Evaluation of the cross-reaction between anti-DNA and anti-cardiolipin antibodies in SLE and experimental animals.

The cross-reaction between anti-DNA and anti-cardiolipin IgG antibodies and its relation to the standard test for syphilis was studied with sera and monoclonal antibodies derived from human patients and mice with systemic lupus erythematosus (SLE). Syphilitic sera of humans and rabbits infected with the spirochete Treponema pallidum were also tested in this study. In addition, rabbits were immunized with ssDNA and cardiolipin and the cross-reactions of the induced antibodies were studied in two different assay systems. The results of these experiments suggest: that the anti-DNA and anti-cardiolipin IgG autoantibodies in SLE sera constitute separate antibody populations and, therefore, cardiolipin cannot play a role in the induction of immune response to DNA in SLE; that in immunized experimental animals there is a significant level of cross-reaction between anti-DNA and anti-cardiolipin-the detection of this cross-reaction depends on highly amplified solid phase assay systems which measure low affinity antibodies and that there is no correlation between the activity of syphilitic sera in the serologic test for syphilis and their binding to pure cardiolipin-this implies that cardiolipin may not be the dominant ingredient in this test as previously proposed.     

Anti-mitochondrial type 5 antibodies and anti-cardiolipin antibodies in systemic lupus erythematosus and auto-immune diseases.

Twenty sera from patients with systemic lupus erythematosus (SLE) and high titre of IgG anti-cardiolipin antibodies (ACA) were studied in order to evaluate the prevalence of anti-mitochondrial type 5 antibodies (AMA 5). None of these sera were found to be AMA 5 positive but five of 18 were positive for VDRL. Twenty sera from patients with AMA 5 were studied in order to evaluate the prevalence of ACA: only six of 20 were positive for ACA. In contrast to this finding, 15 of the 20 sera positive for AMA 5 were also positive for VDRL (P less than 0.001). The six sera positive for ACA and AMA 5 were absorbed with cardiolipin micelles. This absorption eliminated the ACA activity but not the AMA 5 activity. Despite the clinical similarities between the two groups of patients with AMA 5 or ACA, these data suggest that patients with AMA 5 and patients with ACA belong to two different subsets of SLE or SLE-like syndromes and that AMA 5 antigen is different from cardiolipin.     

Use of monoclonal antibodies to identify shared idiotypes on anticardiolipin and anti-DNA antibodies in human sera.

Using hybridoma technology we produced monoclonal antibodies (MoAb) to idiotypic determinants on human anti-cardiolipin antibodies purified from a patient with SLE. Hybridomas were screened by inhibition of cardiolipin binding activity of sera from patients with SLE. Seven hybridomas were selected, two of which were studied extensively. Sera from a number of patients with SLE were found to share idiotypic determinants. This cross-reacting idiotype was not detectable on anti-cardiolipin antibodies in syphilis sera. The cross-reacting idiotype was present in sera with anti-ssDNA antibodies even though some of these sera had no anti-cardiolipin antibodies. We propose that these MoAb may recognize a regulatory idiotype.     

Lymphocyte autoantibodies and alloantibodies in HIV-positive haemophilia patients.

In order to elucidate the fine specificity of anti-cardiolipin antibodies (ACA) in patients with SLE compared to patients with syphilis (SY) various inhibition experiments were performed. Seven SLE sera and eight SY sera positive for ACA were diluted and preincubated with either cardiolipin VDRL-antigen, mitochondial particles, dsDNA, ssDNA or dilution buffer. The sera were subsequently assayed for residual ACA activity of IgG or IgM class using a sensitive ELISA technique. Significant inhibition of IgM ACA activity in SLE sera was found with cardiolipin, VDRL-antigen and mitochondrial particles. Cardiolipin inhibited binding to a significantly higher extent than the other antigens. In SY sera significant inhibition of the IgM ACA activity was found with all antigens used. The strongest inhibition was seen using VDRL-antigen. Inhibition of IgG ACA activity could only be clearly estimated in SY sera where VDRL-antigen was found to be a much stronger inhibitor than the rest, purified cardiolipin being the weakest. Only two out of seven SLE sera were IgG ACA positive which made a clear conclusion impossible but a strong inhibitory capability of pure cardiolipin and a weaker inhibition with VDRL-antigen was found. This study disclosed a difference between SLE and SY sera showing strong reactivity of ACA in SLE sera with purified cardiolipin, contrasting to ACA in SY sera which predominantly reacted with cardiolipin in the liposome environment, as found in the VDRL-antigen and in mitochondrial particles.     

Binding affinity of serum immunoglobulin G to cardiolipin and other phospholipids in patients with systemic lupus erythematosus and syphilis.

Qualitative and quantitative assays for human antibodies to cardiolipin and other phospholipids were used in tests for these reactions in sera from patients with systemic lupus erythematosus (SLE) and syphilis. Of 22 SLE serum samples tested by the qualitative assay, 8 showed positive staining to cardiolipin, phosphatidic acid, and/or phosphatidylserine. All 47 syphilitic sera reacted with these three phospholipids. The apparent affinity of anticardiolipin binding was estimated by normalizing absolute binding levels as a function of serum concentration to the maximum percent bound. It was evident that antibody affinity was four- to fivefold lower in the SLE sera than in the syphilitic sera. Twelve serum samples from patients with one or more features of the anti-cardiolipin syndrome demonstrated mean binding values which were not distinguishable from binding in other SLE sera. In sera from patients with active SLE, binding affinity for cardiolipin was somewhat greater than that in samples from patients with inactive disease, but the differences were not statistically significant. The low anticardiolipin binding affinity which was observed in patients with SLE compared with that in patients with syphilis casts doubt on a pathogenic role for these reactions.     

Heterogeneity of binding reactivity to different phospholipids of antibodies from patients with systemic lupus erythematosus (SLE) and with syphilis.

The binding specificities were investigated of anti-phospholipid antibodies derived from sera from 55 patients with SLE and related diseases, and from 33 patients with syphilis. Antibodies from both these groups of patients bind strongly to cardiolipin in solid-phase immunoassays, but only antiphospholipid antibodies from patients with autoimmune diseases are associated with thrombotic complications and recurrent spontaneous abortions. IgG anti-phospholipid antibodies from both groups of patients cross-reacted with a range of negatively charged phospholipids, but binding to neutral phospholipids was largely restricted to sera from patients with syphilis. A monoclonal IgM lambda anti-cardiolipin antibody, derived from a patient with autoimmunity, was used to inhibit binding of anti-phospholipid antibodies to cardiolipin and to phosphatidic acid. This antibody inhibited the binding of autoimmune sera to cardiolipin more strongly than sera from syphilis patients, but the converse pattern of inhibition of binding to phosphatidic acid was observed. The VDRL titre correlated with anti-phospholipid antibody activity in sera from syphilis patients, but not from those with autoimmunity. Lupus anti-coagulant activity correlated weakly with IgG antibody levels to each of the negatively charged phospholipids among the patients with autoimmunity. Lupus anticoagulant activity did not correlate uniquely with any anti-phospholipid antibody specificity. These results provide further documentation of the great heterogeneity of anti-phospholipid antibodies associated with autoimmune disease and syphilis.     

A common anti-cardiolipin antibody idiotype in autoimmune disease: identification using a mouse monoclonal antibody directed against a naturally-occurring anti-phospholipid antibody

We have recently produced a series of human monoclonal antibodies reacting with cardiolipin. One of these, H3, a polyspecific IgM/k derived from a normal individual, was used to raise mouse monoclonal antibody to its idiotype. Two anti-idiotypic antibodies, S2.9 (IgG2b) and S2.10 (IgM) were selected for their specific reaction with H3.S2.9 did not react with five other human monoclonal antibodies of IgM/k class despite the fact that these shared some antigen-binding characteristics with H3.S2.9 was able to block the binding of H3 to all of its cross-reactive antigens including cardiolipin, while S2.10 was not. S2.9 was equally efficient in blocking the binding of H3 to three of its cross-reactive antigens, cardiolipin, diphtheria and tetanus toxoids; greater than 90% inhibition could be achieved at an equimolar ratio of H3 to S2.9. The anti-idiotype S2.9 was used to demonstrate the presence of the H3 idiotype in serum. This idiotype was found in amounts greater than that seen in 42 normal individuals, in 30 of 36 patients with systemic lupus erythematosus (SLE), eight of 20 patients with rheumatoid arthritis (RA), 8 of 20 patients with Felty's syndrome as well as 10 of 23 patients with syphilis. Not one of nine patients with drug-induced lupus syndrome had abnormal levels. In patients with SLE and Felty's syndrome there was a good correlation between the amount of anti-cardiolipin antibodies and the amount of H3 idiotype (rs = 0.70 and 0.69 respectively). No such correlation was found in syphilitics or in patients with RA. In patients with SLE the H3 idiotype was present on IgM and IgG anti-cardiolipin antibodies. In 15 of 16 SLE sera with high levels of cardiolipin antibody, S2.9 blocked binding of serum antibodies to cardiolipin by 13-72%, with a mean value of 49%. One patient had a high level of anti-cardiolipin antibody which could not be blocked by S2.9. These results indicate that a mouse monoclonal antibody which reacts with an idiotope in the antigen-binding region of a naturally-occurring phospholipid antibody also defines a common idiotype of anti-cardiolipin antibodies in patients with autoimmune disease.     

Binding profiles of anticardiolipin antibodies in sera from patients with SLE and infectious diseases.

Inhibition experiments were performed to study the specificity of IgG-class antibody, binding to cardiolipin immobilized onto a polystyrene surface, in sera from patients with systemic lupus erythematosus (SLE) or infection. Six different phospholipids (three anionic: cardiolipin, phosphatidylserine and phosphatidic acid, and three neutral: phosphatidylcholine, phosphatidylethanolamine and platelet activating factor), lipopolysaccharide from Salmonella Minnesota (ReLPS), strain Re595 and lipoteichoic acid from Streptococcus pyogenes were used as inhibitors, in the form of liposomes. Eight of fifteen SLE sera exhibited strong reactivity to phosphatidylserine liposomes; other anionic phospholipids, cardiolipin and phosphatidic acid, were less effective inhibitors. The binding was inhibited effectively only by cardiolipin in three of the SLE sera, and by none of the anionic phospholipids tested in the remaining four SLE sera. In most sera from patients with bacterial infections (including syphilis), anti-cardiolipin antibodies (ACA) were inhibited only by cardiolipin, but in some cases also by phosphatidic acid. In Gram-negative infections, ACA were inhibited by ReLPS more effectively than by cardiolipin. ReLPS also inhibited ACA in two of five chlamydial sera. Appreciable inhibition of ACA by phosphatidylserine did not occur in infections. Thus, in contrast to previous studies, broad reactivity to anionic phospholipids occurred in only about half of SLE sera. This pattern of polyreactivity was not seen in infections.     

Reactivity patterns of human anticardiolipin and other antiphospholipid antibodies in syphilitic sera.

Sera from patients with proven cases of syphilis were tested for the presence of antibodies to structurally important phospholipids by using qualitative and quantitative assays. All 47 sera examined qualitatively contained antibodies to cardiolipin, phosphatidic acid, and phosphatidylserine, but not antibodies to other selected phospholipids. Such reactivity was not found in normal (Red Cross) sera. Although the degree of antibody binding to phospholipids varied in individual sera, reactivity was almost always greater with cardiolipin than with phosphatidic acid or phosphatidylserine. Binding saturability was found in sera as the cardiolipin concentration was increased over a constant area of nitrocellulose paper. Anti-cardiolipin binding measured by the protein A method gave results similar to the results measured by using anti-immunoglobulin G, which supports the conclusion that binding was to the Fab portion of the immunoglobulin molecule. When measured as a function of serum concentration and plotted in double-reciprocal fashion, the anti-cardiolipin binding data for two syphilitic sera had similar Kd values but different Bmax values. Stoichiometric calculations indicated that approximately 11,000 to 16,000 mol of cardiolipin appeared to be bound per mole of labeled second antibody. These observations may mean that the anti-cardiolipin antibody does not recognize the individual cardiolipin molecule as the antigenic site but recognizes some structural form of the phospholipid or that steric hindrance related to the interaction of the phospholipid with nitrocellulose paper prevented the bulk of cardiolipin molecules from reaching. The structural specificity of the antibodies identified excludes the possibility that these antibodies are directed against the phosphodiester linkage. These findings should give impetus to future study of a potential pathogenic or marker role for these antibodies in syphilis and in other syndromes in which membrane damage may be a primary event.     

The effects of incubation temperature and coating procedure on the measurement of antibodies to cardiolipin

Many laboratories have established ELISAs for the the routine detection of anti-cardiolipin antibodies (ACA). Earlier studies had indicated that assay incubation at 37 degrees C may interfere with the antigen binding capacity of these antibodies. We have reexamined this phenomenon by comparing ACA titers obtained when incubations are performed at either 37 degrees C or at room temperature (RT). In addition, the effect of coating antigen in aqueous or organic solution was compared. The sera tested included a set of recognized ACA standards and samples from 19 patients with SLE, two with primary anti-phospholipid syndrome, 71 patients with a variety of autoimmune and non-autoimmune disorders and 210 blood bank controls. The results show that while some sera do perform better under either incubation temperature there was no correlation between ACA titers and incubation temperature on a population basis either for IgG or IgM isotypes. This was seen both for positive standards and patient sera. For IgG ACA a similar phenomenon was seen if the microplates were coated with cardiolipin either in sodium carbonate or ethanol. For IgM ACA there was a significant increase in ACA titers at RT when cardiolipin was coated in ethanol. The data suggest that for most sera neither the antigen coating medium nor the assay incubation temperature are important variables in the determination of IgG ACA. Factors contributing to the influence of either variable in individual sera could not be identified.     

IgG class anti-cardiolipin antibody as a possible marker for evaluating fetal risk in patients with systemic lupus erythematosus

To identify the correlation between incidence of anti-phospholipid antibodies and fetal prognosis in pregnant SLE patients, we measured the amount of anti-cardiolipin antibody in their sera, using solid-phase enzyme immunoassay (EIA) methods. Findings in the group having poor obstetric results (fetal loss group) and in those with a history of full-term births (live birth group) were compared with regard to other anti-phospholipid antibodies. The incidence of IgG class anti-cardiolipin antibody was 60% in the fetal loss group and 19% in the live birth group, (P less than 0.05). The incidence of the other anti-phospholipid antibodies, including lupus anticoagulant and biological false-positive serological test for syphilis (BFP-STS), did not differ significantly between the two groups. Therefore, the presence of IgG class anti-cardiolipin antibody may prove to be a useful marker for evaluating fetal risk in SLE patients.     

The H3 anti-phospholipid idiotype is found in patients with systemic lupus erythematosus (SLE) but not in patients with syphilis.

The human H3 idiotype, defined by a mouse monoclonal antibody S2.9, is commonly found in patients with SLE where it is correlated with the amount of anti-cardiolipin antibodies. No correlation between the amount of anti-cardiolipin antibody and the H3 idiotype is found in patients with syphilis. Using the S2.9 antibody, serum from each of 10 patients with SLE and eight patients with syphilis was separated into H3-bearing and H3-negative fractions. Comparison of the partition of anti-cardiolipin antibody in these two groups of patients revealed that much of the anti-cardiolipin antibody (44-91%) was found in the H3+ fraction in patients with SLE; in patients with syphilis, virtually none of the anti-cardiolipin antibody was H3+. In patients with SLE, the H3+ fraction contained both IgG and IgM and antibodies of both kappa and lambda light chains. The H3+ fraction was polyspecific and frequently reacted with dsDNA.     

Detection of antiphospholipid autoantibody in systemic lupus erythematosus is temperature-dependent

Non-reactive SLE sera in an ELISA for anticardiolipin antibody (aCL) retested positive in the immunoassay when the sera were first heat-inactivated at 56 degrees C for 30 minutes. This was not a false positive phenomenon since the positive ELISA reactivity of the heated SLE sera was markedly reduced by inhibition with the cardiolipin antigen. Furthermore, the heat-potentiated ELISA reaction was abolished by prior IgG depletion of the SLE sera with Protein A preparation. The unmasked aCL in the heat-treated SLE sera also exhibited selective binding in ELISA to other negatively-charged phospholipids, namely phosphatidylserine and phosphatidic acid but not against either phosphatidylcholine or phosphatidyl-ethanolamine. The data strongly indicate an interaction between antiphospholipid antibodies and heat-sensitive serum component(s), a reduction of the latter resulting in the ELISA detection of the autoantibody.     

Detection of Antiphospholipid Autoantibody in Systemic Lupus Erythematosus is Temperature-Dependent

Non-reactive SLE sera in an ELISA for anticardiolipin antibody (aCL) retested positive in the immunoassay when the sera were first heat-inactivated at 56 C for 30 minutes. This was not a false positive phenomenon since the positive ELISA reactivity of the heated SLE sera was markedly reduced by inhibition with the cardiolipin antigen. Furthermore, the heat-potentiated ELISA reaction was abolished by prior IgG depletion of the SLE sera with Protein A preparation. The unmasked aCL in the heat-treated SLE sera also exhibited selective binding in ELISA to other negatively-charged phospholipids, namely phosphatidylserine and phosphatidic acid but not against either phosphatidylcholine or phosphatidylethanolamine. The data strongly indicate an interaction between antiphospholipid antibodies and heat-sensitive serum component(s), a reduction of the latter resulting in the ELISA detection of the autoantibody.     

Polyspecific reactivity of a murine monoclonal antibody that binds to nuclear matrix-associated, chromatin-bound autoantigens

To investigate polyspecific autoantibody interactions, we have characterized the binding of a cloned murine monoclonal IgM antibody termed (RTE-23) of strain BALB/c origin. By indirect immunofluorescence this antibody displayed a nuclear speckled and peripheral pattern in interphase cells from human and rodent cell lines. In contrast, in mitotic cells, antibody RTE-23 bound to the periphery of individual chromosomes. Immunoblot analysis of soluble and insoluble nuclear proteins from purified rat fibroblast nuclei showed that antibody RTE-23 bound to molecules of 28, 29, and 33 kDa. Furthermore, antibody RTE-23 demonstrated marked polyspecificity and reacted with cytoskeletal proteins (vimentin, keratin, actin), single-stranded DNA, specific synthetic polynucleotides, and cardiolipin. Antibody RTE-23 also showed a lupus anticoagulant-like activity. Screening of sera of autoimmune disease patients with antinuclear antibodies revealed two patients, both with SLE, whose sera blocked antibody RTE-23 binding to nuclei and recognized nuclear proteins identical to those recognized by antibody RTE-23. These results suggested that antibody RTE-23 displays a pattern of self-antigen binding that is represented as well in SLE patient sera.     

Detection of Antiphospholipid Autoantibody in Systemic Lupus Erythematosus is Temperature-Dependent

Non-reactive SLE sera in an ELISA for anticardiolipin antibody (aCL) retested positive in the immunoassay when the sera were first heat-inactivated at 56 C for 30 minutes. This was not a false positive phenomenon since the positive ELISA reactivity of the heated SLE sera was markedly reduced by inhibition with the cardiolipin antigen. Furthermore, the heat-potentiated ELISA reaction was abolished by prior IgG depletion of the SLE sera with Protein A preparation. The unmasked aCL in the heat-treated SLE sera also exhibited selective binding in ELISA to other negatively-charged phospholipids, namely phosphatidylserine and phosphatidic acid but not against either phosphatidylcholine or phosphatidylethanolamine. The data strongly indicate an interaction between antiphospholipid antibodies and heat-sensitive serum component(s), a reduction of the latter resulting in the ELISA detection of the autoantibody.     

A study of immune responses to myelin and cardiolipin in patients with systemic lupus erythematosus (SLE).

Sera from 39 patients with SLE, 20 patients with cerebrovascular disease with no evidence of SLE, and 20 normal controls were tested for antibodies to cardiolipin (CL), brain total upper (UPG) and lower phase (LPG) glycolipids, myelin basic protein (MBP), myelin, and single strand DNA (ssDNA) by ELISA. Binding to the glycolipids and MBP was negative or negligible in all the groups, but significant binding was observed against CL, myelin and ssDNA in some of the SLE patients. Many sera from SLE patients with cerebral disorders and high CL binding also demonstrated high binding to myelin. These sera also labelled cell surface antigens on neonatal mouse neurons and astrocytes by immunofluorescence in tissue culture. A correlation was found to exist between anti-CL and antimyelin antibodies in SLE patients with cerebral lesions, but not between anti-ssDNA and anti-CL antibodies. As much as 80-90% of the specific activity of these antibodies could be absorbed out by the relevant antigens but only partially by the other antigens. In the control groups binding was low and no specific absorption could be demonstrated.     

Opinions on treatment of women with habitual abortion based on investigations for blocking antibody and autoantibodies.

Three hundred and thirty-seven women with habitual abortion of unknown etiology were studied for cellular reactivity and blocking antibody in one-way mixed lymphocyte culture. Their sera were investigated for anti-cardiolipin antibodies, antinuclear antibodies, and antibodies against DNA, and the activated partial thromboplastin time (APTT) and complement levels of their plasma were determined. Increased anti-cardiolipin antibody levels were demonstrated in 77 (22%) of the 337 women, all of whom were considered healthy and had no signs of autoimmune disease. Most patients with high anti-cardiolipin antibody levels displayed lowered values of complement factor C4. According to our experiences, the mere occurrence of anti-cardiolipin antibody in women with habitual abortion is no absolute cause for treatment with prednisolone, not even in cases with greatly elevated anti-cardiolipin values. Therapy with prednisolone and acethylsalicylic acid (ASA) during pregnancy should be given to those women who have high levels of anti-cardiolipin antibodies concomitant with high APTT values, low values of complement C4, and strong blocking antibody. Anti-cardiolipin antibody has been investigated during pregnancy in 136 normal pregnant women, 11 of whom (8%) were positive at any sampling occasion, but only one of whom (1%) had high levels. Evidently the development of anti-cardiolipin antibody is no normal feature of pregnancy among Swedish women and thus the high frequency found among healthy Swedish women with habitual abortion remains unexplained. We have introduced an immunization program of leukocyte transfusions in habitual abortion. The development of previously absent blocking antibody seems to be a valuable prognostic sign of possible success for immunization therapy against habitual abortion.     

Heat inactivation of serum potentiates anti-cardiolipin antibody binding in ELISA

Heat treatment of sera at 56 degrees C for 30 min results in positive ELISA reactions for anti-cardiolipin antibody (aCL) in sera that had undetectable or low levels of aCL before heat inactivation. The positive, potentiated reactivity of the heated sera in the aCL ELISA could be inhibited with the cardiolipin antigen and was abolished by prior IgG depletion using staphylococcal protein A. The heat-potentiating effect of aCL binding in ELISA was evident in both normal human sera and clinical sera including sera from patients with systemic lupus erythematosus and syphilis.     

Anti-phospholipid antibodies in syphilis and a thrombotic subset of SLE: distinct profiles of epitope specificity.

In a study of connective tissue and infectious disease sera, we have demonstrated IgM and IgG anti-cardiolipin activity, in a solid phase radioimmunoassay, in systemic lupus erythematosus (SLE), rheumatoid arthritis, syphilis and in acute malaria caused by four different species of Plasmodium. The highest values were noted in SLE (IgM anti-cardiolipin P less than 0.005, IgG anti-cardiolipin P less than 0.01), but there was no correlation with anti-dsDNA, rheumatoid factor or VDRL titres in any disease group. Anti-cardiolipin binding was significantly associated with the lupus anticoagulant, thrombocytopenia, spontaneous abortions and thromboses in the SLE patients. Ten SLE sera from this thrombotic subset and 10 syphilitic sera with similar anti-cardiolipin activity, were tested against four phospholipid antigens and showed significantly different anti-phosphatidyl ethanolamine/anti-phosphatidyl serine binding ratios (P less than 0.001). These differences in phospholipid epitope specificity could explain the specificity of the VDRL antigen in syphilis serology, and we discuss a putative role for anti-phosphatidyl serine in the thrombotic diathesis of SLE.