. These results suggest that anti-DNA antibodies induced by bacterial DNA bind to DNA structures dependent on both the base and the sugar phosphate moieties of the nucleic acid antigen and may resemble some anti-DNA antibodies expressed in spontaneous autoimmune disease in these binding properties.     

Nitration of H2B histone elicits an immune response in experimental animals

Histone H2B is an autoantigen that appears in circulation due to altered apoptosis/or insufficient clearance and is likely to be involved in the induction and progression of autoimmune diseases since modified-H2B is immunogenic. Our studies demonstrate that tyrosines of H2B histone spontaneously converts to free and nitrotyrosine bound protein in vivo. Commercially available H2B histone was modified with peroxynitrite in vitro. Modified H2B was found to be more immunogenic than native form in experimental animals. Furthermore, the sera of rabbits were analyzed for the native and modified forms of the H2B histone. The binding specificity of autoantibodies was characterized by competitive enzyme-linked immunosorbent assay (ELISA) and band shift assay. The free 3-nitrotyrosine in systemic lupus erythematosus sera was quantified by high-performance liquid chromatography. Peroxynitrite-modified H2B induced high titre antibodies as compared to native form which were directly proportional to the nitrotyrosine content. Furthermore, the induced antibodies showed specificity towards the immunogen and cross-reacted with tyrosine-nitrated proteins. ELISA showed preferential binding of induced anti-peroxynitrite modified H2B antibodies to modified H2B as compared to native H2B. The present study shows that peroxynitrite modification of self-antigen(s) generates neoepitopes capable of inducing modified-H2B autoantibodies in experimental animals.     

Impact of Peroxynitrite Modification on Structure and Immunogenicity of H2A Histone

Peroxynitrite is an extremely reactive entity and has in vivo existence. Its interaction with biomolecules may cause oxidation and nitration. In this study, commercially available H2A histone was exposed to peroxynitrite generated in vitro . The peroxynitrite-mediated structural changes in histone were studied by ultraviolet & fluorescence spectroscopy, high performance liquid chromatography, anilinonaphthalene-8-sulfonic acid binding and polyacrylamide gel electrophoresis. Analysis of results revealed that carbonyl, nitrotyrosine and dityrosine contents were significantly increased in peroxynitrite-modified H2A compared with native H2A. Rabbits challenged with peroxynitrite-modified H2A induced high titre antibodies. The immunogenicity of peroxynitrite-modified H2A was directly proportional to protein nitrotyrosine content and induced antibodies showed specificity for the immunogen and good cross-reaction with nitrated epitopes of other modified proteins. Formation of high molecular weight immune complex with retarded mobility further supports the specificity of induced anti-100 μ m peroxynitrite-modified H2A antibodies for the immunogen. It may be concluded that induction of anti-H2A histone antibodies could be due to protection of peroxynitrite-modified histone from proteolytic breakdown and its subsequent recognition by immunoregulatory cells as foreign molecule.     

Comparative specificity of rabbit antibody populations elicited to chemically modified sDNA

A trinitrophenylation reaction specific for guanosyl residues (2'N position) was used to synthesize a modified deoxyribonucleic acid (DNA) immunogen. Antibodies from a pool of hyperimmune antitrinitrophenyl (TNP)-denatured DNA (sDNA) antisera were resolved into subpopulations by various immunoadsorption and ligand elution schemes. Results indicate that anti-hapten (TNP) and anti-carrier (sDNA) antibodies were present and resolvable by immunoadsorption. Antibodies purified by absorption with trinitrophenyl-deoxyguanosine monophosphate-bovine serum albumin (TNP-dGMP-BSA)-cellulose adsorbent and elution with TNP-dGMP were preferentially specific for the trinitrophenyl immunodominant group. When the antiserum pool was incubated with the same adsorbent and eluted with dGMP, antibodies displaying similar specificities were harvested. Finally, when the pool was adsorbed with an sDNA adsorbent, antibodies to the four constituent bases were eluted. All purified antibody populations were analysed by equilibrium dialysis and ligand inhibition studies to determine hapten binding properties. Based on this model system, the data are discussed in terms of the specificities of antibody subpopulations elicited to chemically modified nucleic acids.     

Surface modified magnetic nanoparticles for immuno-gene therapy of murine mammary adenocarcinoma

Cancer immuno-gene therapy is an introduction of nucleic acids encoding immunostimulatory proteins, such as cytokine interleukin 12 (IL-12), into somatic cells to stimulate an immune response against a tumor. Various methods can be used for the introduction of nucleic acids into cells; magnetofection involves binding of nucleic acids to magnetic nanoparticles with subsequent exposure to an external magnetic field. Here we show that surface modified superparamagnetic iron oxide nanoparticles (SPIONs) with a combination of polyacrylic acid (PAA) and polyethylenimine (PEI) (SPIONs-PAA-PEI) proved to be safe and effective for magnetofection of cells and tumors in mice. Magnetofection of cells with plasmid DNA encoding reporter gene using SPIONs-PAA-PEI was superior in transfection efficiency to commercially available SPIONs. Magnetofection of murine mammary adenocarcinoma with plasmid DNA encoding IL-12 using SPIONs-PAA-PEI resulted in significant antitumor effect and could be further refined for cancer immuno-gene therapy.     

Ureido group-specific antibodies are induced in rabbits immunized with citrulline- or homocitrulline-containing antigens

The specificities and cross-reactions of antibodies induced by citrulline- and homocitrulline-containing proteins may give implications on the role of citrulline- and homocitrulline-binding antibodies in the pathogenesis and progression of rheumatoid arthritis (RA). Here we use rabbits as an experimental model of antibody development in RA. Thirty-two animals were immunized with peptide antigens containing either homocitrulline or citrulline. The sera were tested for binding to CCP and MCV antigens and to peptide sequences related to carboxyterminal telopeptides of type I and II collagens and containing arginine, citrulline, or homocitrulline. The binding of CCP and MCV antigens to antisera against homocitrulline-containing immunogens could be inhibited by human serum albumin containing homocitrulline, whereas similar binding to sera against citrulline-containing immunogens was not inhibited. The antisera induced with citrulline-containing collagen telopeptides recognized type I collagen-related antigens in a sequence-specific manner, as antibody binding to both citrulline- and homocitrulline-containing peptides was inhibited by corresponding citrullinated and native peptides. In contrast, type II collagen-related peptides were recognized by the antisera in a ureido group-specific manner, as their binding to homocitrulline-containing peptide was inhibited by both citrulline- and homocitrulline-containing, but not native peptide. Binding of the citrullinated type II collagen peptide could only be inhibited by the similarly citrullinated peptide. In conclusion, antibodies induced with citrulline or homocitrulline-containing antigens bound antigens in a ureido group-specific manner, recognizing citrulline and homocitrulline also in other sequences than those used in the original immunization. In competitive situations the amino acid present in the immunization antigen was favored.     

Demonstration of IgE antibodies to nucleic acid antigens in patients with SLE.

Using a combination of avidin-biotin microELISA and solid phase radioimmunoassay, we examined sera from 23 patients with systemic lupus erythematosus (SLE), two patients with established sensitivity to ingested shrimp, and 15 healthy normal subjects. In addition to IgG antibodies, varying amounts of IgE antibodies specific for native DNA (nDNA), denatured or single-stranded DNA (dnDNA), RNA, and tRNA were demonstrable in the sera of SLE patients, but not in the sera of normal subjects. A comparison of the specificity of nucleic acid-specific IgE antibodies present in the sera of shrimp-sensitive patients with those present in the sera of seven SLE patients revealed that the IgE antibodies in the sera of shrimp-sensitive patients specifically recognized shrimp tRNA but not yeast tRNA, calf thymus RNA, or calf thymus DNA, while those present in the sera of patients with SLE recognized all these nucleic acid antigens. The IgE antibodies directed against nDNA, dnDNA, RNA, and tRNA may mediate mast cell and basophil degranulation and thus contribute both to immediate-type hypersensitivity phenomena including hives seen in patients with SLE and to the localization of IgE-nucleic acid complexes in target tissues.     

Cloning and expression of the major 47-kilodalton surface immunogen of Treponema pallidum in Escherichia coli.

Monoclonal antibodies directed against the 47-kilodalton (kDa) major outer membrane surface immunogen of virulent Treponema pallidum subsp. pallidum were used to select Escherichia coli recombinant clones expressing the 47-kDa immunogen. The phenotype of the clones was dependent on the presence of recombinant plasmid in the host cell. Southern hybridization revealed that the cloned T. pallidum subsp. pallidum DNA sequence was an accurate representation of the T. pallidum subsp. pallidum genomic DNA arrangement. Purified immunoglobulin G from rabbits experimentally infected with T. pallidum subsp. pallidum and human secondary syphilitic sera specifically reacted with the clones, while normal human serum or immunoglobulin G from normal rabbit serum did not. Results of Southern hybridization indicated that a homologous 47-kDa immunogen gene was absent in at least four species of nonpathogenic treponemes tested, as well as from total rabbit genomic DNA. Rabbit anti-T. phagedenis biotype Reiter (treponemal nonpathogen) antiserum and a monoclonal antibody directed against a common treponemal determinant were unreactive with the clones. Western blotting and radioimmunoprecipitation experiments with specific monoclonal antibodies revealed that the recombinant (E. coli) and native (T. pallidum subsp. pallidum) forms of the antigen had identical electrophoretic mobilities. The availability of recombinant 47-kDa immunogen provides a new opportunity for biochemical analysis of the protein, structure-function studies, examination of its role in microbial pathogenesis, and assessment of its diagnostic and vaccinogenic potentials.     

Antibodies against 4-hydroxy-2-nonenal modified epitopes recognized chromatin and its oxidized forms: role of chromatin, oxidized forms of chromatin and 4-hydroxy-2-nonenal modified epitopes in the etiopathogenesis of SLE

Objectives: This study was undertaken to investigate the role of lipid oxidative-by-product 4-hydroxy-2-nonenal (HNE)-modified human serum albumin (HSA), chromatin, reactive oxygen species (ROS)-modified chromatin and nitric oxide (NO)-modified chromatin in systemic lupus erythematosus (SLE). Methods: HSA was modified by HNE. Immunogenicity of modified HSA was probed by inducing polyclonal antibodies in rabbits. Chromatin was isolated from goat liver and modified by ROS or NO. Immunocross-reactions of Protein-A purified anti-HNE-HSA-IgG with chromatin, ROS-chromatin and NO-chromatin were determined. Autoantibodies from 74 SLE patients were screened. HSA was isolated from SLE patients (SLE-HSA) and immunocross-reactions of isolated SLE-HSA with HNE-specific antibodies were investigated. Results: HNE-HSA was found to be highly immunogenic in rabbits. The notable feature of anti-HNE-HSA-IgG showed cross-reactions with chromatin, ROS-chromatin and NO-chromatin (p < 0.01). High degree of specific binding to HNEHSA, chromatin, ROS-chromatin or NO-chromatin was observed with antibodies from 55% of SLE patients. SLE anti-native/oxidized chromatin antibodies showed specificity towards HNE-HSA. Furthermore, SLE-HSAshowed binding with HNE-specific antibodies. Conclusions: This is the first study to demonstrate that chromatin and its oxidized forms have been recognized by antibodies against HNE modified epitopes. Our results provide an important insight into the immunological basis of the reported HNE-modified epitopes in SLE.