Analysis of natural and disease-associated autoantibody repertoires: anti-endothelial cell IgG autoantibody activity in the serum of healthy individuals and patients with systemic lupus erythematosus

The present study demonstrates that natural IgG with anti-endothelialcell activity is present in the serum of healthy individualsand in pooled normal human Ig. By using a novel method thatallows for the simultaneous and quantitative assessment of reactivitiesof antibodies with a large number of antigens in tissues, weobserved that natural anti-endothelial cell antibody (AECA)recognizes a restricted set of self antigens in endothelialcells that is conserved among healthy individuals. The extentto which natural AECA activity is expressed in serum and thepattern of reactivity of AECA with endothelial cell antigensshowed little variability between individuals. Analysis of AECAin the serum of patients with systemic lupus erythematosus (SLE)revealed a higher amount of activity and a wider spectrum ofantigentic specificities than that recognized by natural antibodiesin endothelial cell extracts. AECA activity of IgG in wholeserum was lower than that of purified IgG in the case of healthyindividuals and showed little variation among individuals. incontrast, no difference was found between AECA activity of purifiedIgG and that of IgG in patients' serum, suggesting that SLEsera lack the factors that control expression of AECA activityin the serum of healthy individuals. Our results indicate thatnatural autoantibodies recognize a restricted and conservedset of self antigens. Our observations further suggest thatdefective regulation of the expressed autoreactive B cell repertoireis the basis for expansion of novel clonal specificities andenhanced autoantibody activity in serum of patients with autoimmunedisease.     

Autoantibody patterns in diabetes-prone NOD mice and in standard C57BL/6 mice.

Autoantibodies are commonly found in healthy individuals and strains of mice that are not prone to autoimmunity. The present study was undertaken to identify self antigens recognized by serum autoantibodies from unimmunized mice of two strains: NOD mice prone to spontaneously develop autoimmune diabetes and C57BL/6 mice known to be relatively resistant to autoimmune disease. IgM and IgG autoantibodies detected in the sera of NOD and C57BL/6 mice manifested different patterns of reactivity. The IgM autoantibodies from C57BL/6 serum reacted with more self antigens and showed higher OD values than the IgM autoantibodies from NOD mice. In contrast, the IgG autoantibodies from NOD serum reacted with more antigens and displayed higher OD readings than did IgG autoantibodies from C57BL/6 mice. Among the antigens recognized by the autoantibodies, particularly of the IgG class, were self antigens known to induce experimental autoimmune diseases in NOD and C57BL/6 mice. In addition, IgG autoantibodies from NOD mice reacted with self antigens reported to mark the spontaneous autoimmune diabetes that characterizes this strain of mice. These results suggest that naturally occurring IgG autoantibodies reflect susceptibility to induction of specific autoimmune diseases. In addition, the results suggest that IgM autoantibodies may by associated with mechanisms that might prevent autoimmune disease.     

Antibodies against Trypanosoma cruzi alkaline antigens are elicited in sera from acute but not chronic human chagasic patients.

The aim of this work was to study the antibody response of acute and chronic chagasic patients against a Trypanosoma cruzi alkaline fraction (FI) in comparison with the reactivity against a T. cruzi acidic antigen, the main cystein proteinase of the parasite named cruzipain, and "natural" antigens. FI-specific antibodies were detected only during the acute phase of the infection and IgM was the main isotype produced, whereas cruzipain-specific antibodies were detected during all phases of the infection. By means of immunoblot and sequencing analysis we identified a 47-kDa FI proteic band recognized by IgM from acute chagasic patients as the T. cruzi glutamate dehydrogenase (GluDH). Furthermore, the antibody response against isolated GluDH showed similar characteristics as the one against FI. We also observed a strict association between the reactivity of IgM against FI and GluDH and IgM natural antibodies. However, reactivity against these alkaline antigens was not modified after absorption of natural antibodies in sera from acute chagasic patients, indicating that these parasite antigens are not recognized by the polyspecific natural antibodies. The most important goal of this report is that for the first time the T. cruzi antigen isoelectric point has been associated with its ability to trigger immunological memory, raising a novel antigen property that should be considered in the selection of antigens used in Chagas' disease diagnostic test and in the design of a vaccine against T. cruzi infection.     

A high incidence of cross-reactive idiotypes among murine natural autoantibodies

Anti-idiotypic (anti-Id) antibodies were produced in rabbits against two natural monoclonal IgM autoantibodies (NmAb), D23 and E7, which exhibited a broad reactivity and were derived from fusions of spleen cells from adult unprimed BALB/c mice and nonsecreting myeloma cell lines. They were used to test the reactivities of 12 NmAb obtained from adult and newborn unprimed mice. Both anti-Id recognized cross-reactive idiotopes frequently shared by NmAb; 8 out of the 12 NmAb reacted with anti-IdD23, while 5 of them also reacted with anti-IdE7. All of the Id-bearing antibodies possessed widespread reactivity with structurally dissimilar self and nonself antigens. In most cases, their cross-reactive Id determinants seemed to be located outside of their antigen-binding sites. Furthermore, the presence in normal mouse sera of significant levels of D23 and E7 idiotopes correlated with the presence of natural antibody activity and was mainly associated with IgM and IgG2b fractions. Finally, D23 idiotope(s) were also found on induced murine anti-myosin antibodies. The high incidence of cross-reactive idiotopes found among NmAb produced by clones derived from different mice and their presence in normal BALB/c mouse serum Ig fractions suggest that families of germ-line genes may encode for at least a part of them.     

B-cell and T-cell immune responses to experimental Helicobacter pylori infection in humans

The acute antibody and T-cell immune response to Helicobacter pylori infection in humans has not been studied systematically. Serum from H. pylori-naive volunteers challenged with H. pylori and cured after 4 or 12 weeks was tested by enzyme-linked immunosorbent assays for anti-H. pylori-specific immunoglobulin M (IgM) and IgA established using bacterial lysates from homologous (the infecting strain) and heterologous H. pylori. Proteins recognized by IgM antibody were identified by mass spectrometry of immunoreactive bands separated by two-dimensional gel electrophoresis. Mucosal T-cell subsets (CD4, CD8, CD3, and CD30 cells) were assessed by immunohistochemistry. All 18 infected volunteers developed H. pylori-specific IgM responses to both homologous or heterologous H. pylori antigens. H. pylori antigens reacted with IgM antibody at 4 weeks postinfection. IgM Western blotting showed immunoreactivity of postinfection serum samples to multiple H. pylori proteins with molecular weights ranging between 9,000 (9K) to 150K with homologous strains but only a 70K band using heterologous antigens. Two-dimensional electrophoresis demonstrated that production of H. pylori-specific IgM antibodies was elicited by H. pylori flagellins A and B, urease B, ABC transporter binding protein, heat shock protein 70 (DnaK), and alkyl hydroperoxide reductase. Mucosal CD3, CD4, and CD8 T-cell numbers increased following infection. IgM antibody responses were detected to a range of homologous H. pylori antigens 2 to 4 weeks postchallenge. The majority of H. pylori proteins were those involved in motility and colonization and may represent targets for vaccine development.     

Demonstration and partial characterization of parasite-specific immunoglobulin A responses in human strongyloidiasis.

By using an enzyme-linked immunosorbent assay, serum immunoglobulin A (IgA) responses directed against Strongyloides stercoralis larvae antigens were measured in 104 presumably immunocompetent individuals with chronic uncomplicated strongyloidiasis and in 15 immunocompromised patients with S. stercoralis infection. Fifty healthy North American adults and 18 patients with other helminthic parasites served as controls. All 50 healthy controls were negative for antibody responses (mean absorbance +/- standard deviation = 0.0724 +/- 0.040). The mean absorbance of the 18 parasitized controls was 0.230 +/- 0.087; two individuals parasitized by Ascaris lumbricoides showed positive antibody responses. The mean absorbance of the immunocompetent patients with strongyloidiasis was 0.680 +/- 0.364, with 91 subjects (87.5%) having a positive value (greater than 0.300). Of the immunocompromised patients (mean absorbance +/- standard deviation = 0.735 +/- 0.538), 11 (73%) had a positive antibody response test. When the IgA responses of these two groups were compared, they were not significantly different. There was no correlation between the levels of total serum IgA and the concentration of specific IgA in the infected patients. Both IgA and IgG immunoreactive bands were detected on immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated larval antigen protein blots. Nineteen bands were recognized by IgG, and 13 were recognized by IgA from sera of infected patients. Several bands displayed specific IgG or IgA reactivity. The present work shows that most patients with strongyloidiasis mount specific IgA responses against filariform larval antigens. These responses are, for the most part, directed against antigens that are different from those recognized by IgG. The lack of correlation between the magnitude of the specific serum IgA responses and the clinical aspects of the infection suggests that these antibodies may not play a central role in the regulation of this parasitosis.     

Production, characterization, and species specificity of five monoclonal antibodies to Mycobacterium tuberculosis.

The production and characterization of five monoclonal antibodies to Mycobacterium tuberculosis are described. Specificity of the monoclonal antibodies was tested against other mycobacterial species by an enzyme-linked immunosorbent assay and immunoblots. HGT 3a, an immunoglobulin M (IgM) antibody, recognizes a molecule of 38,000 molecular weight present only in the tuberculosis complex of M. tuberculosis and Mycobacterium bovis BCG. HGT 6, an IgG1 antibody, recognized two molecules with molecular weights of 43,000 and 45,000 and showed limited cross-reactivity. Three other antibodies, HGT 1, HGT 2, and HGT 4, all belonging to the IgG1 type, recognized multiple bands and showed broad reactivity among all mycobacterial antigens tested, Escherichia coli and Nocardia asteroides.     

HHV-6 A- or B-specific P41 antigens do not reveal virus variant-specific IgG or IgM responses in human serum.

The etiology of multiple sclerosis (MS) remains unknown, but there are indications of a role of human herpesvirus 6 (HHV-6), especially variant A, in the pathogenesis. Higher serum antibody reactivity against an HHV-6 early protein, p41, has been found in MS cases than in controls. The antigen, however, was purified from infected cells with a monoclonal antibody also reactive with a protein (p38) likely to be of cellular origin. To avoid serological crossreactivity with the cellular protein, recombinant p41 proteins from HHV-6A strain GS and HHV-6B strain Z29 were expressed as glutathione-S-transferase fusion proteins (p41-GST), and used as antigens in an enzyme-linked immunosorbent assay (ELISA). p41 variant specific monoclonal antibodies reacted strongly with the respective recombinant proteins. Serum IgM and IgG reactivities with the recombinant p41 antigens were analysed in patients with manifest MS, patients with optic neuritis, patients with other neurological diseases, and in one group of healthy controls. All sera were HHV-6 IgG seropositive by immunofluorescence. The serum IgM or IgG reactivities against the recombinant p41 antigens did not differ significantly between the groups, and the reactivities against the variant A and B antigens were identical. In many samples, the reactivity was very low. The results indicate that p41 is not an optimal target for HHV-6 serology studies, and that the data obtained with the p41 antigen prepared from infected cells (possibly including also p38) should be interpreted with caution.     

Specific and natural antibody production during Salmonella typhimurium infection in genetically susceptible and resistant mice.

Genetically susceptible (C57BL/6) and resistant (CBA) mice were infected with an avirulent strain of Salmonella typhimurium and studied over a 35-day period for the production of antibodies directed against bacterial antigens including lipopolysaccharide (LPS) (specific antibodies) and antibodies directed against self antigens [natural antibodies (NAb)]. Antibodies directed against LPS and self antigens were detected by enzyme immunoassay (EIA) and those directed against other bacterial antigens by immunoblotting. We found that serum natural antibody titres in C57BL/6 and CBA mice were similar and correlated with the bacterial load in the spleen and liver. In C57BL/6 mice, anti-LPS antibodies remained polyreactive and of the IgM isotype. In contrast, CBA mice, after an early increase in polyreactive IgM anti-LPS antibodies, mounted a specific anti-LPS IgG antibody response. The immunoblotting results demonstrated that the IgM polyreactive antibodies in the resistant and susceptible mice recognized bacterial antigens of different molecular weights and that CBA, but not C57BL/6 mice, were able to produce IgG antibodies recognizing bacterial components. Our results suggest that the synthesis of antibodies directed against bacterial antigens and natural antibodies follow, at least partially, distinct pathways, but they do not allow us to determine whether these two antibody populations are produced by the same or distinct B-cell subpopulations.     

The Self-Reactive Antibody Repertoire of Normal Human Serum IgM is Acquired in Early Childhood and Remains Conserved Throughout Life

The authors have used a quantitative immunoblotting technique to analyse the antibody repertoire of IgM in cord blood and in the serum of young children, young adult males and aged males directed towards antigens in homologous tissues utilized as sources of self antigens. The reactivities of IgM with self antigens exhibited striking homogeneity and invariance among newborns. Self-reactive IgM repertoires of children, young adults and aged males were markedly conserved among individuals and comprised most of the anti-self reactivities that prevailed in neonates. Reactivities of IgM with bacterial antigens showed a high degree of homogeneity among newborns but were more diverse in children, young adults and elderly individuals. Diversity of IgM reactivities with self and non-self antigens did not vary significantly with aging. Principal component analysis (PCA) and linear discriminant analysis (LDA) of the data discriminated between self-reactive IgM repertoires of newborns and children, but failed to discriminate between repertoires of children, young adults and aged males. The data indicate that the self-reactive antibody repertoire of IgM differentiates during the first years of life and remains relatively constant thereafter.     

Negative selection of multireactive B cell clones in normal adult mice

In the absence of intentional immunizations, normal mice produce natural antibodies that react with a variety of self and foreign antigens. We have now addressed the putative physiological selection of such reactivities and some of their clonal characteristics, by analyzing antibodies produced by B cells at different stages of differentiation. Using an antigen-specific spot-enzyme-linked immunosorbent assay (ELISA) with a panel of self and foreign antigens, we found that newly formed B cells, either from adult bone marrow or from newborn spleen, contain the highest frequencies of IgM antibodies with reactivities towards the panel. Resting peripheral B cells show lower frequencies of such antibodies, that are lowest among naturally activated splenic plasma cells. Analyses of monoclonal IgM antibodies derived from lipopolysaccharide-stimulated bone marrow and spleen cell hybridomas in normal mice show that the majority of reactivities scored in spot-ELISA originate from multireactive IgM clones. In Western blots against a large number of self antigens, each multireactive IgM antibody studied shows a unique and specific pattern of reactivity. We conclude that multireactive B cell clones are very frequent in the emergent repertoires of newborns and adults, but are subsequently negatively selected from bone marrow to periphery, and from the available repertoire to that of natural plasma cells. It, thus, seems that multireactivity of natural antibodies is not a positively selected property, but represents the sum of unique multireactive clones that have escaped inactivation or deletion.     

Human humoral responses to antigens of Mycobacterium tuberculosis: immunodominance of high-molecular-mass antigens.

The selection of antigens of Mycobacterium tuberculosis for most studies of humoral responses in tuberculosis patients has been restricted to molecules that were either immunodominant in immunized animals or amenable to biochemical purification rather than those that were reactive with the human immune system. Delineation of antigens that elicit humoral responses during the natural course of disease progression in humans has been hindered by the presence of cross-reactive antibodies to conserved regions on ubiquitous prokaryotic antigens in sera from healthy individuals and tuberculosis patients. The levels of cross-reactive antibodies in the sera were reduced by preadsorption with Escherichia coli lysates, prior to studying their reactivity against a large panel of M. tuberculosis antigens to which the human immune system may be exposed during natural infection and disease. Thus, reactivity against pools of secreted, cellular, and cell wall-associated antigens of M. tuberculosis was assessed by an enzyme-linked immunosorbent assay (ELISA). Initial results suggested that the secreted protein preparation contained antigens most frequently recognized by the humoral responses of pulmonary tuberculosis patients. The culture filtrate proteins were subsequently size fractionated by preparative polyacrylamide gel electrophoresis, characterized by reaction with murine monoclonal antibodies to known antigens of M. tuberculosis by an ELISA, and assessed for reactivity with tuberculous and nontuberculous sera. Results show that a secreted antigen of 88 kDa elicits a strong antibody response in a high percentage of patients with pulmonary tuberculosis. This and other antigens identified on the basis of their reactivity with patient sera may prove useful for developing serodiagnosis for tuberculosis.     

Selective impairment of serum antibody repertoires toward muscle and thymus antigens in patients with seronegative and seropositive myasthenia gravis

We analyzed the antibody (Ab) repertoires of IgM and IgG of patients with seropositive and patients with seronegative myasthenia gravis (MG) toward self antigens by means of a quantitative immunoblotting technique using normal human tissue extracts as sources of self antigens. Repertoires of reactivities of IgG and IgM with liver, kidney and stomach antigens were conserved between myasthenic patients and controls. IgG and IgM Ab repertoires toward muscle antigens differed significantly between patients with seropositive MG and healthy donors, as assessed by multiparametric statistical analysis. Patterns of Ab reactivities to muscle antigens were similar in patients with seronegative MG and healthy controls. Antibody repertoires of IgG and IgM toward thymus antigens of both seropositive and sero negative MG patients, differed significantly from those of healthy individuals. Our results indicate that MG is characterized by a selective impairment of self-reactive Ab repertoires toward muscle and thymus antigens. The observation that self-reactive Ab repertoires toward thymus antigens are similar in patients with seropositive and seronegative MG suggests that both forms of MG share common immunopathological features.     

Analysis of the natural human IgG antibody repertoire: life-long stability of reactivities towards self antigens contrasts with age-dependent diversification of reactivities against bacterial antigens

We used a quantitative immunoblotting technique to analyze the repertoires of IgG antibody reactivities in the serum of healthy young children, young adult males and aged males with self and non-self antigens. Densitometric patterns of reactivity of purified IgG with self antigens were highly conserved between individuals within a given age group and across age groups. Inter-individual differences were observed, however, upon analysis of self reactivities of IgG in whole serum. A striking heterogeneity between individuals within a given age group and across age groups characterized the reactivity of purified IgG and of IgG in whole serum with bacterial antigens. Inter-individual differences were more marked among aged individuals than among individuals of other age groups. Analysis of variances of reactivities of IgG with bacterial antigens further demonstrated an increased diversity of repertoires of aged donors compared with those of young adults and children. Our results document the stability of the self-reactive repertoires of IgG throughout life, which contrasts with the diversification of the repertoire of IgG antibody reactivities directed toward foreign antigens with aging. These findings support the concept that self-reactive antibody repertoires are positively selected throughout life by a restricted set of self antigens shared by all individuals.     

Antibody responses in recipients of varicella vaccine assayed by immunoblotting

Sera taken from 35 children with cancer who had been vaccinated with live varicella vaccine were assayed using immunoblotting for the presence of IgG class antibodies to proteins present in varicella-zoster virus (VZV)-infected cells. Sera from 23 of these patients were also assayed for IgM class antibodies. The patterns of immunoreactivity observed for these patients following vaccination were substantially weaker and more variable than those detected following natural VZV infection in otherwise healthy individuals. The IgG responses detected following vaccination involved up to 10 protein bands between 28 and 188 kDa. Bands were particularly frequent in the 78–96 kDa region. IgM responses involved up to 10 bands between 28 and 114 kDa, with the bands in the 78–96 kDa region and at 32–36 kDa being detected most frequently. Repeated vaccination generally produced a stronger IgG antibody response than did single vaccination, and subsequent exposure of vaccinees to natural VZV infection resulted in an increased level of reactivity for IgG antibodies, but not for IgM. Similar reaction patterns were obtained with sera from vaccinees when the vaccine virus and wild-type VZV were used as antigens. Immunoblotting showed good correlation with indirect radioimmunoassay for the detection of a vaccine-induced IgG response.     

Multiple Antigen Recognition by Individual Human Monoclonal Antibodies of Rheumatic Disease Patient Origin

We tested by Western blot several thousand antibody-secreting human cell lines immortalized by hybridoma fusion or Epstein-Barr virus transformation of peripheral blood lymphocytes from patients with systemic lupus erythematosus or mixed connective tissue disease. The blots utilized total human Jurkat cell extract as the antigen. More than 20% of these established lines produced antibodies which recognized multiple bands on the blots, frequently 50 bands or more. Experiments were performed to rule out the possibility of the bands being the result of mixed cell populations or nonspecific antibody-antigen binding. Cloning of these cell lines failed to alter the Western blot patterns produced, indicating that the populations were monoclonal. Antibody eluted from a number of the different single blot bands showed the same ability to reproduce the multiple band pattern, thus revealing the presence of only one antibody. Western blots performed in the presence of specific and nonspecific inhibitors demonstrated the ability of the antibody to specifically recognize and bind to certain antigens. Binding did not result from indiscriminate sticking of IgM molecules to the nitrocellulose paper. The patterns of multiple antigen recognition were not due to antigen degradation. Additionaly, enzyme linked immunosorbant assays revealed binding of the monoclonal antibodies to specific antigens, and the antibodies failed to recognize commonly crossreactive antigens such as DNA, histone, poly-L-lysine, glycophorin, and serum glycoproteins. The patterns of multiple antigen binding to a large number of polypeptides are therefore due to single antibodies, and the binding is specific.     

Serum or breast milk immunoglobulins mask the self‐reactivity of human natural IgG antibodies

B cells producing IgG antibodies specific to a variety of self‐ or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein‐destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab’)₂ fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self‐reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients.     

Lymphocytes of persons with the acquired immunodeficiency syndrome and related conditions express reactivity with the monoclonal antibody 4D12 reflective of in vivo lymphocyte activation

We observed that lymphocytes obtained from healthy persons generally expressed infrequent reactivity with the monoclonal antibody 4D12, an antibody raised against a cell line infected by the human T-lymphotropic virus type I. As had been observed previously, persons bearing HLA-B5 cross-reactive antigens and certain other allotypes had frequent lymphocyte reactivity with 4D12. Lymphocytes obtained from persons infected by the human immunodeficiency virus were highly reactive with 4D12 as were lymphocytes obtained from persons with other viral or bacterial infections. Flow cytometric studies revealed greater 4D12 reactivity by larger lymphocytes, and in vitro studies demonstrated that lectin-stimulated lymphocytes acquired 4D12-reactive antigens. There was also a significant correlation between expression of 4D12-reactive antigens and the presence of the interleukin-2 receptor as recognized by the monoclonal antibody anti-Tac. Thus, the monoclonal antibody 4D12 recognizes a lymphocyte surface antigen frequently expressed among persons with various acute and chronic infections. This antigen is a marker of lymphocyte activation.     

Expression and control of the natural autoreactive IgG repertoire in normal human serum

We have investigated the autoreactive repertoire expressed by serum IgG of healthy individuals of various age groups using a large panel of self antigens. Natural IgG autoantibodies against all self antigens of the panel were found in the purified IgG fraction of the serum of all donors that were tested. The mean binding activity to self antigens of IgG of pregnant women was higher than that of IgG purified from the serum of infants, young adults and aged individuals. No increase in IgG autoreactivity was observed with aging neither in the purified IgG fraction of serum nor in whole serum. Whereas autoantibody activity was easily detectable in purified IgG, it was low in serum. No difference was observed, however, between the binding activity of purified IgG and of IgG in serum in the case of foreign antigens nor in the case of anti-thyroglobulin autoantibodies of patients with Hashimoto's thyroiditis. Purified IgM from normal serum bound to F(ab')₂ fragments of autologous IgG in a dose-dependent fashion and inhibited the binding of autologous IgG to self antigens. Our results thus indicate that autologous IgM contributes to regulate expression of the natural IgG autoreactive repertoire through V region-dependent interactions, resulting in low levels of IgG autoreactivity in serum under physiological conditions.     

Production and characterization of a murine monoclonal antibody to Aspergillus fumigatus antigen having IgG- and IgE-binding activity

By employing hybridoma technology, a monoclonal antibody against Aspergillus fumigatus was produced. This antibody, isotyped as IgM k, reacted with 12 of 16 antigens extracted from 9 different A. fumigatus strains. This antibody also reacted with all 3 Aspergillus flavus antigens studied, but not with Aspergillus niger, Aspergillus terreus, Penicillium notatum or Candida albicans antigens. Western blot analysis indicated that this antibody reacted with two concanavalin A (Con-A) binding bands of the A. fumigatus antigen extract. The specific binding antigens were isolated using monoclonal antibody affinity chromatography. When used in immunoassay this fraction demonstrated strong IgG and IgE antibody-binding activities against patient sera. The antibody levels against the purified fraction were significantly higher in patients' sera than in normal controls. The purified fraction demonstrated comparable reactivity with the crude A. fumigatus extract against patient sera, but the former has the added advantage of being pure and standardizable for dependable and reproducible results.