Anti-La antibody production by MRL-1pr/1pr mice. Analysis of fine specificity

In evaluating the origin of autoantibodies, patterns of self-Ag recognition have been interpreted to reflect the relative role of Ag in stimulating a response. Few studies, however, have assessed whether human autoantibodies display patterns of autoantigen recognition similar to those of SLE-prone mice. In previous studies, anti-La antibodies from humans have been shown to bind multiple epitopes on recombinant human La Ag, including immunoreactivity with a large fragment, termed La C, representing the middle portion of the La sequence. We report herein for the first time that MRL-1pr mice also spontaneously produce antibodies to recombinant human La protein and resemble human autoantibodies in their reactivity with La C. To further investigate the fine specificity of this response, we tested for antibody binding to six synthetic La peptides representing sequences within La C. Whereas two of the synthetic La peptides reacted with MRL-1pr sera containing anti-La binding, low reactivity was observed with a large panel of human anti-La sera. Our results therefore show that patterns of La antigen recognition displayed by MRL-1pr antibodies differ from those of human autoantibodies, possibly reflecting differences between mouse and man in the induction of these responses.     

Two induced anti-Z-DNA monoclonal antibodies use VH gene segments related to those of anti-DNA autoantibodies

The cDNA for H and L chain V regions of two anti-Z-DNA mAb, Z22 and Z44, were cloned and sequenced. These are the first experimentally induced anti-nucleic acid antibody sequences available for comparison with autoantibody sequences. Z22 and Z44 are IgG2b and IgG2a antibodies from C57BL/6 mice. They recognize different facets of the Z-DNA structure. They both use VH10 family genes and share 95% sequence base sequence identity in the VH and leader sequences; however, they differ in the 5'-untranslated region of the VH mRNA, indicating they arise from different germline genes. Both use JH4 segments. They differ from each other very extensively in the CDR3 of both H and L chains. The most closely related H chains in the current GenBank/EMBL data base are two mouse IgG anti-DNA autoantibodies, one from an MRL-lpr/lpr mouse (MRL-DNA4) and one from an NZB/NZW mouse (BV04-01). Z22 and Z44 share 95% sequence identity with these antibodies in the VH segment. In addition, Z22 is identical to MRL-DNA4 at 91% of the positions in the 5'-untranslated region of the H chain mRNA. The two antibodies share 95% base sequence identity in the V kappa segment. The most closely related L chains, with 97 to 98% sequence identity, are the V kappa 10b germline gene for Z22 and the V kappa 10a germ line gene, which is associated with A/J anti-arsonate antibodies and BALB/c anti-ABO blood group substance antibodies, for Z44. Z22 and Z44 share several structural features (similarities in VH, JH, and V kappa) but differ very markedly in the L chain CDR1 and both H and L chain CDR3 sequences; these regions may determine the differences in their specific interactions with Z-DNA.     

Polyreactive autoantibodies are nephritogenic in murine lupus nephritis

To characterize the antibodies that form glomerular immune deposits in lupus nephritis, immunoglobulin (Ig) was eluted from the perfused kidney cortices of female MLR-lpr/lpr mice with early nephritis. The eluted Ig was predominantly IgG with antibody activity against DNA, multiple polynucleotides, SmRNP, gp70, and levan that was greater than the serum antibody activity of age- and sex-matched mice. Of particular interest, both kidney eluate and serum anti-DNA antibodies were observed to cross-react with multiple polynucleotides; however, only the kidney eluate Ig cross-reacted with phospholipids and RNA. Furthermore, the anti-DNA antibodies in the kidney eluate also cross-reacted with SmRNP and gp70; these ligand-binding properties were shared by the Ig in the kidney eluate that did not bind to DNA; and both kidney eluate fractions shared Id-H130 activity (a high frequency MRL-1pr/1pr idiotype). In contrast, the spectrotypes of Ig in the kidney eluate were found to be similar to serum, and they were observed to be between isoelectric points 6.5 to 7.8. Both the anti-DNA antibodies and the Ig that did not bind to DNA had similar isoelectric points throughout this entire range. These findings indicate that polyreactivity is a distinguishing feature of nephritogenic autoantibodies. They also raise the possibility that these ligand-binding properties influence the capacity of autoantibodies to form immune deposits. This influence could occur because polyreactive antibodies cross-react with antigenic determinants within the normal glomerular capillary wall. Alternatively, polyreactive antibodies may more readily form circulating immune complexes that are, in turn, passively trapped within the glomerulus.     

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.     

Human insulin autoantibody fine specificity and H and L chain use

Fine specificity and H and L chain isotypes of insulin autoantibodies in sera from 11 subjects were examined. None of these 11 subjects was treated with exogenous insulin. Two patterns of fine specificity were found. In one, the autoantibodies were specific for human insulin, with a requirement for threonine at B30. The conservative substitution in pork insulin (threonine to alanine) abrogated IgG binding by these sera. Insulin autoantibodies in other sera cross-reacted with beef, pork, and human insulin; not requiring threonine at B30. Reciprocal competitive inhibition experiments showed that epitopes recognized by the human specific insulin autoantibodies were exclusively on the B chain, whereas the cross-reactive sera contain autoantibodies that recognize both the B chain and combinatorial (A and B chain) epitopes. The fine specificity of cross-reactive insulin autoantibodies are thus similar to insulin antibodies from insulin-treated subjects. When IgG subclasses and L chains of insulin autoantibodies were examined, however, restricted C region usage was found. The hierarchy was IgG3 greater than G1 greater than G2 greater than G4; with one subclass dominant in each serum, although others were used. L chain use was similarly restricted. There was no correlation between isotype and fine specificity or between H and L chain type. It is concluded that heterogeneity of insulin autoantibodies is restricted. The response is probably more oligo- or pauciclonal than insulin antibody from insulin-treated subjects.     

Autoimmunization in murine graft-vs-host disease. I. Selective production of antibodies to histones and DNA

A lupus-like disease characterized by a severe immune complex glomerulonephritis and IgG autoantibody production was induced in (C57BL/6 X DBA/2)F1 mice by injection of parental DBA/2 lymphoid cells. The ensuing graft-vs-host (GVH) reaction resulted in a 10- and a 100-fold increase in serum IgG antibody levels to denatured DNA and total histones, respectively, compared with that in F1----F1 control mice. The level of anti-DNA antibodies peaked 2 wk after injection of DBA/2 cells and preceded peak anti-histone levels by approximately 2 wk. Anti-histone antibodies were generated predominantly to histones H1, H2A, and H2B, a profile different from that observed in NZB/NZW and MRL-lpr/lpr mice. The marked increase in IgG antinuclear antibodies did not correlate with increases in total IgG serum levels and was not associated with comparable increases in antibodies to transferrin, hemoglobin, fibrinogen, or thyroglobulin. Selective autoantibody production was also observed in vitro, wherein GVH spleen cells produced high levels of IgG antibodies to total histones and denatured DNA but not to these non-nuclear protein antigens. In contrast, spleen cells stimulated in vitro with lipopolysaccharide produced equivalent amounts of antibodies to all antigens tested. Our results are in agreement with those of other investigators and collectively suggest that IgG autoantibodies in GVH disease, and possibly in spontaneous lupus-like disease, are not secondary to a generalized B cell activation, but may be selectively generated in response to self antigens with unique configurational properties.     

Cloning of genomic sequences of human prointerleukin 1 beta

The human brain library carried in the EMBL3 vector was employed for isolating prointerleukin 1 beta genomic sequences using three synthetic 20-member oligonucleotides. Oligonucleotides were homologous to the following mRNA regions: 3'-nontranslated region/C1/, 3'-translated region of mRNA/C2/ and the sequence coding N-terminal of mature protein/N1/. The oligonucleotide labeling utilized the terminal nucleotidyltransferase and [alpha-32P] dATP and specific activity of labeled oligonucleotides reached 1.6.10(10) cpm. The sizes of the synthesized labeled sequences (tails) were about 10 b.p. Hybridization probe C1 was used for the first screening and 24 hybridization positive clones were detected. For the next screening probe C2 was used and only 2 hybridization positive clones with different level of hybridization were detected from 24 clones. Probe N1 was used for the third screening and allowed to identify the only positive clone. The characterization by restriction mapping and Southern blot analyses have shown that recombinant phage DNA contains all three exons, coding the mature interleukin 1 beta. Some fragments were recloned to tg130 vector phage and nucleotide sequences of exon 5 (completely) and exon 7, intron 4 and 5 (partially) were determined.     

Autoimmune mice and stimulated normal mice make lambda 1 with increased V region diversity

Previous studies of the genetic bases of murine SLE have defined gene segments that encode the H chain and the kappa L chain of anti-DNA, anti-Sm, and anti-IgG autoantibodies. As a result of these studies, the genetic origins of autoantibody H chains and kappa L chains are better understood, but little remains known about the genetic bases of autoantibody lambda-chains. Thus, we have analyzed serologically the germ-line and somatic origins of lambda 1 L chains in antibodies of normal mice and in both antibodies and autoantibodies of autoimmune mice. This study finds an increased lambda 1 diversity in both Ag-stimulated mice and autoimmune mice. This study also finds that the lambda 1 L chains in antibodies of unstimulated normal mice have the gene segment-encoded variable region, V lambda 1. In contrast, additional genetic processes appear to make the lambda 1 V regions of antibodies in Ag-stimulated normal mice and the lambda 1 V regions of both antibodies and autoantibodies in autoimmune mice. The increased lambda 1 diversity that we found in both Ag-stimulated mice and autoimmune mice might be caused by mutational processes creating antibody diversities. Therefore, the same somatic processes might be able to make both antibody and autoantibody lambda 1 diversities.     

Monoclonal antibodies to streptococcal group A carbohydrate. I. A dominant idiotypic determinant is located on Vk

In an effort to better define the antibody repertoire to streptococcal group A carbohydrate (GAC), somatic cell hybrids were prepared from A/J mice immunized with streptococcal vaccine. Most antibodies were IgG3K and IgMK, while 2 of 26 antibodies were lambda type. Each of the IgG3 antibodies had a distinct isoelectric point consistent with previous estimates of clonal repertoires of approximately 200. IEF analysis of the L chains, however, showed that about half of the antibodies produce a common L chain, called VK1GAC, previously identified in A/J anti-GAC serum antibodies. Additional support for the structural similarity of these L chains was gained by developing an idiotype antiserum to VK1GAC. All proteins with the common L chain spectrotype react strongly with anti-VK1GAC. Thus, it appears that anti-GAC antibodies are composed of H chains bearing a few VH regions pairing with a few L chains.     

Lupus prone (SWR x NZB)F1 mice produce potentially nephritogenic autoantibodies inherited from the normal SWR parent

The incidence of nephritis in autoimmune NZB mice is low, but when they are crossed with normal SWR mice, almost 100% of the female F1 hybrids (SNF1) develop lethal glomerulonephritis. To define the contribution of the normal SWR strain to the development of nephritis, we analyzed 65 monoclonal anti-DNA autoantibodies derived from SNF1 mice and compared them with those obtained from the NZB parent. The majority of the SNF1-derived anti-DNA antibodies were IgG and cationic in charge. By contrast, 77% of the NZB-derived antibodies were IgM. Moreover, all three NZB-derived IgG anti-DNA antibodies were anionic. The cationic property of the SNF1-derived IgG autoantibodies was not restricted to any particular antigenic specificity pattern or IgG subclass, nor was there a preference for the allotype of either parent. However, we identified a set of highly cationic (pI at 8.2 to 8.8 pH) IgG2b anti-DNA antibodies from SNF1 hybrids that had the SWR allotype. Isoelectric focusing of intact antibodies and isolated heavy and light chains showed that the highly cationic charge of these antibodies was determined by the variable regions of their heavy chains. Because IgG anti-DNA antibodies with cationic charge are especially pathogenic, those antibodies bearing the allotype of the normal SWR parent may account for the high incidence of severe nephritis in the F1 hybrids. The results indicate that pathogenic autoantibodies, which are encoded by genes of the nonautoimmune SWR parent, are expressed in the SNF1 mice due to some cellular and genetic regulatory influence of the NZB parent.     

Independent expression of a regulatory idiotype on heavy and light chains. A further immunochemical analysis with anti-heavy and anti-light chain antibodies

The heavy (H) and light (L) chains of murine monoclonal autoantibody 62 reacting with thyroglobulin independently express idiotypic (Id) determinants that are very similar if not identical with the Id62 expressed on the intact protein. In this report, we describe the production and characterization of rabbit antibodies to isolated H62 and L62 chains to further prove that individual chains express Id62 in an immunogenic form. The results demonstrate that both chains are capable of eliciting antibodies that, after appropriate adsorption, behave like conventional anti-Id62 antibodies prepared against the intact antibody molecule. By direct radioimmunoassay binding, competition of Id binding and Western blot anti-H62 and anti-L62 antibodies identify as Id-positive the same group of IgG1, bind in a reciprocal fashion to H- and L-chains of parental monoclonal antibody 62, and detect Id62-positive polyclonal serum autoantibodies to thyroglobulin. We conclude that monoclonal antibody 62 expresses independently a similar Id on both polypeptide chains and the intact antibody molecule, or its isolated chains, induce qualitatively similar anti-Id responses. These results are discussed in light of the possible structure/function implication such autoantibodies may have within the Id network.     

IgG subclasses of autoantibodies in systemic lupus erythematosus, Sjogren's syndrome, and drug-induced autoimmunity

The IgG subclasses displayed by autoantibodies were examined in patients with systemic rheumatic diseases. Solid-phase assays performed with purified antigens were combined with a set of four mouse monoclonal antibodies specific for each human subclass to provide quantitative data for all the major autoantibody specificities. IgG1 accounted for an average of 55% of the total antibody activity to native and denatured DNA, Sm antigen, and histone and constituted significantly more anti-SS-B and anti-nRNP (84% and 92%, respectively). The remaining antibody activity consisted largely of IgG3, and this subclass was particularly prominent with anti-histone and anti-Sm in patients with systemic lupus erythematosus. In contrast, IgG2 constituted 3 to 12% of the anti-native and anti-denatured DNA and less than 5% of the anti-SS-B/La activity in only three patients with Sjogren's syndrome. IgG2 was essentially undetectable in antibodies to Sm and RNP antigens. IgG4 was also uncommon, although this isotype was significantly more prevalent in anti-histone from patients treated with procainamide showed that the isotype distribution of anti-histone and anti-denatured DNA remained remarkably constant. However, during periods of large increases in autoantibody activity, a shift from predominantly IgG3 to predominantly IgG1 occurred, consistent with the interpretation that there might be a sequential activation of heavy chain constant regions as the immune response matures. The disproportionately high levels of IgG1 and IgG3 displayed by all the autoantibody specificities examined may indicate that a common immunogenic feature of autoantigens or a common control mechanism underlies the regulation of autoantibody expression.     

Alteration in H chain V region affects complement activation by chimeric antibodies

Chimeric antibodies to the synthetic polypeptide (Tyr, Glu)-Ala-Lys ((T,G)-A-L) were used to examine C activation by human IgG1. Two IgG1 antibodies, which contained mouse L chains and H chains with mouse V domains and human C domains, differed only in their VH domain. Ag binding and C activation by these antibodies were analyzed by ELISA. When limiting amounts of Ag were used in the assays, the antibodies required different quantities of Ag for optimal binding, suggesting that the antibodies bind to different epitopes on the (T,G)-A-L molecule. However, when competitive inhibition assays were performed with an optimal concentration of Ag, there were no differences in relative binding affinities for (T,G)-A-L or dissociation characteristics of the antibodies. C activation was examined at optimal Ag concentration to ensure equivalent binding of two IgG1 antibodies to Ag. After combination with immobilized Ag, these two antibodies bearing different V regions exhibited marked differences in the binding of C components C1q and C3d. When present in equal amounts in the assay, antibody 10B activated C and bound more C1q and C3d than antibody B11. These results indicate that V region differences can affect C activation by IgG.     

Pepsin digestion of a mouse monoclonal antibody of IgG1 class formed F(ab')(2) fragments in which the light chains as well as the heavy chains were truncated

In the preparation of F(ab')(2) fragments of monoclonal antibodies (mAbs) of IgG class, heavy (H) chains are truncated by pepsin and light (L) chains are remained intact. However, F(ab')(2) fragments formed by pepsin-digestion of a mouse mAb PM373, which was of the IgG1 class and raised against human prostate specific antigen (PSA), indicated that the L chains of 31 kDa were cleaved into 23-kDa fragments as well as the cleavage of H chains of 50 kDa into 28-kDa fragments. On the other hand, F(ab')(2) fragments formed by digesting the mAb by cathepsin D showed that the L chains were intact and the H chains were truncated. The immunoreactivities against PSA of the F(ab')(2) fragments containing the intact L chains and those containing the truncated L chains were almost the same as that of the parental mAb, suggesting that the truncation of the L chains does not affect the interaction of the mAb with its specific antigen.     

Total synthesis of a tyrosine suppressor transfer RNA gene. XIII. Synthesis of deoxyribopolynucleotide segments corresponding to the nucleotide sequence -1 to -29 in the promoter region.

Chemical syntheses of two tridecanucleotides, d(G-C-A-T-C-A-T-A-T-C-A-A-A) and d(G-C-G-T-C-A-T-T-T-G-A-T-A), and three undecanucleotides, d(G-G-A-A-G-C-G-G-G-G-C), d(T-G-A-T-G-C-G-C-C-C-C), and d(T-G-A-C-G-C-G-C-C-G-C), are described. These deoxyribo-oligonucleotide segments together represent the DNA duplex corresponding to the previously determined nucleotide sequence -1 to -29 of the promoter region of the tyrosine tRNA gene (Sekiya, T., van Ormondt, H., and Khorana, H.G. (1975) J. Biol. Chem. 250, 1087-1098). Chemical syntheses used the principles of stepwise addition of protected mono- and oligonucleotides to the 3'-hydroxyl end of growing oligonucleotide chains. The desired condensation products were isolated by solvent extraction methods in the case of di- and trincleotides and by anion exchange chromatography in the case of longer chains. All the five synthetic oligonucleotides were characterized by chromatographic and radioactive fingerprinting methods after labeling at the 5'-ends with a [32P]phosphate group.     

Development of non-electrophoretic assay method for DNA ligases and its application to screening of chemical inhibitors of DNA ligase I

A new rapid assay method for DNA ligases has been developed, which allows direct quantification of enzyme activity without using the traditional polyacrylamide gel electrophoretic technique. In this method, the 5'-biotinylated nicked duplex was used as a substrate for the ligase reaction, in which the 5'-end of the first oligonucleotide (19-mer) on the nicked strand is biotinylated and the second oligonucleotide (20-mer) on the same strand is labeled with radioactive 32P at the 5'-end. After ligation of the biotinylated 19-mer oligonucleotide into the second oligonucleotide with the reaction of DNA ligases, the biotinylated 19-mer oligonucleotide is converted into the radioactive 39-mer oligonucleotide. The ligase reaction products were heat-denatured to release both ligated and unligated biotinylated oligonucleotides. The biotinylated oligonucleotides were then captured on a streptavidin-coated microtiter plate and counted. The results obtained using this method correlated very well with those from the standard assay method using electrophoresis. Using this assay method, we were able to screen a chemical library and identify new DNA ligase inhibitors structurally related to resorcinol, which has growth inhibitory effects on the human breast cancer cell, MCF-7. The method described here is anticipated to be very useful for screening DNA ligase inhibitors from chemical libraries.     

V region sequences of an idiotypically connected family of pathogenic anti-DNA autoantibodies

The H and L chain V region sequences of nine anti-DNA mAb that are representative of a pathogenic population of autoantibodies produced by the nephritis prone (SWR x NZB)F1 (SNF1) mice, were determined. These nine anti-DNA autoantibodies were idiotypically connected members of a cross-reactive Id family called the Id564 cluster. Moreover, these autoantibodies were all cationic in charge, IgG2b in isotype, and their H chain C regions had the normal SWR parent's allotype. Although derived from two different SNF1 animals, these pathogenic autoantibodies possessed highly homologous Leader-VH sequences that could account for their idiotypic cross-reactivity. Furthermore, the VH region sequences of these anti-DNA antibodies contained numerous basic residues that could impart their cationic charge. The Leader-VH sequences of these autoantibodies were also highly homologous to that of an anti-NP antibody-related germ-line gene of C57BL/6 mice, called VH-23. Among these nine pathogenic autoantibodies, three sets of clonally related anti-DNA antibodies could be identified. Thus the Id564 cluster of cationic anti-DNA autoantibodies of SNF1 mice are encoded by highly related VH genes, and this idiotypically connected population of pathogenic autoantibodies are selected to undergo an oligoclonal expansion in the lupus-prone SNF1 mice.     

Determination of the poliovirus RNA polymerase error frequency at eight sites in the viral genome.

The poliovirus RNA polymerase error frequency was measured in vivo at eight sites in the poliovirus genome. The frequency at which specific G residues in poliovirion RNA changed to another base during one round of viral RNA replication was determined. Poliovirion RNA uniformly labeled with 32Pi was hybridized to a synthetic DNA oligonucleotide that was complementary to a sequence in the viral genome that contained a single internal G residue. The nonhybridized viral RNA was digested with RNase T1, and the protected RNA oligonucleotide was purified by gel electrophoresis. The base substitution frequency at the internal G residue was measured by finding the fraction of this RNA oligonucleotide that was resistant to RNase T1 digestion. A mean value of 2.0 x 10(-3) +/- 1.2 x 10(-3) was obtained at two sites. A modification of the above procedure involved the use of 5'-end-labeled RNA oligonucleotides. The mean value of the error frequency determined at eight sites in the viral genome by using this technique was 4.1 x 10(-3) +/- 0.6 x 10(-3). Sequencing two of the RNase T1-resistant RNA oligonucleotides confirmed that the internal G was changed to a C, A, or U residue in most of these oligonucleotides. Thus, our results indicated that the polymerase had a high error frequency in vivo and that there was no significant variation in the values determined at the specific sites examined in this study.     

Antibody-nucleic acid complexes. Oligo(dG)n and -(dT)n specificities associated with anti-DNA antibodies from autoimmune MRL mice

The specificity of anti-DNA antibodies in the sera of unimmunized autoimmune MRL mice was initially assessed via an enzyme-linked immunosorbent assay (ELISA). Antibody binding profiles to a panel of immobilized antigens (AMP-, GMP-, CMP-, UMP-, and TMP-BSA, ss- and dsDNA) demonstrated high levels of immunoglobulins reacting with GMP and ssDNA and intermediate levels with AMP, TMP, and dsDNA. Fractionation of serum anti-DNA antibodies into subsets on the basis of their binding to GMP- and TMP-agarose indicated that the resulting GMP- or TMP-reactive antibodies bound to their homologous nucleotides and ssDNA. Competition-inhibition studies with soluble mono-, oligo-, and polynucleotides revealed that GMP- and TMP-reactive antibodies were highly specific for oligo(dG)n and -(dT)n sequences, respectively. Whereas the relative affinity of TMP-reactive autoantibodies to oligo(dT)n increased with oligonucleotide length (n = 2, 4, 6, 8, 10, 15), GMP-reactive antibodies preferentially recognized oligo(dG)10 (Ka congruent to 1 x 10(7) M-1). While neither antibody recognized oligo(dA)8 and -(dC)8 competitors, mixed-base oligonucleotides were inhibitory at concentrations approximately 10-fold greater than similarly sized oligo(dG)n and -(dT)n sequences. Similar characterizations of both pooled and individual MRL sera indicated that anti-DNA antibodies represent 8-10% of the total serum IgG. More importantly, GMP-reactive autoantibodies predominated and accounted for 60-70% of the entire unbound anti-DNA antibody population.