Immunoregulatory T cell epitope peptides: the new frontier in allergy therapy.

Allergen immunotherapy (AIT) has been practised since 1911 and remains the only therapy proven to modify the natural history of allergic diseases. Although efficacious in carefully selected individuals, the currently licensed whole allergen extracts retain the risk of IgE-mediated adverse events, including anaphylaxis and occasionally death. This together with the need for prolonged treatment regimens results in poor patient adherence. The central role of the T cell in orchestrating the immune response to allergen informs the choice of T cell targeted therapies for down-regulation of aberrant allergic responses. Carefully mapped short synthetic peptides that contain the dominant T cell epitopes of major allergens and bind to a diverse array of HLA class II alleles, can be delivered intradermally into non-inflamed skin to induce sustained clinical and immunological tolerance. The short peptides from allergenic proteins are unable to cross-link IgE and possess minimal inflammatory potential. Systematic progress has been made from in vitro human models of allergen T cell epitope-based peptide anergy in the early 1990s, through proof-of-concept murine allergy models and early human trials with longer peptides, to the current randomized, double-blind, placebo-controlled clinical trials with the potential new class of synthetic short immune-regulatory T cell epitope peptide therapies. Sustained efficacy with few adverse events is being reported for cat, house dust mite and grass pollen allergy after only a short course of treatment. Underlying immunological mechanisms remain to be fully delineated but anergy, deletion, immune deviation and Treg induction all seem contributory to successful outcomes, with changes in IgG4 apparently less important compared to conventional AIT. T cell epitope peptide therapy is promising a safe and effective new class of specific treatment for allergy, enabling wider application even for more severe allergic diseases.

The immunoregulatory and fibrotic roles of activin A in allergic asthma.

Activin A, a member of the TGF-ß superfamily of cytokines, was originally identified as an inducer of follicle stimulating hormone release, but has since been ascribed roles in normal physiological processes, as an immunoregulatory cytokine and as a driver of fibrosis. In the last 10-15 years, it has also become abundantly clear that activin A plays an important role in the regulation of asthmatic inflammation and airway remodelling. This review provides a brief introduction to the activin A/TGF-ß superfamily, focussing on the regulation of receptors and signalling pathways. We examine the contradictory evidence for generalized pro- vs. anti-inflammatory effects of activin A in inflammation, before appraising its role in asthmatic inflammation and airway remodelling specifically by evaluating data from both murine models and clinical studies. We identify key issues to be addressed, paving the way for safe exploitation of modulation of activin A function for treatment of allergic asthma and other inflammatory lung diseases.

Ara h 1 CD4+ T cell epitope-based peptides: candidates for a peanut allergy therapeutic.

BACKGROUND: Peanut allergy is a life-threatening condition; there is currently no cure. While whole allergen extracts are used for specific immunotherapy for many allergies, they can cause severe reactions and even fatalities in peanut allergy. OBJECTIVE: To identify short, HLA-degenerate CD4(+) T cell epitope-based peptides of the major peanut allergen Ara h 1 that target allergen-specific T cells without causing IgE-mediated inflammatory cell activation, as candidates for safe peanut-specific immunotherapy. METHODS: Ara h 1-specific CD4(+) T cell lines (TCL) were generated from peripheral blood mononuclear cells (PBMC) of peanut-allergic subjects using CFSE-based methodology. T cell epitopes were identified using CFSE and thymidine-based proliferation assays. Epitope HLA-restriction was investigated using blocking antibodies, HLA-genotyping and epitope prediction algorithms. Functional peanut-specific IgE reactivity to peptides was assessed by basophil activation assay. RESULTS: A total of 145 Ara h 1-specific TCL were generated from 18 HLA-diverse peanut-allergic subjects. The TCL recognized 20-mer peptides throughout Ara h 1. Nine 20-mers containing the most frequently recognized epitopes were selected and their recognition confirmed in 18 additional peanut-allergic subjects. Ten core epitopes were mapped within these 20-mers. These were HLA-DQ and/or HLA-DR restricted, with each presented on at least two different HLA-molecules. Seven short (≤ 20 aa) non-basophil-reactive peptides encompassing all core epitopes were designed and validated in peanut-allergic donor PBMC T cell assays. CONCLUSIONS AND CLINICAL RELEVANCE: Short CD4(+) T cell epitope-based Ara h 1 peptides were identified as novel candidates for a safe, T cell targeted peanut-specific immunotherapy for HLA-diverse populations.

Functional immunoglobulin E cross-reactivity between Pas n 1 of Bahia grass pollen and other group 1 grass pollen allergens.

BACKGROUND: Grass pollens are major triggers of allergic rhinitis and asthma, but the immunological relationships between pollen allergens of the subtropical Bahia grass, Paspalum notatum, and temperate grasses are unresolved. OBJECTIVE: To assess serum IgE cross-reactivity between subtropical P. notatum and temperate Lolium perenne (Ryegrass) pollen allergens. METHODS: Serum IgE reactivities of grass pollen-allergic patients with P. notatum, L. perenne and Cynodon dactylon (Bermuda grass) pollen extracts and their respective purified group 1 allergens, Pas n 1, Lol p 1 and Cyn d 1, were compared by immunoblotting, ELISA and basophil activation. RESULTS: In a cohort of 51 patients from a temperate region, a high frequency of IgE reactivity with each grass pollen was detected, but reactivity with L. perenne pollen was substantially greater than with P. notatum and C. dactylon pollen. Similarly, serum IgE reactivity with Lol p 1 was greater than with Pas n 1 or Cyn d 1. For seven of eight sera studied in detail, asymmetric serum IgE cross-reactivity was observed; L. perenne pollen inhibited IgE reactivity with P. notatum pollen but not the converse, and IgE reactivity with Pas n 1 was inhibited by Lol p 1 but IgE reactivity with Lol p 1 was not inhibited by Pas n 1 or Cyn d 1. Importantly, P. notatum pollen and Pas n 1 activated basophils in grass pollen-allergic patients from a temperate region, although stimulation was greater by pollen of L. perenne than P. notatum or C. dactylon, and by Lol p 1 than Pas n 1 or Cyn d 1. In contrast, a cohort of 47 patients from a subtropical region showed similar IgE reactivity with P. notatum and L. perenne pollen, and reciprocal cross-inhibition of IgE reactivity between L. perenne and P. notatum. CONCLUSIONS: Pollen allergens of the subtropical P. notatum, including Pas n 1, show clinically relevant IgE cross-reactivity with pollen allergens of L. perenne but also species-specific IgE reactivity.

Latex allergy: a model for therapy.

Allergy to natural rubber latex products emerged as an important clinical condition following an increase in the use of latex gloves for barrier protection in the early 1980s. In addition to latex glove users, other high-risk groups with different latex exposure include spina bifida patients and others with multiple surgical procedures. Subjects with fruit and vegetable allergy are also at risk due to cross-reactive allergens. Following the significant advances in the identification and characterization of common aeroallergens, latex allergy was well placed to become an excellent model of therapy. Awareness of latex allergy and modes of sensitization enabled epidemiological studies to inform allergen avoidance initiatives, substantially reducing inadvertent exposure in major hospitals in Western countries. Spina bifida is often identified in utero or soon after birth, allowing vigorous latex allergen avoidance with enhanced efficacy of primary prevention. However, changing demographics of latex allergy and technological revolution in countries such as China and India are predicted to unleash a second wave of latex allergy reemphasizing the incentive for improved manufacturing procedures for latex products. The desirable high tensile strength and elasticity of natural rubber latex have made the commercial identification of good alternatives very difficult but this would also be attractive for primary prevention. In addition, an effective specific immunotherapy regimen would be valuable for selected high-risk atopic individuals. Current subcutaneous and sublingual immunotherapy schedules have been tested for treatment of latex allergy with evidence of efficacy but the risks of adverse events are high. For such potent allergens as latex, hypoallergenic but T cell-reactive preparations are required for clinical use. Identification of allergenic components of latex products, with generation of monoclonal antibodies and recombinant allergens, allowed sequence determination and mapping of T cell and B cell epitopes. Together, these reagents and data facilitated improved diagnostics and investigation of novel-specific therapeutics. Potential hypoallergenic latex preparations identified include modified non-IgE-reactive allergen molecules and short T cell epitope peptides. The co-administration of adjunct therapies such as anti-IgE or corticosteroids and of appropriate adjuvants for induction of regulatory T cell response offers promise for clinically effective, safe latex-specific vaccines.

IgE cross-reactivity between the major peanut allergen Ara h 2 and tree nut allergens.

Allergy to peanut and tree nuts is characterised by a high frequency of life-threatening anaphylactic reactions and typically lifelong persistence. Although peanut is the most common cause of nut allergy, peanut allergic patients are frequently also sensitive to tree nuts. It is not known if this is due to cross-reactivity between peanut and tree nut allergens. In this study, the major peanut allergen Ara h 2 was cloned from peanut cDNA, expressed in E. coli cells as a His-tag fusion protein and purified using a Ni-NTA column. Immunoblotting, ELISA and basophil activation indicated by CD63 expression all confirmed the IgE reactivity and biological activity of rAra h 2. To determine whether or not this allergen plays a role in IgE cross-reactivity between peanut and tree nuts, inhibition ELISA was performed. Pre-incubation of serum from peanut allergic patients with increasing concentrations of almond or Brazil nut extract inhibited IgE binding to rAra h 2. Purified rAra h 2-specific serum IgE antibodies also bound to proteins present in almond and Brazil nut extracts by immunoblotting. This indicates that the major peanut allergen, Ara h 2, shares common IgE-binding epitopes with almond and Brazil nut allergens, which may contribute to the high incidence of tree nut sensitisation in peanut allergic individuals.

Sublingual allergen immunotherapy: immunological mechanisms and prospects for refined vaccine preparation.

Allergic diseases constitute a major health issue worldwide. Mainstay treatment constitutes allergen avoidance and pharmacotherapy for symptom relief, but allergen immunotherapy offers advantages of specific treatment with long lasting efficacy, and being able to modify the course of the disease. Conventional immunotherapy involves the subcutaneous injection of gradually increasing amounts of allergen extract but the use of current whole allergen extracts is restricted by the risk of adverse IgE-mediated events especially for potent allergens such as peanut and latex and for asthmatics. This has lead to interest in alternative routes of immunotherapy. Oral tolerance is a well-documented immune process and the sublingual route of administration of allergen immunotherapy is attracting interest. Recent meta-analyses show that sublingual allergen immunotherapy for grass pollen and house dust mite allergy is clinically effective and safer than injection immunotherapy. Some studies show SLIT induces changes of T cell anergy, immune deviation, blocking antibodies, and induction of regulatory T cells, as described for injection immunotherapy pointing to the need to target allergen-specific T cells, there is emergent evidence that the oral mucosa presents distinct regulatory features. Evidence suggests that oral dendritic cells play a key role in inducing tolerance especially when allergen is taken up via Fc receptor bound IgE. This suggests that although both would target allergen-specific T cells, allergen formulations may differ with respect to IgE epitopes for optimal SLIT compared with SCIT. Identification of the molecular nature of the allergen-DC receptor interaction is required to determine whether short peptides or recombinant allergen preparations and of suitable adjuvants specifically tailored for the sublingual route will allow the development of improved allergen formulations and delivery strategies for efficacy of treatment whilst decreasing IgE-mediated adverse effects.

Significance of lymphocyte fluorescence polarization changes after phytohemagglutinin stimulation in cancer and noncancer conditions.

The double-zone fluorescein fluorescence polarization (FFP) "cancer" test was used to study 540 blood lymphocyte samples from 341 donors, of whom 158 had confirmed cancer: The other donors were noncancer patients, pregnant women, and normal individuals. The FFP response in cancer patients was the reverse of that in normal individuals, but an abnormal response was also obtained in some noncancer conditions, including the chronic inflammatory disorders--rheumatoid arthritis, cholecystitis, and diverticulitis--and in early pregnancy or pregnancy-induced hypertension. Thus the cancer discriminatory value of the test is limited. Examination of its biologic basis suggests that the positive FFP response in cancer and other conditions is due to altered immune status of blood lymphocytes, with associated change in cytoplasmic fluidity affecting the polarization of fluorescence. Incubation of normal blood lymphocytes with cyclic GMP induced an abnormal, cancer-like FFP response.

Lymphocyte fluorescence polarization changes after phytohemagglutinin stimulation in the diagnosis of colorectal carcinoma.

A lymphocyte fluorescence polarization test that measures reactivity to phytohemagglutinin (PHA) has permitted discrimination of a majority of patients with colorectal carcinoma from noncancer individuals. The test involves separation of two lymphocyte fractions from 20 ml venous blood on a modified leukocyte separation gradient, incubation with PHA for 45 minutes, addition of fluorescein diacetate, and analysis of change in fluorescence polarization. Of 19 colorectal patients tested preoperatively, 13 had a positive stimulation index, 3 a zero index, and 3 a negative index. Of 7 patients with other malignant neoplasms, a positive value was obtained in 6 and a negative value in 1. Of 14 patients with other diseases, a negative value was obtained in 9, zero in 3, and positive in 2. Of 31 normal donors a negative value was obtained in 27, zero in 2, and weak positive in 2. This rapid test promises to develop into a useful cancer-diagnostic method, and elucidation of its biological basis should identify a new cancer marker.

Molecular basis of IgE-recognition of Lol p 5, a major allergen of rye-grass pollen.

Grass pollen, especially of rye-grass (Lolium perenne). represents an important cause of type I allergy. Identification of IgE-binding (allergenic) epitopes of major grass pollen allergens is essential for understanding the molecular basis of interaction between allergens and human IgE antibodies and therefore facilitates the devising of safer and more effective diagnostic and immunotherapy reagents. The aim of this study was to identify the allergenic epitopes of Lol p 5, a major allergen of rye-grass pollen, immunodissect these epitopes further so that the amino acid residues critical for antibody binding can be determined and investigate the conservation and nature of these epitopes within the context of the natural grass pollen allergens. Peptides, 12-13 amino acid residues long and overlapping each other by 4 amino acid residues, based on the entire deduced amino acid sequence of the coding region of Lol p 5, were synthesised and assayed for IgE-binding. Two strong IgE-binding epitopes (Lol p 5 (49-60) and (265-276), referred to as peptides 7 and 34, respectively) were identified. These epitopes were further resolved by truncated peptides and amino acid replacement studies and the amino acid residues critical for IgE-binding determined (Lol p 5 (49-60) residue Lys57 and (265-276) residue Lys275). Sequences of these epitopes were conserved in related allergens and may form the conserved allergenic domains responsible for the cross-reactivity observed between pollen allergens of taxonomically related grasses. Furthermore, due to its strong IgE-reactivity, synthetic peptide Lol p 5 (265-276) was used to affinity-purify specific IgE antibodies which recognised proteins of other clinically important grass pollens. further indicating presence of allergenic cross-reactivity at the level of allergenic epitope. Moreover, Lol p 5 (265 276) demonstrated a strong capacity to inhibit IgE-binding to natural rye-grass pollen proteins highlighting the antibody accessibility to these sequences within the context of the natural allergens. Strong IgE-binding epitopes of Lol p 5 have been identified down to single critical amino acid residues and are shown to occur as linear or continuous domains in the natural conformation of natural Lol p 5 and other group 5 grass pollen allergens. The fact that such an allergenic synthetic epitope has the capacity to strongly inhibit IgE-binding to natural allergens highlight its potential for use as a candidate in future therapeutics to treat pollen-associated allergies.

Occult ovarian failure: a syndrome of infertility, regular menses, and elevated follicle-stimulating hormone concentrations.

Ten women with infertility, regular menses, and elevated plasma FSH concentrations after a failed in vitro fertilization attempt were studied throughout a spontaneous menstrual cycle. Plasma estradiol, progesterone, inhibin, LH, and FSH concentrations were measured by RIA on days 1, 8, 15, and 22 and compared with the ovarian steroid and gonadotropin profiles obtained from seven endocrine-normal women. The elevated FSH concentrations in the hypergonadotropic group were not associated with significant changes in E2 and P4, but an increase in LH concentrations was found on days 1, 8, and 22 (medians of 18 and 4, 17 and 6, and 7 and less than 3 U/L for the hypergonadotropic and normal groups, respectively; P less than 0.01). Their plasma inhibin concentrations [213, 242, 747, and 561 U/L (median values on days 1-7, 8-14, 15-21, and 22-28)] were normal. Autoantibodies to adrenal, thyroid, or ovary were present in five (50%) women, and antiovarian antibodies were present in 4. Two women gave a family history of thyroid disease, and one woman was hypothyroid. Repeat assessment 3-6 months revealed persistently elevated FSH concentrations in five (63%) of eight women; the other three had normal ovarian steroid and gonadotropin concentrations. The triad of infertility, regular menses, and elevated plasma FSH concentrations describes a group of women with occult ovarian failure, a condition of compensated granulosa cell function, which may be an early stage of premature ovarian failure. These women with occult ovarian failure had an impaired response to ovarian hyperstimulation and may be at increased risk of developing polyglandular autoimmunity.

Molecular cloning, expression and immunological characterisation of Lol p 5C, a novel allergen isoform of rye grass pollen demonstrating high IgE reactivity.

A novel isoform of a major rye grass pollen allergen Lol p 5 was isolated from a cDNA expression library. The new isoform, Lol p 5C, shares 95% amino acid sequence identity with Lol p 5A. Both isoforms demonstrated shared antigenic activity but different allergenic activities. Recombinant Lol p 5C demonstrated 100% IgE reactivity in 22 rye grass pollen sensitive patients. In comparison, recombinant Lol p 5A showed IgE reactivity in less than 64% of the patients. Therefore, Lol p 5C represents a novel and highly IgE-reactive isoform allergen of rye grass pollen.

Lymphoid cell fractionation by aggregated immunoglobulin-agarose columns.

Fractionation by columns of aggregated rat immunoglobulin (Agg Ig)-agarose was investigated as a method of separating different populations of lymphoid cells. With rat spleen cells, Agg Ig columns retained phagocytes, IgM- and IgG-antibody-forming-cells, cells mediating antibody- or PHA-induced lysis of chicken erythrocytes, and specifically immune splenocytes lytic to chicken erythrocytes without exogenous antibody. Agg Ig columns did not selectively remove 'B lymphocytes' (surface-Ig-bearing lymphocytes with or without EAC' receptors), or T lymphocytes capable of PHA-induced proliferation or graft-versus-host reactivity. With mouse spleen cells, Agg Ig columns retained alloimmune cytotoxic T cells.

Detection of early lymphocyte activation by the fluorescent cell membrane probe N-phenyl-1-naphthylamine.

N-phenyl-1-naphthylamine (NPN) becomes fluorescent after binding to hydrophobic regions of cell membranes. Rat and mouse lymphoid cell suspensions stained with NPN showed changes in fluorescence emission 30 min after stimulation with mitogen or antigen, detected by microfluorimetry. Incubation of NPN-labelled mouse and rat thymocytes with phytohaemagglutinin or concanavalin A (Con A) caused an increase in mean cell fluorescence intensity. The response to Con A was inhibited by sodium azide and alpha-methyl mannoside. Stimulation of spleen cells from mice by allogeneic cells, or from tumour-bearing rats by tumour antigen consistently resulted in decreased fluorescence. The 'mixed lymphocyte response' detected only certain genetic differences between mouse strains and was proportional to the ratio of stimulator to responder cell number. The NPN staining procedure offers a simple and rapid assay of immunoreactivity and a means of studying early subcellular changes following lymphocyte activation.

Acridine orange fluorescence cytochemistry for detecting lymphocyte immunoreactivity.

Acridine orange staining reveals changes within 3 hours of in vitro stimulation of normal rat lymphocytes with mitogens, and of immune rat lymphocytes with the sensitizing antigen. An increased number of red fluorescent cytoplasmic organelles, presumably lysosomes are seen by fluorescence microscopy. Fluorimetry of the supernatants from stained cell suspensions suggests an overall decreased cell uptake of the dye. The microscopy and fluorimetry detected early events in the reaction of lymphocytes from tumour-bearing rats with the target tumour cells. It would appear that the changes in intracellular behaviour of the dye and in overall cell uptake after immune stimulation are a reflection of dissociated variations in internal and external cell membrane permeability, and may provide simple general means for recognizing cellular immune reactions.

Fluorescence polarization assay by flow cytometry.

Fluorescence polarization measurement on cell suspensions provides a highly sensitive means for detecting subtle changes in the cells, such as occur early after lymphocyte activation or on malignant transformation. We review here the principles of fluorescence polarization, its measurement by a commercially available flow cytometer and application of such assays especially in cellular immunology.

Fluorescent probe assay of early mixed lymphocyte reaction to predict allograft survival in mice.

A rapid fluorescent probe assay of mixed lymphocyte reactivity has permitted prediction of allograft survival in mice. The assay is based on detection of early membrane events in stimulated cells by decreased fluorescence intensity of cell-bound N-phenyl-l-napthylamine (NPN) 30 min after allogenic cell interaction. The NPN-mixed-lymphocyte reaction (NPN-MLR) detected antigenic differences coded for by the whole H-2 complex or by the I region but not by the M locus. The probe assay correlated better with graft survival than did conventional 3H-thymidine assay, which also detects differences at the M locus that are less relevant to allograft rejection. Investigation of the cell types needed to obtain the response detected by the NPN-MLR assay revealed a requirement for mature T cells and plastic-surface-adherent monocytes and macrophages in the responding cell population. The monocytes and macrophages had an essential role in the generation of soluble factors that mediated the NPN-detected response. The NPN-MLR assay offers a reliable, rapid test of recipient-donor compatibility for allograft survival, and a system for studying early events in allogeneic cell interaction.

Allergen immunotherapy: current and new therapeutic strategies.

Allergic individuals respond to an environmental allergen encounter by producing T-cell cytokines, predominantly IL-4 and IL-5, which in turn drive the production of allergen-specific IgE antibodies and recruitment of an eosinophil-rich inflammatory infiltrate. Allergen-specific immunotherapy (SIT) involves the repeated injection of the allergen to specifically downregulate this predominantly Th2-type immune response. SIT is a clinically proven effective treatment for allergic diseases, including rhinoconjunctivitis and asthma. However, despite having been in clinical practice since early this century, its use remains empirical. Best practice protocols are based on clinical experience and include recommendations for selecting patients for treatment, SIT regimes and avoidance of adverse events. More rational and safer SIT regimes will result from new insights into the underlying immune mechanisms for allergic disease, in particular the critical role of helper T-cells in orchestrating this response. The development of recombinant techniques for producing purified allergens and allergen derivatives has led to a dramatic improvement in the ability to standardise allergen preparations and to develop novel vaccines for allergy treatment. Potential vaccines include short peptides based on dominant T-cell epitopes of allergens, allergen fragments and mutant allergens. All of these preparations are designed to target T-cells without binding IgE and inducing local and systemic side effects. Additional strategies under consideration include DNA vaccines and fusion protein constructs incorporating immunomodulatory elements such as bacterial cell proteins, cytokines and immunostimulatory sequences of DNA. Different forms of allergens are being evaluated for the more practical mucosal administration of allergy vaccines. The identification of recombinant allergens suitable for diagnostic use and the development of reliable laboratory assays, based on T-cell function to monitor clinical efficacy of SIT, are important practical outcomes from this research.

Autoantibodies to neurons and to the cytoskeleton in small cell carcinoma with paraneoplastic sensory neuropathy.

Autoantibodies to neurons and to the cytoskeleton were demonstrated in the serum of a patient with small cell carcinoma of the lung (SCCL) and paraneoplastic sensory neuropathy. The serum reacted by immunofluorescence with the nuclei of neurons, and with cytochalasin B-sensitive "stress fibres" of cultured cells. The serum also reacted by immunofluorescence with the nuclei of some cultured cell lines. Immunoblotting experiments with brain tissue, with SCCL, HeLa and other cultured cells showed that the serum reacted with 1-4 bands of 35-39 kDa apparent m.w. Two dimensional immunoblotting showed that these molecules had a neutral pI. Antibodies, affinity-purified by elution from the 35-39 kDa bands, gave staining of the nuclei of neurons and of cultured cells and immunoblotted the same 35-39 kDa antigens. These observations show that the anti-neuronal autoantibody reacts not only with neurons and with SCCL but also with some cultured cell lines. Molecular mimicry has been invoked as the basis for the reactivity of this autoantibody with exposed epitopes on SCCL and on neurons.

A comparison of commercial and in-house ELISAs for antineutrophil cytoplasmic antibodies directed against proteinase 3 and myeloperoxidase.

This study compares the concordance of results in different ELISAs for antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) or myeloperoxidase (MPO). Sera were considered "true positives" if they were positive according to the manufacturer's criteria in a least three of the five PR3-ANCA ELISAs, or in at least four of the six MPO-ANCA ELISAs. Of the 26 sera that demonstrated cytoplasmic fluorescence (C-ANCA), 23 (89%) contained PR3-ANCA and three (11%) had MPO-ANCA. Two sera that were negative by indirect immunofluorescence (IIF) contained PR3-ANCA. Of the 26 sera with perinuclear fluorescence (P-ANCA), 19 (73%) contained MPO-ANCA, and one (4%) had PR3-ANCA. Six sera with P-ANCA did not have PR3- or MPO-ANCA. No serum that was negative by IIF contained MPO-ANCA. For the different PR3-ANCA ELISAs, sensitivities ranged from 88 to 100%, and specificities from 91 to 100%. For the MPO-ANCA ELISAs, sensitivities varied from 59 to 100% and specificities from 83 to 100%. The highest sensitivity and specificity for both the PR3- and MPO-ANCA ELISAs were obtained with the IBL and Eurodiagnostica assays. The in-house PR3-ANCA ELISA performed slightly less well than the commercial assays, but the performance of the in-house MPO-ANCA assay was comparable or better.