Antibody to poly(ADP-ribose) is an indicator of obstetric complications in pregnant patients with systemic lupus erythematosus.

Anti-poly(ADP-ribose) antibodies were measured by an enzyme-linked immunosorbent assay (ELISA) in 6 pregnant women with systemic lupus erythematosus (SLE), 11 normal pregnant women and 6 randomly selected female SLE patients. Four pregnant SLE patients who had either an abortion after 13 weeks of gestation or a premature delivery showed very high titers of anti-poly(ADP-ribose) antibodies in week 8 of pregnancy. However, the titers of anti-poly(ADP-ribose) antibodies of all the normal pregnant women were similar to those of non-pregnant female SLE patients, being slightly higher than those of normal non-pregnant women. The isotype of anti-poly(ADP-ribose) antibodies in pregnant SLE patients was IgG, which did not crossreact with either DNA or cardiolipin. The number of pregnant SLE patients tested was small, but the coincidence of abortion with high titers of anti-poly (ADP-ribose) antibody was very close. Therefore, a high titer of anti-poly(ADP-ribose) antibody in pregnant SLE patients seems useful as an indicator of abortion or fetal distress.

A dot-blot method for screening polyclonal and monoclonal antisera to poly(ADP-ribose).

ADP-ribosylation reactions play a key role at several points in cellular regulation and repair of DNA damage. The use of polyclonal or monoclonal antisera to poly(ADP-ribose) as probes to localize the site(s) of action of the polymer offers a promising tool for these studies. We report here a simple, sensitive method for detection and titration of these antisera to poly(ADP-ribose) using nitrocellulose membrane (NC) as a support for a dot-blot analysis. We take advantage of the fact that a highly labeled poly(ADP-ribose) preparation can be obtained by incubation of a 0.3 M KCl extract prepared from calf thymus nuclei with 32P-NAD. Such a preparation of labeled antigen is used as a reagent to detect the positive antibody spots on the NC with negligible background. Subsequent titration of the antisera and their semi-quantitative evaluation are also feasible using the dot-blot method. The sensitivity of the assay is only limited by the specific activity that can be achieved for the labeled polymer prepared as the antigen probe. The advantage of this method is that it eliminates the need to prepare pure, highly radiolabeled polymer as well as the fact that several samples can be handled on the membrane simultaneously. We demonstrate application of this technique for screening sera from patients with systemic lupus erythematosus (SLE) for anti-poly(ADP-ribose) antibodies. Further, we also extend the use of these sera for immunoquantitation of ADP-ribosylated proteins in six human tumor cells in tissue culture.

Shared idiotypes on anti-DNA and anti-poly (ADP-ribose) antibodies.

Hed 10 is a ssDNA-specific mAb derived from an NZB/W autoimmune mouse. ADP 1 is a poly(ADP-ribose)-specific mAb derived from a C57BL/6 mouse. Rabbit anti-idiotypic sera were raised against Hed 10 and ADP 1. By affinity chromatography it was shown that at least 20 to 30% of DNA-binding antibodies contained these idiotypes. The sera were also used to evaluate idiotypic cross-reactivity among 26 diverse, predominantly anti-nucleic acid, murine mAb. Each serum bound directly to several mAb and in addition inhibited the binding of several antibodies to their appropriate Ag. The anti-Hed 10 serum detected an idiotype which was restricted to antibodies that bound to poly(dT) and/or poly(ADP-ribose). The anti-ADP 1 serum detected a more widely distributed idiotype contained in antibodies which bound to a variety of nucleic acids. Although both sera bound directly to Hed 10, only the anti-Hed 10 serum could compete for the binding of Hed 10 to poly(dT). On the other hand, both sera could compete for the binding of ADP 1 to poly(ADP-ribose) as well as bind directly to ADP 1. In addition anti-ADP 1 serum appeared to enhance, rather than inhibit, the binding of one mAb to native calf thymus DNA and poly[d(AT)] but had no effect on the binding to several ssDNA. These results demonstrate that many antibodies which recognize DNA and poly(ADP-ribose) have shared idiotypes. This may be of relevance to the development of autoimmunity because poly(ADP-ribose) is ubiquitous and immunogenic.

Antibody to poly(ADP-ribose) as a predictor of obstetric complications in autoimmune MRL/Mp-lpr/lpr mice: basis for its application to pregnant patients with systemic lupus erythematosus.

In pregnant autoimmune MRL/Mp-lpr/lpr (MRL/l) mice with many fetuses the level of anti-poly(ADP-ribose) antibodies was found to be the same as that of age-matched non-pregnant female mice, whereas in mice with few fetuses the level of anti-poly(ADP-ribose) antibodies was high in the early period of pregnancy and rapidly returned to control level at puerperium. The anti-poly(ADP-ribose) antibody that increased during pregnancy seemed to be mono-specific for its antigen, whereas the antibody that increased with age was polyspecific. The isotype/subclass of the former was mainly IgG2a. The marked increase in anti-poly(ADP-ribose) antibodies in the early period of pregnancy suggests endogenous sensitization to poly(ADP-ribose), which may be synthesized abnormally or stored during pregnancy, and is a predictive sign in pregnant lupus mice of a low litter size. This finding is applicable to pregnant patients with systemic lupus erythematosus (SLE); that is, it is a predictive sign of fetal loss and/or maternal risk. This was confirmed in the human cases examined so far.

Antibodies to the five histones and poly(adenosine diphosphate-ribose) in drug induced lupus: implications for pathogenesis.

Certain drugs are a frequent source of antinuclear antibody (ANA) induction, and ANA is invariably present in the few patients who progress to the drug induced lupus syndrome. This report concerns the fine specificity of the ANA response to hydralazine, penicillamine, and sulphasalazine therapy. Using highly purified individual histones in fluorimetric assays, antihistone antibodies are always detectable, often in large amounts, but the pattern of response to individual histones is variable and not drug specific. In addition to the response to the three histones H1, H2B, and H3 reminiscent of idiopathic systemic lupus erythematosus, antibody to histone H2A predominates in some drug induced cases. Contrary to previous thought, histones are not the sole target of the antinuclear response: we also demonstrate a significant correlation between ANA titre and antibody to poly(adenosine diphosphate-ribose). Like the histones, this is a macromolecule that can bind to deoxyribonucleic acid (DNA). It is proposed that drug induced damage to chromatin leads to ANA production, while drug induced impairment of complement activity may then enable these autoantibodies to mediate the lupus syndrome.

The production of antibodies to DNA in normal mice following immunization with poly(ADP-ribose).

Monoclonal antibodies were prepared from C57/black mice which had been immunized with poly(ADP-ribose). As expected, some of these antibodies were very specific for poly(ADP-ribose) but, surprisingly, several bound to DNA as well. Analysis of the sera of these mice also showed elevated levels of antibodies to DNA. Monoclonal antibodies against poly(ADP-ribose) were also recovered from unimmunized NZB/W mice which provided an animal model for systemic lupus erythematosus(SLE). These antibodies also cross-reacted with DNA. Comparison of the specificities of the monoclonal antibodies from the two groups of mice showed some striking similarities. In particular, three out of 11 antibodies from the C57/black mice preferred poly(dT) as judged by a solid phase radioimmunoassay. Similarly, 10 out of 17 antibodies from the NZB/W group showed the same type of specificity pattern. These results demonstrate that anti-DNA antibodies can be induced by poly(ADP-ribose) and that some of the autoimmune DNA-binding antibodies found in SLE may result from exposure to poly(ADP-ribose).

Specificity of naturally occurring antibodies to poly(ADP-ribose) in patients with systemic lupus erythematosus: determination by an enzyme linked immunosorbent assay.

Serum autoantibodies to poly(ADP-ribose), single-stranded (ss) DNA and double-stranded (ds) DNA in 145 patients with systemic lupus erythematosus (SLE) were measured by an enzyme-linked immunosorbent assay (ELISA). The specificities of the antibodies for poly(ADP-ribose) or for ssDNA in 14 serum samples from different patients, who had relatively high antibody titers to either or both these antigens, were tested by competitive ELISA with poly(ADP-ribose) and ssDNA as inhibitors. The IgG class anti-poly(ADP-ribose) antibodies of 4 serum samples (cases 9, 11, 13 and 14) preferred poly(ADP-ribose) and those of 2 samples (cases 2 and 4) cross-reacted preferentially with ssDNA, while the IgG class anti-ssDNA antibodies of 2 serum samples (cases 9 and 11) significantly cross-reacted with poly(ADP-ribose). Hence, the nature of the antibodies to poly(ADP-ribose) in SLE patients seemed to be different from that of the anti-poly(ADP-ribose) antibodies in autoimmune MRL/Mp-lpr/lpr (MRL/l) mice, which seem to be subpopulations of anti-ssDNA antibodies and react equally well with poly(ADP-ribose) and ssDNA.

Production of anti-(ADP-ribose) antibodies with the aid of a dinucleotide-pyrophosphatase-resistant hapten and their application for the detection of mono(ADP-ribosyl)ated polypeptides.

Previous attempts to produce anti-(ADP-ribose) antibodies by immunization of rabbits with ADP-ribose conjugated to serum albumin had resulted in the production of 5'AMP-specific antibodies [Bredehorst et al. (1978) Eur. J. Biochem. 82, 105-113]. To obtain true anti-(ADP-ribose) antibodies an antigen was constructed that was resistant to enzymic degradation at the pyrophosphate group. The enzymically active beta-methylene derivative of NAD (NAD[CH2]) was synthesized from ADP containing a methylene bridge (CH2) instead of an oxygen in the diphosphate group. NAD[CH2] was converted to its N6-[(2-carboxyethyl)thiomethyl] derivative and hydrolyzed to the corresponding ADP[CH2]-ribose derivative which was then coupled to bovine serum albumin. The antibodies obtained with this antigen were specific for free or protein-bound ADP-ribose groups, except for a cross-reaction with FAD, AMP, ADP, ATP or poly(ADP-ribose) interfered with [3H]ADP-ribose tracer binding only at higher concentrations. No interference was observed with poly(A), RNA and DNA at 6000-fold excess. The antibodies were purified on a novel type of affinity matrix. This was formed from NAD and guanidinobutyrate by a cholera-toxin-catalyzed reaction and the product, ADP-ribosyl guanidinobutyrate, was bound to Affi Gel by carbodiimide-aided condensation. The purified antibodies allowed the detection of ADP-ribose conjugated to polypeptides in amounts lower than 1 pmol as demonstrated by immunoblotting of [14C]ADP-ribosylated elongation factor 2. They also could be used to observe in situ, by indirect immunofluorescence, the increased mono(ADP-ribosyl)ation of nuclear proteins in dimethyl-sulfate-treated cells, and to show that histone H2B was the principal histone acceptor of single ADP-ribose groups in alkylated 3T3 cells.

MRL/Mp-lpr/lpr mouse derived monoclonal antibodies that recognise determinants shared by poly (ADP-ribose), single stranded DNA and left handed Z-DNA.

MRL/Mp-lpr/lpr (MRL/l) mice spontaneously produce antibodies to poly(ADP-ribose) that are cross-reactive with single stranded DNA (ssDNA). Spleen cells from these animals were used for fusion with murine plasmacytoma cells to prepare hybridomas that produce autoantibodies to poly(ADP-ribose). Monoclonal antibodies (MoAb) produced by the selected hybridomas not only preferred ssDNA to poly(ADP-ribose), but also reacted with left handed Z-DNA; the MoAb reflected the nature of serum antibodies to poly (ADP-ribose) in MRL/l mice. These results suggest that similar antigenic determinants exist in poly (ADP-ribose), ssDNA and left handed Z-DNA and that the cross-reactive nature of autoantibodies to poly (ADP-ribose) in MRL/l mice may be the results of expansion of such clones as selected in this experiment.

Naturally occurring antibodies to poly(ADP-ribose) in autoimmune MRL/Mp-lpr/lpr mice.

The presence of antibodies to poly(ADP-ribose) was demonstrated in the sera of MRL/Mp-lpr/lpr (MRL/l) mice by an enzyme linked immunosorbent assay (ELISA). Antibody to poly(ADP-ribose) was strongly inhibited by single stranded DNA (ssDNA) and poly(ADP-ribose), and less by double stranded DNA (dsDNA). Affinity purified anti-poly(ADP-ribose) antibodies bound more with immobilized ssDNA than with poly(ADP-ribose), and were significantly inhibited by soluble ssDNA, although poly(ADP-ribose) was the best soluble inhibitor. On the contrary, affinity purified anti-ssDNA antibodies bound best with ssDNA and significantly but less with poly(ADP-ribose); however, they were scarcely inhibited by poly(ADP-ribose). These results suggest that similar antigenic determinants exist in poly(ADP-ribose) and ssDNA. It is conceivable, however, at the present moment that 'naturally occurring antibodies to poly(ADP-ribose)' in MRL/l mice are subpopulations of anti-ssDNA antibodies that react equally well with poly(ADP-ribose) and ssDNA.

Monoclonal antibodies to poly(adenosine diphosphate ribose) recognize different structures.

Two hybridomas producing monoclonal antibodies to poly(adenosine diphosphate ribose) [poly(ADP-Rib)] were established. One antibody, 10H (IgG3, kappa), bound to most of the poly(ADP-Rib) preparation, which consisted of molecules of various sizes of more than 20 ADP-Rib residues. The binding of this antibody was inhibited by not only poly-(ADP-Rib) but also a monomer unit of poly(ADP-Rib), Ado(P)-Rib-P. The sites protected by antibody 10H were isolated and analyzed by hydrolysis with alkaline phosphomonoesterase and then snake venom phosphodiesterase. The sites contained the same amounts of monomer units and branched portions [Ado(P)-Rib(P)-Rib-P] as the original poly(ADP-Rib) molecules but a lower average number of branched portions per molecule than in the original molecules. The other antibody, 16B (IgM, lambda), reacted with only 50% of the radioactive poly(ADP-Rib), and its binding was not inhibited by a monomer unit. This antibody protected 25% of all the poly(ADP-Rib) molecules from hydrolysis by snake venom phosphodiesterase. The protected sites contained twice as many branched portions per molecule as the original poly(ADP-Rib) molecules. These results show that the two monoclonal antibodies recognize different structures of poly-(ADP-Rib); 10H antibody recognizes the linear structure with ribose-ribose linkages, and 16B antibody may recognize specific structures, including the branched portions of poly-(ADP-Rib).

Measurement of antibody to poly (adenosine diphosphate-ribose): its diagnostic value in systemic lupus erythematosus.

Poly (ADP-ribose) and dsDNA binding activity have been measured in sera from 61 patients with systemic lupus erythematosus (SLE) and 188 control sera from 20 normal individuals, 144 patients with clinically similar diseases and 24 patients with drug-induced anti-nuclear antibodies (ANA). Elevated poly (ADP-ribose) binding was not observed with normal sera. Five of 144 samples from diseases entering the differential diagnosis of SLE gave raised poly (ADP-ribose) binding compared with 12 in the 125I-dsDNA binding. Only two of these false positive samples gave elevated binding in the 14C-dsDNA assay. The apparent high specificity of the poly(ADP-ribose) assay was not observed with samples containing drug-induced ANA where 62% had elevated binding values. The frequency with which the poly(ADP-ribose) assay was positive with SLE sera (sensitivity) was lower than either of the dsDNA assays. This low sensitivity and the high rate of false positives in patients with drug-induced ANA limit the value of the poly(ADP-ribose) assay as a diagnostic test for SLE. However the restriction of poly(ADP-ribose) antibody to SLE and patients with drug-induced ANA together with the known role of poly(ADP-ribose) in DNA excision repair suggest that the antibody may be of fundamental significance.

Poly-adenosine diphosphate ribose autoantibody in systemic lupus erythematosus and other related autoimmune diseases.

The binding activities of poly-adenosine diphosphate-ribose (ADPR) and ds-DNA were measured in the sera of patients with systemic lupus erythematosus (SLE) and other collagen diseases in comparison with normal subjects. High polyADPR binding activity was detected in the SLE sera. The polyADPR binding assay was as sensitive as the DNA binding assay for diagnosing SLE. In SLE sera, the increased polyADPR binding activity was correlated with that of ds-DNA and negatively with the complement (CH50) titer. With improvement of clinical symptoms of SLE, the binding activity of polyADPR decreased in parallel to the binding activity of ds-DNA and opposite to the CH50 titer. The polyADPR binding activity was occasionally high in other collagen diseases. Effects of steroid treatment of SLE on the binding activities of polyADPR and ds-DNA, and CH50 titer were examined over half a year, indicating that both binding activities are reliable parameters for judgment of the clinical course.

Monoclonal antibodies against poly(ADP-ribose) recognize different structures of poly(ADP-ribose).

The characteristics of two monoclonal antibodies to poly(ADP-Rib) and the various structures of poly(ADP-Rib) recognized by these monoclonal antibodies have been examined. One antibody, IgG monoclonal antibody 10H, reacted with most parts of poly (ADP-Rib) molecules, and its binding was only slightly inhibited by Ado(P)-Rib-P, a monomer unit of the polymer. On hydrolysis of poly(ADP-Rib) protected by the antibody hydrolytic products, such as Ado(P)-Rib-P and Ado(P)-Rib(P)-Rib-P, were almost the same as those from control poly(ADP-Rib). The other antibody, IgM monoclonal antibody 16B, recognized only some parts of poly(ADP-Rib) molecules of larger size, and its binding was not inhibited by Ado(P)-Rib-P. Branched portions were considerably concentrated in parts protected by this antibody. These two monoclonal antibodies are suggested to recognize different structures of poly(ADP-Rib): possibly IgG antibody 10H recognizes linear portions whereas IgM antibody 16B recognizes branched portions of the polymer. These monoclonal antibodies should be useful in studies on the structures, including unknown structures and the locations of these structures in poly(ADP-Rib).

Profiles of antibodies to poly(ADP-ribose), left-handed Z-DNA, and other nuclear constituents in systemic lupus erythematosus and progressive systemic sclerosis.

A simple and sensitive method was developed for detection of antibodies to histones using a modification of the enzyme-linked immunosorbent assay (ELISA) for antibodies to nucleic acids. The antigens used for antibody assay were poly(ADP-ribose), left-handed Z-DNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and calf thymus histones. These five antigen-antibody systems were used to examine the antibodies in patients with systemic lupus erythematosus (SLE) and progressive systemic sclerosis (PSS). A positive correlation was found between the antibody titers in patients with PSS. However, weak correlation was found between the titers of antibodies to these nuclear antigens in the SLE patients, except for correlations between the titers of antibodies to left-handed Z-DNA and histones, and those to ssDNA and dsDNA. From these data, it is suggested that presentation of modified chromatin to immune systems is involved in antinuclear antibody formation in PSS patients, whereas spontaneous expansion of clones for antinuclear antibodies may occur in SLE patients under certain conditions.

A simple and rapid microenzyme-linked immunosorbent assay for antibodies to poly(ADP-ribose) in systemic lupus erythematosus.

A simple and rapid microenzyme-linked immunosorbent assay has been developed for determination of anti-poly(ADP-ribose) antibodies in humans using a combination of protein A-alkaline phosphatase conjugates and poly(ADP-ribose)-coated polyvinyl microplates. After a 1-h treatment of the plates with 100 microliters of poly L-lysine (PLL) solution (50 micrograms/ml), an aliquot of the solution containing 100 ng poly(ADP-ribose) (50 microliters) was added to the PLL-treated plates and evaporated at 37 degrees C overnight to facilitate the adherence of poly(ADP-ribose) to the plates. Nonspecific binding of diluted test sera from patients with systemic lupus erythematosus (SLE) or from normal individuals to the PLL-coated plates was minimized by exposure of the plates for 1 h to Tris-buffered saline (pH 7.4) containing 0.01% bovine serum albumin (BSA). This method was also applicable to the determination of anti-double-stranded DNA antibodies in humans. The present assay is advantageous over those reported so far as it saves time and antigen.

Studies on autoantibodies to poly (adenosine diphosphate-ribose) in SLE and other autoimmune diseases.

Sera from 41 patients with systemic lupus erythematosus (SLE), 87 controls with various diseases, and 30 normal subjects were examined for poly (adenosine diphosphate-ribose) and ds DNA binding. Elevated levels of poly (ADP-ribose) binding were found in 73% of the SLE patients compared with 58% who had raised ds DNA binding. In a further study of 160 sera from 27 patients with SLE, levels of antipoly (ADP-ribose) antibodies were shown to correlate with clinical activity better than either anti-ds DNA or ss DNA antibodies.

Comparative studies on antibodies to poly(ADP-ribose) in rabbits and patients with systemic lupus erythematosus.

Immunochemical studies were made on the antibodies induced in rabbits against poly(ADP-ribose) and naturally-occurring antibodies in patients with systemic lupus erythematosus. Antibodies against poly(ADP-ribose) could also be induced in rabbits by oligo(ADP-ribose) associated with rat liver histones and by a complex of poly(ADP-ribose) with methylated bovine serum albumin (MBSA). The two types of antibody were inhibited to the same extent by poly(ADP-ribose). However, the antibody induced by oligo(ADP-ribose) associated with histones was inhibited by oligo(ADP-ribose) with an average chain length if 4 ADP-ribosyl units and by phosphoribosyl adenosine monophosphate (PR-AMP) but not by mono ADP-ribose, whereas that induced by poly(ADP-ribose) was practically not inhibited by these related compounds even in excess amounts. The sera of ten cases of systemic lupus erythematosus showing high antibody activity against poly(ADP-ribose) were also examined immunochemically. It was found that the antibodies of three patients showed a similar inhibitory pattern to that of antibody induced in rabbits by oligo(ADP-ribose) associated with histones, those of three patients showed a similar pattern to that of antibody produced in rabbits by poly(ADP-ribose), and the remainder did not show either pattern. These findings suggest that oligo(ADP-ribose) associated with histones may serve as antigen to elicit naturally-occuring antibodies to poly(ADP-ribose) in patients with systemic lupus erythematosus.

The significance of antibodies to poly(adenosine diphosphate-ribose) in systemic lupus erythematosus.

Poly(adenosine diphosphate-ribose) and ds-DNA binding activity have been measured in thirty-nine systemic lupus erythematosus (SLE) sera, nineteen rheumatoid arthritis sera, fourteen sera from non-SLE rheumatic and non-rheumatic diseases and in ten normal sera. Antibodies to poly(ADP-ribose) were found only in the SLE and in three SLE-like rheumatic diseases. Anti-DNA antibodies, on the other hand, were found not only in the SLE and SLE-like diseases, but also in rheumatoid arthritis and chronic active hepatitis. Estimation of poly(ADP-ribose) binding was, therefore, more specific for, and more discriminatory of SLE from other diseases, than the estimation of ds-DNA binding. The results indicate that the estimation of poly(ADP-ribose) binding in serum may be more useful in the diagnosis of SLE than the presently employed estimation of DNA binding using the Amersham kit. DNA-anti-DNA immune complexes are detected in some of the SLE sera after deoxyribonuclease I digestion, confirming earlier reports of the existence of circulating DNA-anti-DNA complexes in SLE patients. Snake venom phosphodiesterase treatment of some of the SLE sera also resulted in increased poly(ADP-ribose) binding activity, suggesting the existence of poly(ADP-ribose)-anti-poly(ADP-ribose) immune complexes in the circulation of SLE patients. This observation raises the possiblity that poly(ADP-ribose) immune complexes may play some part in the pathogenesis of some cases of SLE.