Antiidiotypic suppression of autoantibodies with normal polyspecific immunoglobulins

A mechanism by which therapeutic normal polyspecific immunoglobulins (IVIg) may suppress autoimmune responses in vivo is that of antiidiotypic suppression of autoantibodies mediated by anti-idiotypes present in IVIg. In vitro incubation with IVIg of either the plasma or the IgG fraction from plasma of patients with autoantibodies against procoagulant factor VIII (VIII:C), DNA, thyroglobulin, peripheral nerve and intrinsic factor resulted in dose-dependent inhibition of autoantibody activity. The pattern of inhibition curves showed a prozone phenomenon. Maximal inhibition was achieved at a ratio of patient's IgG to IVIg which was specific for each antibody tested. Inhibition was dependent on idiotypic/antiidiotypic interactions between autoantibodies and IVIg since: 1) F(ab')2 from IVIg inhibited autoantibody activity in F(ab')2 fragments from patients' IgG; 2) IVIg contained no antigen-like activity and no antibodies against the commonest allotypes expressed in F(ab')2 fragments of human IgG; 3) autoantibody activity in F(ab')2 fragments from patients' IgG was specifically retained on affinity columns of Sepharose-bound F(ab')2 fragments from IVIg. The presence of antiidiotypes against autoantibodies in pooled normal IgG supports the concept of a functional idiotypic network regulating autoimmune responses in man.     

Intravenous γ-globulin in myasthenia gravis: Interaction with anti-acetylcholine receptor autoantibodies

Clinical improvement has been observed in myasthenia gravis patients treated by intravenous immunoglobulin (IVIg). In order to investigate the mechanism of action of these IVIg, we looked for anin vitro interaction between IVIg and the anti-acetylcholine receptor autoantibodies. Significant inhibition by IVIg of anti-acetylcholine receptor autoantibody activity from 30 MG sera was observed and binding of anti-acetylcholine receptor autoantibodies on IVIg was found for four of five myasthenia gravis sera. These observations suggest that IVIg contains Ig directly binding to and inhibiting pathogenic autoantibodies.     

Detection of antibodies to gangliosides and glycolipids in various intravenous immunoglobulin (IVIg) preparations

The aim of this study was to examine the presence of antibodies to GM1 and sulfatide in various IVIg preparations. Five brands of commercially available human IVIg (Sandoglobulin, Isiven, Cytogam, Omrigam and Cutter) were examined and compared. Serial dilutions of each of the above preparations were prepared at a working range of 0.009 to 25.0 mg/ml IVIg, and screened by a standard 96-well microplate EIA for autoantibodies to the ganglioside GM1 and to the glycolipid sulfatide. The various IVIg preparations (Omrigam, Cytogam, Sandoglobulin, Isiven), except for Cutter IVIg, contained low to medium titers of the autoantibodies tested. Omrigam and Cytogam IVIg contained low titer of antibodies to GM1, and medium-titer of antibodies to sulfatide, whereas Sandoglobulin and Isiven contained only low-titer of autoantibodies to sulfatide. The presence of natural autoantibodies to myelin in human sera may explain the presence of the tested antibodies within IVIg preparations. Measurements of antibodies to ganglioside and glycolipid in sera of Guillain-Barré patients immediately following IVIg, would probably not reveal antibody decrease. Alternatively, long-term (several weeks) follow-up of titers might result in their modification due to inhibition of antibodies production by IVIg.     

The autoreactivity of therapeutic intravenous immunoglobulin (IVIG) preparations depends on the fractionation methods used

Natural immunoglobulin G (IgG) autoantibodies are present in the plasma of healthy individuals and, as a result, in pooled therapeutic intravenous immunoglobulin (IVIg) preparations. The production processes of commercial IVIg preparations involve different fractionation and virus-inactivation steps that include in some cases treatments at extreme conditions. Different physical and chemical treatments are known to augment greatly the reactivity of natural autoantibodies to self-antigens. It is not clear to what extent the self-reactivity of IVIg preparations is due to the presence of natural IgG antibodies in the plasma pools used for fractionation, and to what extent it is due to the treatments that the IgG molecules have been subjected to during the fractionation process. We compared the binding of seven different commercial IVIg preparations to human liver antigens. All studied IVIg's could be clearly separated into two distinct groups: those that possess significant self-reactivity and those with low binding to self-antigens. Increased self-binding was seen in the preparations produced using a fractionation step at low pH. The treatment of IVIg at low pH resulted in increasing the inhibitory effect of the pooled IgG on PHA-induced proliferation of human peripheral blood mononuclear cells. IVIg's with high and low self-binding may have different immunomodulating activities when infused to autoimmune patients.     

Significance of thyroglobulin antibodies cross-reactive with thyroperoxidase (TGPO antibodies) in individual patients and immunized mice.

Thyroglobulin (TG) and thyroperoxidase (TPO), both involved in thyroid hormone synthesis, represent major autoantigens in thyroid autoimmune disease. Despite numerous studies, the emergence, pathophysiological significance and role of autoantibodies to TG and TPO remain elusive. The recent identification of a new category of thyroid-specific autoantibody interacting with both TG and TPO (TGPO autoantibodies) offers a new opportunity in the study of thyroid autoimmunity. To gain a better insight into the significance of these TGPO autoantibodies, measurement in individual samples appeared necessary. The unique property of TGPO autoantibodies, simultaneous binding to TG and TPO, was used to set up a sandwich method which combined coated TG and radio-iodinated TPO. This method was found to be strictly specific for TGPO autoantibodies and sensitive enough to assay TGPO autoantibodies in serum. In humans, TGPO autoantibodies were found in most of the sera with high TG and TPO autoantibody titres, but not in sera negative for TG autoantibodies, whatever the TPO autoantibody titre. Furthermore, high TGPO autoantibody titres were found in sera strongly cytotoxic for cultured porcine thyroid cells. However, significant correlation of TGPO autoantibody titre was observed neither with TG and TPO autoantibody titres (n = 48) nor with complement-dependent cytotoxicity (n = 50). TGPO antibody assay was also performed in individual plasma of CBA/J mice immunized with either human TG (n = 6) or human TPO (n = 6). Immunization with TG induced high levels of not only TG but also TGPO antibodies, which exhibited a strong reactivity for TPO and whose binding to TG and TPO was fully inhibited by TG. In contrast, immunization with TPO induced high levels of only specific TPO antibodies accompanied by low levels of specific TG antibodies. In this case TGPO antibodies were not detected. Of note, TG- and TPO-immunized mice mounted an immune response against their own TG, but did not exhibit histological signs of thyroiditis. Large panels of TG and TPO MoAbs were also investigated with this method: 18/25 TG MoAbs and only 1/13 TPO MoAbs were found cross-reactive. Taken together, these data provide evidence that TGPO antibodies are effectively present in individual patients and TG-immunized mice, are different from specific TG and TPO antibodies, and may derive from natural B cell repertoire by autoimmune processes involving TG and not TPO.     

Genetic and epitopic analysis of thyroid peroxidase (TPO) autoantibodies: markers of the human thyroid autoimmune response

TPO autoantibodies, the hallmark of human autoimmune thyroid disease, are of IgG class and are associated with thyroid destruction and hypothyroidism. Using the immunoglobulin gene combinatorial library approach, a panel of human monoclonal TPO autoantibodies (expressed as Fab) has been generated from thyroid tissue-infiltrating B cells. TPO-specific Fab closely resemble patients' serum autoantibodies in terms of L chain type, IgG subclass, affinities for TPO as well as epitopes recognized by > 80% of TPO autoantibodies in an individual's serum. TPO autoantibody V region genes are not unique; H chain V genes are usually mutated, while L chain V genes are sometimes in germ-line conformation. The autoantibodies recognize an immunodominant region involving conformational, overlapping epitopes in domains A and B. Finally, TPO autoantibody epitopic fingerprints are distinctive for individual sera, are not associated with hypothyroidism, but are conserved over time (indicating a lack of B cell epitope spreading). Evidence for conservation as well as inheritance of the fingerprints in some families, together with V_H gene polymorphisms, may provide insight into the genetic basis of human autoimmune thyroid disease. Furthermore, monoclonal human TPO autoantibodies will be invaluable for B cell presentation of TPO to determine the T cell epitopes involved in TPO autoantibody production.     

Anti-idiotypes against autoantibodies and alloantibodies to VIII:C (anti-haemophilic factor) are present in therapeutic polyspecific normal immunoglobulins.

Therapeutic, polyspecific, normal immunoglobulins (IVIg) suppress anti-factor VIII (VIII:C) activity of anti-VIII:c autoantibodies in vivo and in vitro. In the present study anti-VIII:C activity was found to be inhibited by two different preparations of IVIg in the plasma of three of four patients with autoantibodies and two of three patients with alloantibodies. F(ab')2 fragments from IVIg inhibited anti-VIII:C activity in F(ab')2 fragments from the plasma of the patients. In patients in whom anti-VIII:C activity was inhibited by IVIg, anti-VIII:C F(ab')2 antibodies were specifically retained on an affinity column of Sepharose-bound F(ab')2 from IVIg. In patients in whom anti-VIII:C activity was not suppressed by IVIg in vitro, no binding of anti-VIII:C antibodies to Sepharose-bound IVIg was observed. In a patient in whom anti-VIII:C activity was only suppressed by one preparation of IVIg, specific binding of anti-VIII:C antibodies was only observed with that preparation but not with another. These results indicate that IVIg contain anti-idiotypes against autoantibodies and alloantibodies to VIII:C. The capacity of IVIg to inhibit anti-VIII:C activity in vitro is directly related to the presence of demonstrable anti-idiotypes against anti-VIII:C antibodies. The finding of anti-idiotypes against anti-VIII:C alloantibodies in IVIg suggests that, in addition to autoantibodies, some alloantibodies may be suppressed in vivo by IVIg.     

Intravenous immunoglobulin: exploiting the potential of natural antibodies

Antibodies present in healthy conditions in the absence of deliberate immunization or infections are called natural antibodies. A significant proportion of natural antibody pool is believed to interact with self-antigens, and thus is called natural autoantibodies. Natural autoantibodies belong to IgG, IgM and IgA subclasses, and are encoded by V(D)J genes in germline configuration and bind to self molecules with varying affinities. In addition to serving in first line defense mechanism, natural antibodies participate in the homeostasis of the immune system. Intravenous immunoglobulin (IVIg) is a therapeutic preparation that contains substantial amount of natural antibodies exclusively of IgG subclass. In addition to its role in protection against pathogens in primary and secondary immunodeficiency patients, IVIg exerts a number of immunoregulatory functions through its interaction with innate and adaptive immune system and thereby imposing immune homeostasis.     

Natural anti-Siglec autoantibodies mediate potential immunoregulatory mechanisms: implications for the clinical use of intravenous immunoglobulins (IVIg)

Human intravenous immunoglobulins (IVIg) contain natural autoantibodies against the inhibitory lectin-receptors Siglec-8 and Siglec-9. These two members of the Siglec family are known to mediate both inhibitory and death signals. Here, we discuss recent findings regarding the cytotoxic effects of natural anti-Siglec autoantibodies on both neutrophils and eosinophils, and present the concept of a novel regulatory mechanism exhibiting anti-inflammatory properties. Consequently, IVIg may amplify this regulatory pathway by increasing the concentration of natural anti-Siglec autoantibodies in blood and tissues.     

Selection of the expressed B cell repertoire by infusion of normal immunoglobulin G in a patient with autoimmune thyroiditis

In the present study we have analyzed the changes in the expressed antibody repertoire and in temporal fluctuations of antibody levels in serum that followed infusion of normal IgG (IVIg) in a patient with autoimmune thyroiditis. Administration of IVIg resulted in the stimulation of IgM production, in alterations of expressed antibody activity in serum that could not merely be accounted for by the passive transfer of antibody specificities contained in IVIg, in transient down-regulation of B cells clones expressing a specific disease-related idiotype and in the increase in serum in recipient's autoantibodies specifically reactive with F(ab′)₂ fragments of IVIg. In addition, infusion of IVIg shifted the pattern of spontaneous fluctuations of autoantibody activities in the patient's serum from a pattern indicative of disconnected events in the immune network to a pattern similar to that which is consistently observed in healthy controls. These results suggest that normal IgG may modulate autoreactivity by selecting expressed antibody repertoire through V region-dependent interactions with antibodies.     

Common idiotypes expressed on human, monoclonal, abnormal immunoglobulins and cryoglobulins with polyreactive autoantibody activities.

Several human monoclonal immunoglobulins with the same autoantibody activity have been shown to have cross-reactive idiotypes (CRI). In this study, using polyclonal anti-idiotypic antibodies, we found that 28% of human monoclonal immunoglobulins with polyreactive autoantibody activity from myeloma, Waldenström's macroglobulinaemia and cryoglobulinaemia patients shared common idiotype(s). Furthermore, the latter were expressed on human and murine natural MoAbs (respectively in 12% and 22% of the clones tested) and on human IgG preparations used for therapeutic intravenous injections (IVIg) and which contain natural antibodies. These findings suggest that monoclonal immunoglobulins could arise from the proliferation of a clone that normally produces a natural antibody. The existence of common idiotype(s) between monoclonal immunoglobulins and IVIg could be relevant to the improvement noted after treatment with IVIg in patients suffering from peripheral neuropathies associated with monoclonal gammopathy.     

Intravenous immunoglobulin preparations contain anti-Siglec-8 autoantibodies

BACKGROUND: Human intravenous immunoglobulin (IVIg) preparations are used for the treatment of autoimmune and allergic diseases. Natural autoantibodies are believed to contribute to IVIg-mediated anti-inflammatory effects. OBJECTIVE: To address the question of whether IVIg preparations contain anti-sialic acid-binding Ig-like lectin-8 (anti-Siglec-8) autoantibodies. METHODS: The presence of possible anti-Siglec-8 autoantibodies in IVIg preparations was first examined by functional eosinophil death and apoptosis assays. Specificity of IVIg effects was shown by depleting anti-Siglec-8 autoantibodies from IVIg. Binding of purified anti-Siglec-8 autoantibodies to recombinant Siglec-8 was demonstrated by an immunodot assay. RESULTS: IVIg exerts cytotoxic effects on purified human blood eosinophils. Both potency and efficacy of the IVIg-mediated eosinophil killing effect was enhanced by IL-5, granulocyte/macrophage colony-stimulating factor, IFN-gamma, TNF-alpha, and leptin. Similarly, inflammatory eosinophils obtained from patients suffering from the hypereosinophilic syndrome (HES) demonstrated increased Siglec-8 cytotoxic responses when compared with normal blood eosinophils. Pharmacologic blocking experiments indicated that the IVIg-mediated additional eosinophil death in the presence of cytokines is largely caspase-independent, but it depends on reactive oxygen species. Anti-Siglec-8 autoantibody-depleted IVIg failed to induce caspase-independent eosinophil death. CONCLUSION: IVIg preparations contain natural anti-Siglec-8 autoantibodies. CLINICAL IMPLICATIONS: Anti-Siglec-8 autoantibodies present in IVIg preparations may have therapeutic relevance in autoimmune and allergic diseases, respectively, such as Churg-Strauss syndrome.     

Profile of autoantibody to basement membrane zone proteins in patients with mucous membrane pemphigoid: long-term follow up and influence of therapy

Mucous membrane pemphigoid (MMP) is an autoimmune mucocutaneous blistering disease characterized by autoantibodies to components within the basement membrane zone. In this study, we report the titers of autoantibodies to antigens in the BMZ, in the sera of 13 patients, treated with intravenous immunoglobulin as monotherapy over a consecutive 18-month period. Using bovine gingiva lysate as substrate in an immunoblot assay, autoantibodies to human bullous pemphigoid antigens (BPAg1 and BPAg2), human beta4 integrin, and laminin 5 were measured. A statistically significant (P < 0.05) decline in the autoantibody titers to beta4-integrin was observed after 3.42 months of initiating the IVIg therapy. These titers were undetectable after 13 months of therapy. The titers of antibodies to BPAg1 and BPAg2 did not correlate with disease activity or response to therapy. Antibodies to laminins were not detected. In patients with MMP, autoantibody titers to beta4-integrin correlate with disease activity and response to therapy.     

Idiotypic interactions between normal human polyspecific IgG and natural IgM antibodies

Pooled normal human polyspecific IgG (IVIg) contain anti-idiotypes against a variety of autoantibodies from patients with autoimmune diseases and IgG autoantibodies present in IVIg. The present study indicates that IVIg may also react through idiotypic/anti-idiotypic interactions with human natural IgM antibodies. Sixty-four percent of IgM secreted by B lymphoid cell lines derived from B cells of healthy elderly donors and 18% of IgM secreted by cloned EBV-transformed cord B cells that were tested, bound through their variable region to F(ab')2 fragments of IVIg. The binding to 2,4,6-trinitrophenyl (TNP) of a polyreactive IgM with anti-TNP specificity, was inhibited by F(ab')2 fragments from IVIg, indicating the presence in IVIg of anti-idiotypes that may interfere with the antibody-combining site of polyreactive IgM antibodies. The ability of IgM antibodies to interact with idiotypes on IVIg was not related to the degree of polyreactivity of natural antibodies. Our observations further document that IVIg contain antibody specificities against Ig from normal individuals and suggest that IgG originating from the physiologically expressed repertoire may modulate the expression of the potential B cell repertoire. The results may be relevant to the suppressive effect of IVIg in autoimmune diseases.     

A human anti-cardiolipin autoantibody is encoded by developementally restricted heavy and light chain variable region genes

Based on recent structural analyses of monoclonal autoantibodies, it appears that a number of these antibodies express germ-line immunoglobulin variable region (V) genes with little or no somatic mutation. In addition, our group and others have noted the identity or near identity of some autoantibody-associated V genes to V genes apparently expressed preferentially in the fetal pre-B cell repertoire. To extend these data, we now report that the heavy and light chain V genes of an anti-cardiolipin antibody derived from a healthy individual display 99% nucleotide sequence homology with V genes expressed in early B cell ontogeny. Sequence comparisons indicate the likely use of fetal-restricted V genes by this autoantibody. Taken together with other data on autoantibody V gene usage, these findings provide further evidence for overlap between the autoantibody-associated and early ontogeny expressed V gene repertoires and suggest that natural autoreactivity may be instrumental in the development and maintenance of the normal immune repertoire.     

Serologic and clinical response to treatment of systemic vasculitis and associated autoimmune disease with intravenous immunoglobulin.

BACKGROUND: Autoimmune vasculitides cannot always be controlled by steroids and immunosuppressive drugs. Intravenous immunoglobulin (IVIg) treatment was found beneficial in several vasculitides including systemic and organ-specific diseases. In this article we tested whether the beneficial clinical response of IVIg treatment in vasculitides was accompanied by a decrease in vasculitis-associated autoantibody levels. METHODS: Ten patients diagnosed as having vasculitis were treated with high-dose (2 g/kg) IVIg monthly, in a 5-day schedule. In all the patients, other therapeutic measures failed to control disease progression prior to IVIg treatment. Each patient received between 1 and 6 treatment courses. All patients were evaluated for the levels of 5 autoantibodies (Abs) related to vasculitis before and after each treatment course. RESULTS: In 6 out of the 10 patients, a beneficial clinical response followed IVIg treatment. Moreover, no treatment-related adverse effects were observed in any of the patients. Anti-myeloperoxidase antibodies and cytoplasmic-antineutrophil cytoplasmic antibodies levels decreased concomitantly with the clinical improvement observed in the patients with Churg-Strauss vasculitis and Wegener's granulomatosis, respectively. Levels of cytoplasmic-antineutrophil cytoplasmic antibodies (ANCA) with specificity for bacteridial/permeability-increasing protein and human lysosomal-associated membrane protein increased after each treatment course, but returned to normal values before the following one. CONCLUSIONS: When other therapeutic measures, such as immunosuppressive therapy, fails to control disease manifestations in patients with vasculitides, IVIg is a possible effective intervention method with a high response rate. IVIg probably exerted its effects on disease progression via different mechanisms. Among these mechanisms, a decrease in relevant Ab levels is often found (probably by anti-idiotypes in IVIg), and thus ANCA levels are expected to associate with disease activity.     

Influence of IVIg therapy on autoantibody titers to desmoglein 1 in patients with pemphigus foliaceus

Pemphigus foliaceus (PF) is an autoimmune skin disease characterized by the presence of a pathogenic autoantibody to desmoglein 1, an epidermal cadherin molecule. Antibody titers to the desmoglein 1 protein can be used to monitor disease activity and severity in patients with PF. The purpose of this study is to report the influence of IVIg therapy on anti-desmoglein 1 antibody titers, in eight patients with severe PF, over a period of 18 consecutive months on each patient. This prospective study consisted of eight patients with severe widespread active PF at the time of entry into the study. The levels of autoantibody to desmoglein 1 were measured by an ELISA, at monthly intervals. Sera of all eight had high titers of the autoantibody to desmoglein 1, prior to initiating of IVIg therapy. A statistically significant reduction in the autoantibody titer index to desmoglein 1 was seen after 4 months of IVIg therapy. All eight patients were observed to have a progressive decline in autoantibody titer index while they were receiving IVIg. Patients on IVIg therapy had nondetectable titers after a mean period of 13 months and continued to remain in a serological remission for an additional observation period of 5 months. In the context of this study, autoantibody titers to desmoglein 1 can be used to monitor the serological response to treatment in patients with PF. Patients receiving IVIg therapy achieve serological remission.     

Characterization of human monoclonal autoantibody Fab fragments against oxidized LDL

Oxidized low-density lipoprotein (oxLDL) is a key autoantigen in atherosclerosis. The genetic structures and pathogenic roles of autoantibodies against this protein remain to be established. In this study, we cloned several monoclonal IgG autoantibody Fab fragments specific for oxLDL from peripheral blood lymphocytes of atherosclerosis patients, using phage display technology. The sequences of their variable regions were determined at the cDNA level. The closest germline counterparts for the heavy chains belonged to the V(H)3 or V(H)1 family. The sequences and lengths of complementarity-determining regions (CDR)3-V(H) were diverse, and frequent mutations of positively charged amino acids (particularly arginine) over entire V(H) and V(L) sequences were observed. It is proposed that anti-oxLDL autoantibody formation is driven by antigens. Among the Fabs, P2-8 and P3-175 bound to both MDA-LDL and Cu-oxLDL, and inhibited the uptake of oxLDL by macrophages, suggesting the epitope(s) recognized by the Fabs is a part of ligands on oxLDL that is involved in uptake by macrophage scavenger receptor. These human autoantibody Fabs require detailed investigation to ascertain their potential as agents for clinical applications.     

Autoantibodies directed against labile epitopes on cell surface proteins in autoimmune disease patients: proposal of a novel ELISA for the detection of anti-endothelial cell antibodies

This article describes a novel method for detecting anti-endothelial cell antibodies (AECAs). Sera from patients with systemic vasculitis or inflammatory conditions have been reported to contain antibodies (Abs) that bind to endothelial cells (EC), i.e., AECAs. AECAs are known to play immunogenic effects by triggering EC activation and vascular damage, but the immunopathological role of AECAs is not clear. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting have previously been used for detecting target antigens of AECAs. However, we assumed that these methods are not appropriate for searching genuine target antigens (Ags) on cell surface, and developed a novel solubilized cell surface protein-capture ELISA (CSP-ELISA). Ags were obtained as cell surface proteins from the plasma membrane of human umbilical vein endothelial cells (HUVECs); these cell surface proteins were biotinylated, solubilized with detergent, and captured on ELISA wells coated with NeutrAvidin? biotin binding protein (NeuAvi). AECA titers in serum from 126 autoimmune disease patients and 122 healthy donors were tested. AECAs were detected in 28 of 36 (78%) of systemic lupus erythematosus (SLE) patients; in 13 of 16 (81%) of mixed connective tissue disease (MCTD) patients; and in 5 of 9 (56%) of systemic sclerosis (SSc) patients. Relatively weak denaturation of antigens on ELISA wells caused loss of binding of these autoantibodies (autoAbs). Thus, this newly developed CSP-ELISA method enables the detection of Abs to the labile epitopes of autoantigens (autoAgs) such as membrane proteins, and this method is generally applicable to various kinds of membrane proteins and the Abs against them. We propose CSP-ELISA for measuring AECAs in serum samples for routine laboratory testing.     

Detection of one VH antibody sequence in both healthy donors and urticaria patients

We have previously isolated anti-FcepsilonRIalpha autoantibodies from phage libraries of healthy donors and urticaria patients. Strikingly, the same antibody, LTMalpha15, was isolated from both libraries. Sequence analysis revealed a germline configuration of the LTMalpha15 variable heavy (V(H)) chain with a slightly mutated variable light (V(L)) chain supporting its classification as a natural autoantibody. Distribution analysis of anti-FcepsilonRIalpha autoantibodies by functional or serological tests delivered conflicting data. For this reason we have developed a new real-time PCR to analyse the distribution of LTMalpha15V(H) in healthy donors and urticaria patients. Our new bioinformatic program permitted the design of a minor groove binder (MGB) TaqMan probe that specifically detected the LTMalpha15V(H). We were able to demonstrate a broad range of rearranged V(H) gene copy number without any correlation to the state of health. Monitoring LTMalpha15V(H) gene copy number in a single donor over a period of 70 days revealed a time-related fluctuation of circulating B cells carrying LTMalpha15V(H). We propose that our real-time PCR may serve as a model for the quantification of natural antibody sequences at a monoclonal level.