Characterization of immunologically important antigens and allergens of Aspergillus fumigatus

Using a variety of immunochemical methods, including quantitative immuno-electrophoretic techniques, combined with gel filtration and iso-electric focusing, and production of monospecific antisera for identification and affinity purification, 4 major components of Aspergillus fumigatus have now been partially characterized. Numbering of these was derived from a reference allergic bronchopulmonary aspergillosis (ABPA) self-crossed radio-immuno-electrophoresis pattern of reactivity. Two major intracellular/cytoplasmic, concanavalin A (Con A)-binding antigens, Ag 7 and Ag 13, of molecular weights 150-200 and 70 kilodaltons (kD), respectively, were confirmed to be of importance for both ABPA and aspergilloma in specific sandwich enzyme-linked immunosorbent assays. A rapidly released component, Ag 5, of molecular weight 35 kD, proved both antigenic and allergenic, with aspergilloma patients having especially high-titre IgG antibodies. The major allergenic component Ag 3, of molecular weight 24 kD by gel filtration and 18 kD by SDS-PAGE was, like Ag 5, relatively heat-labile and non-Con-A-binding. Interestingly, T cell clones have been identified which respond primarily to an 18-kD fraction.     

The stoichiometry of J chain in human secretory dimeric IgA

Dimeric human secretory IgA was completely reduced with mercaptoethanol and alkylated with [14C]iodoacetamide. The component polypeptide chains were separated by high performance gel filtration in 5 M guanidine HCl into two fractions: one containing secretory component (SC) + heavy (H) chains; and the second containing light (L) + J chains. L and J chains were subsequently separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) or in alkaline urea. Calculations of the J chain stoichiometry in the dimeric secretory IgA (S-IgA) molecule were based on: the measurement of the ratio of radioactivities of SC + H chain and L + J chain-fractions or L chain- and J chain-fractions; the known stoichiometry of SC, H and L chains; and the known number of half-cystine residues in the component polypeptide chains of S-IgA molecule. The data demonstrated that one molecule of dimeric S-IgA contains approx. one J chain.     

Restricted specificity of the autoantibody response in Goodpasture''s syndrome demonstrated by two-dimensional western blotting.

The autoantigen in Goodpasture's syndrome is known to be contained within the non-collagenous (NC1) domain of type IV collagen. We have examined the specificity of autoantibodies to glomerular basement membrane (GBM) using the technique of 2-D electrophoresis followed by Western blotting. Protein stains of 2-D gels of collagenase-digested human GBM revealed extensive charge and size heterogeneity. Major components were of mol. wt 24-30 kD and 43-56 kD, corresponding to monomeric and dimeric subunits of NCl. Western blotting of 2-D gels with IgG from patients with anti-GBM disease demonstrated that the most antigenic components migrated as cationic 28-kD monomers (pI 10) and similarly charged dimers, although other components were recognized less strongly. The mobility of the strongly antigenic polypeptides was different to that of the known alpha 1 and alpha 2 chains of type IV collagen. Autoantibodies from all 20 patients studied showed the same pattern of reactivity, regardless of their clinical features (in particular, the presence or absence of pulmonary haemorrhage) or HLA type. A monoclonal antibody (P1) to human GBM bound in a similar pattern, particularly recognizing the cationic components. 2-D gels of affinity-purified GBM from a P1 column showed enrichment of the 28-kD monomers, which were recognized by human autoantibodies on Western blotting. These results demonstrate that the autoimmune response in Goodpasture's syndrome is of restricted specificity, and support the suggestion that the major autoantigenic determinant is present on the novel alpha 3 chain of type IV collagen.     

Genetic polymorphism of IgG in mink. I. Identification of 8 allotypes

By means of intraspecific immunization of domestic mink (Mustela vison Schr.), 8, in all probability, complex IgG allotypes were detected in their sera. Based on the results of analysis of the preparations of the IgG heavy (H) and light (L) chains, as well as proteolytic IgG fragments, we assigned the allotypes detected to three groups: (1) marker of the L chain, L1; (2) allotypes of the C region of gamma-chains (H2, H3, H4, H6, and H8) and conformational allotype H7; (3) conformational allotype 5 with unknown location on the chains.     

Rapid molecular weight estimation and separation of selected immunoglobulin chains by high speed gel filtration

Rapid and reliable molecular weight estimations of reduced and alkylated immunoglobulin heavy or light chains were performed by high speed gel filtration in 6 M guanidinium chloride using a short (30 cm X 7.5 mm) TSK 3000 SW type column. Molecular weight estimations based on Kav values of eluted polypeptides and glycopolypeptides were generally unaffected by protein bound carbohydrate. Rapid separation of immunoglobulin H and L chains was also achieved during high speed gel filtration.     

Allergy to Rabbits

Investigations have been carried out into the presence of antibody light chains in rabbit allergenic extracts and the interference in RAST and crossed-radioimmunoelectrophoresis (XRIE) caused by antibodies directed against them- A "non-specific" uptake of radioactivity in XRIE has been demonstrated to be caused by direct cross-linking of the ^{12 5}I rabbit anti-human IgE by the sheep antibodies in the Immunoprecipitate of rabbit light chains. Preincubation with normal rabbit serum blocked this direct uptake of the labelled antibody and enabled specific IgE uptake on the light chains to be demonstrated for rabbit allergic sera. Verification of the allergenicity of the light chains was obtained from a specific light chain RAST. Elution from a Sephacryl S-200 gel filtration column indicated a MW of approx. 50 Kd and confirmation of the component as light chain dimers, not Fab fragments, was obtained by allotyping for loci present on heavy chains and light chains in the Fab region. Light chains were detected in urine from rabbits of all ages and in extract of dust collected in a rabbit housing area. No background staining was observed in XRIE using rabbit antisera, either with rabbit allergic sera with specific IgE or with a human serum containing specific IgG antibodies to rabbit IgG. This latter serum also showed no evidence of uptake on all immunoprecipitates in systems using rabbit antisera, and did not give false positive RAST results when the labelled rabbit anti-human IgE contained unlabelled rabbit IgG. Those sera with specific IgE to light chains showed no uptake in XRIE using rabbit antisera, indicating that the IgE was possibly specific for epitopes revealed by the dissociation of the whole IgG molecule.     

Reassembly of Immunoglobulin M Heavy and Light Chains In Vitro

Reduced and alleviated monoclonal IgM was fractionated into μ and light (L) chains by gel chromatography in 1N acetic acid. Equimolar mixtures of the chains formed a noncovalently bonded structure in 0.01M sodium acetate buffer, pH 4.1, that had the properties of a half subunit. The latter reassociated into a subunit-like structure after transfer into 0.08M sodium phosphate buffer, pH 7.5. The similarity of the reconstituted IgM subunit (IgMs) to that of the native molecule was established by its physicochemical and immunochemical properties. Comparable products were obtained on reassembly of the alkylated μ and L chains from several other monoclonal IgM. The presence of active binding sites for IgG on subunits reconstituted from the chains of proteins with anti-IgG activity further indicated correct assembly of the μ and L chains. High yields of subunit-like products were also obtained by assembly of μ chains from one protein and L chains from another. Evidence was obtained that L chains of appropriate specificity can substitute for the homologous chain in the formation of the active site. Heterogeneous mixtures of high molecular weight products were generated from μ and L chains that were not alkylated. Reduction and alkylation demonstrated that the products represented polymers of reconstituted IgMs. Significant levels of anti-IgG activity were detected in the polymeric IgM generated from the chains of active proteins by precipitation with aggregated IgG.     

Non-Immune IgG F(ab'')2 Binding to Group C and G Streptococci Is Mediated by Structures on γ Chains

The present investigation was designed to determine whether the heavy or the light immunoglobulin chain is involved in the non-immune binding of IgG F(ab')2 fragments to specific surface receptors on human group C and G streptococci. Purified human polyclonal IgG was mildly reduced with dithiothreitol and alkylated with iodoacetamide. Light (L) and heavy (H) chains were separated. Intact IgG and purified L and H chains of polyclonal immunoglobulin G were tested in an inhibition assay for non-immune IgG F(ab')2-mediated binding to group C and G streptococci. H chains inhibited the uptake of isotope-labelled IgG F(ab')2 fragments. Isolated L chains were non-reactive. Intact IgG molecules were more potent inhibitors than isolated H chains tested in equimolar concentrations. These results indicate that the non-immune interaction between human group C and G streptococci and F(ab')2 fragments of human IgG is mediated by reactive sites exposed on the immunoglobulin G H chains. The observation that intact IgG on a molar basis was more inhibitory than purified gamma chains suggests that the L chains may contribute to the reactivity, presumably by passive stabilization of the immunoglobulin molecule.     

Characterization of a 25,000-dalton Helicobacter pylori protein, cross-reacting with a Campylobacter jejuni protein.

Extract obtained by ultrasonic disruption of Helicobacter pylori bacteria contained a protein with subunit molecular mass of 25 kD which bound antibodies in sera from patients with H. pylori-associated disease. The protein was purified by gel permeation and elution from SDS-polyacrylamide gel slices, and was used to raise an anti-25-kD protein-specific rabbit serum. Using the antiserum in experiments, the results indicated the following: The protein exists as covalently linked dimers (45 kD) of the 25-kD subunits. Variable numbers of non-covalently linked copies of the dimers make up the native protein. The protein was susceptible to digestion by papain, pronase, and trypsin. Pepsin cleaved off a fragment of approximately 2 kD. A small share of the protein was exposed at the bacterial cell surface, the greatest share being localized internally. The protein was not secreted and it was probably not an integral part of the outer membrane. It was produced in variable quantity by all of 11 H. pylori strains tested and was a major protein in some strains. A cross-reacting protein with subunit size of 25 kD was also produced by Campylobacter jejuni strains, but not by any of a variety of other bacteria. Since both H. pylori and C. jejuni infection occur with a high frequency. the cross-reacting 25-kD protein may interfere unfavourably with the diagnostic specificity of serological tests for infection caused by these bacteria.     

Both VH and VL chains of polyreactive IgM antibody are required for polyreactivity: expression of Fab in Escherichia coli.

Monoclonal polyreactive antibodies can bind to many structurally dissimilar self and non-self antigens. Neither the precise antigen-binding site on the polyreactive antibody molecule nor the molecular basis of polyreactivity has been elucidated. The present study was initiated to see whether antibody genes encoding the Fab fragment of a human monoclonal polyreactive IgM antibody (MoAb 67) could be efficiently expressed in Escherichia coli, and whether the bacterially expressed Fab fragments possessed biological activity. cDNA encoding the variable domains of the heavy and light chains of MoAb 67 were cloned, amplified by polymerase chain reaction (PCR) and expressed in E. coli. Neither the recombinant heavy nor light chain showed antigen-binding activity. In contrast, the recombinant Fab 67 fragment showed the same antigen-binding reactivity profile as the native IgM antibody. It is concluded that the antigen-binding activity of polyreactive antibodies resides in the Fab fragment, and that both the heavy and light chains are required for activity.     

Anti-IL-1 alpha autoantibodies in patients with rheumatic diseases and in healthy subjects.

Antibody reactivity against the 'mitochondrial M2 antigen' was determined in sera from 10 patients with primary biliary cirrhosis (PBC), using Western blotting after SDS-PAGE separation of rat liver mitochondria (RLM) and plasma membrane proteins. The molecular weights of the major M2 antigens in rat liver mitochondria were 67 and 50 kD. Two of the 10 PBC patients did not react to any of these major antigens, eight reacted to the 67-kD and four of those also to the 50-kD antigen. The 67- and 50-kD antigens were present in both plasma membrane and RLM and had affinity to concanavalin A. Antibody reactivity against the 67-kD antigen could be detected in both IgG and IgA as well as in the IgM class. The reactive IgG subclasses to both types of antigen preparations were mainly of the G1 and G3 isotypes. This reactivity was always stronger with antigens from the plasma membrane preparations. Sera from two patients with high antibody titres against mitochondria also reacted with IgG2 against the 50-kD antigen from plasma membrane, but not to the corresponding antigen in mitochondria. Reactivity of antibodies in PBC sera to the periphery of viable hepatocytes and radioactive surface labelling of the 50-kD component are both consistent with a plasma membrane localization of M2. Serum from healthy controls and several patients with different diseases did not contain antibodies reactive against any of the antigens described. We suggest that antigens, partly identical to the mitochondrial M2, are located in the plasma membrane compartment. The PBC pathogenetical consequences of these findings are discussed.     

Antigenic markers of V domain of mouse MOPC 21 IgG1 L chain. The conformational basis of VL domain markers and content of MOPC 21 VL-like immunoglobulins in sera of inbred mouse strains

Antisera were raised in rabbits against L chains, isolated from mouse myeloma protein MOPC21 (gamma 1, chi V chi 15 group). Specific antibodies for the V and C domain of MOPC21 L chain were obtained by cross-immunoadsorption of the antisera. The pure anti-V and anti-C antibodies were fixed on diazocellulose and used as immunosorbents. The inhibitory capacity of L chin-monomers and dimers isolated from the L chain preparation was compared to that of intact IgG1 using binding inhibition of 125I-labeled IgG1 on the antibody-containing immunosorbents. It was established that changes of IgG1 quaternary structure influences the conformational state of the L chain V domain only. The inhibitory capacity of the V domain is 1000-fold lower in L monomers, if compared with native IgG1, and only 10-fold lower than in L dimers. The inhibiting capacity of the C domain, however, does not differ in L monomers and intact IgG1. Thus the conformational rigidity of the C domain co-exists with conformational flexibility of the V domain on the same polypeptide chain. We tried to estimate the content of MOPC21 V1-like normal IgG in mouse serum of 6 inbred strains using antibodies against the V1 domain. Data obtained by inhibition of radioimmunoadsorption, indicate that in C57BL/6 mice 0.08% of normal serum Ig carries a V1 region which is idiotypically related to the V1 of MOPC21. In serum Ig of BALB/c mice the percentage is 0.16.     

Sequential serological responses to Aspergillus fumigatus in patients with cystic fibrosis. Use of antigen ''stretching'' to delineate IgG and IgE activity.

Immunogens from Aspergillus fumigatus were fractionated on the basis of molecular weight. Nine fractions ranging from 900 to 10 kDa were used in ELISA and in a radioallergosorbent test (RAST) with sera from cases of allergic bronchopulmonary aspergillosis (ABPA) and from cystic fibrosis (CF) patients with ABPA or other Aspergillus involvement and compared with control subjects. The profile of IgG reactivity to the nine fractions did not vary substantially for all Aspergillus-involved groups producing peaks at greater than 900 kD and 170 kD whereas the profile for control subjects had a peak at greater than 900 kD only. The IgE profile for CF patients with ABPA did not differ from the profile of the RAST-positive CF patients without ABPA and provided only one peak of activity at 24 kD. Recovery from an episode of ABPA in CF patients was accompanied by a fall in both IgG and IgE antibody levels to all nine fractions, whereas increases in IgG and IgE to all fractions were seen during an episode of ABPA. Although there was an exaggerated IgG increase to antigens in the 43-170 kD range during ABPA, a meaningful increase was also observed to unfractionated A. fumigatus antigen preparations. With IgE in one detailed study the 24-kD fraction provided a better indication of Aspergillus involvement than the unfractionated A. fumigatus antigens. Sequential studies of IgG and IgE levels were not able to predict an episode of ABPA but were useful in conjunction with clinical assessment in following the course of the illness.     

Purification of the major allergen of red soft coral (Dendronephthya nipponica).

Red soft coral (RSC; Dendronephthya nipponica, a marine coelenterate) causes spiny lobster fishermen living along the Pacific coast of Miyazaki Prefecture in Japan to develop occupational allergies, such as conjunctivitis, rhinitis, dermatitis and bronchial asthma. The aim of this study was to purify and to characterize RSC allergen, which causes occupational asthma in spiny lobster fishermen. The allergic responsiveness of spiny lobster fishermen to RSC was examined. The examinations included specific IgE production, skin test responses, lymphocyte stimulation tests and specific IgG production. We found that RSC has a strong sensitizing activity in humans at a molecular weight of 10 kD or more, while it has no IgE-producing activity at a molecular weight of less than 10 kD. Neither the nonatopic controls nor the atopic non-coral-allergic controls exhibited any RAST-binding activity to any fraction. For the purification and the identification of this new allergen component, repeated gel filtration of the RSC extract was performed on a Sephacryl S-200 column, followed by gel filtration on a Superose-6 column. The purified major allergen component Den n 1, which is separated on a Mono-Q column, showed intradermal responses, lymphocyte stimulating activity and specific IgG-producing activity in RSC-induced bronchial asthma patients. The 53-kD component was electroblotted on a polyvinylidene difluoride membrane. The N-terminal amino acid sequence of this new allergen component (Den n 1) was determined as Asp-Asp-Ile-Asn-Arg-Tyr-Ala-Phe-Asp-Asn-Lys-Ile-Asn- Asp-Lys-Leu-Phe-Asp-His-Trp-Gln-Ser.     

Immunological studies on Kawasaki disease: II. Isolation and characterization of an immunosuppressive factor in acute phase sera

Acute phase serum from a patient with Kawasaki disease possessed strong inhibitory activity for the proliferative response of Con A-stimulated peripheral blood mononuclear cells. The inhibitor was fractionated step-wise by means of DEAE-Sephadex A-50 ion exchange chromatography followed by Sephadex G-100 gel filtration and high performance liquid chromatography. A major protein of 140 kD with 2000 times greater inhibitory activity than the original serum was identified in the final fraction. Immunization of mice with this fraction resulted in the production of three hybridoma clones secreting monoclonal antibodies (MoAb) of IgG1 class which blocked the inhibitory activity of the fraction. Two of these MoAb recognized the same epitope of the inhibitory factor, while the remaining MoAb was directed to a different epitope. Western blot analysis of acute phase sera by the MoAb demonstrated the presence of 140 kD molecules in 43 of 55 patients.     

Primary structure of a monoclonal kappa chain in myeloma with light chain deposition disease.

Previous data suggest that structural abnormalities of immunoglobulin light chains may be responsible for non-amyloid light chain deposition disease (LCDD). We report on the complete primary sequence deduced from complementary (c)DNA analysis of a normal-sized kappa chain in a case of myeloma-associated LCDD. The patient's urine contained a kappa type Bence-Jones protein made of monomers and dimers of an unglycosylated kappa chain. The bone marrow myeloma cells contained intracellular kappa and gamma chains by immunofluorescence. Biosynthesis experiments showed the production of normal-sized gamma chains and of kappa chains with the same apparent molecular mass (Mr) in SDS gels as the urinary kappa chain (26,000-27,000). These kappa chains were secreted as assembled IgG molecules and as a large excess of free monomers and dimers. The complete sequence of two identical cDNA clones derived from a normal-sized kappa messenger RNA indicated that this kappa chain belonged to the rare V kappa IV subgroup. The kappa mRNA had an overall normal structure made up of the V kappa IV sequence rearranged to J kappa 1 and followed by a normal constant exon of the Km(3) allotype. The variable region differed from the V kappa IV-J kappa 1 germline sequence by 17 amino acid substitutions. The peculiar sequence of the variable region of this kappa chain of a rare subgroup might relate to its tissue deposition.     

A serum antibody in patients with rheumatoid arthritis stimulates cathepsin B activity in peritoneal mouse macrophages.

Mouse peritoneal macrophages were stimulated by sera from patients with active rheumatoid arthritis (RA) to increased intracellular cathepsin B activity. By gel filtration of three RA sera, the stimulatory activity was found in the IgG and to a lesser extent in the IgM containing fraction. The DEAE-cellulose purified IgG preparations of five additional RA patients stimulated intracellular cathepsin B activity significantly above IgG from healthy controls. IgG and IgM antibodies to macrophages were detected in sera from RA patients but not from controls by indirect immunofluorescence (IIF) technique. Pepsin F (ab')2 fragments of IgG from the RA patients also gave clearcut membrane fluorescent staining of the macrophages which demonstrated the antibody nature of the binding. A good correlation between the cathepsin B assay and the IIF was found when serial dilutions of serum were compared.     

Characterization of serum immunoglobulins in a chondrostean fish, Acipenser baeri

The euglobulin fraction of sturgeon (Acipenser baeri) serum was analyzed using electrophoretic and immunoblotting techniques. The major protein of this fraction is an IgM-like molecule composed of equimolar 70-kDa glycosylated H chains and 26–30 kDa L chains. In the absence of a reducing agent, the L and H polypeptides may form (μ2L2)n high molecular weight polymers, μ2L2 170-kDa units or L2 dimers. These different bonding patterns suggest some structural heterogeneity in the distribution of cysteine residues along the sturgeon Ig chains. The H chain N-terminal sequence indicates significant homologies with the conserved V_HIII subgroup. Heavy chains antigenically different from the 70-kDa H chain were not detected, suggesting that IgM is the only Ig class synthesized by this sturgeon species.     

Structural studies of the Xenopus 19S immunoglobulin and 7S immunoglobulin and two immunoglobulin-like proteins.

Xenopus laevis 19S and 7S immunoglobulins (Ig) were extensively reduced and alkylated, their H and L chains spearated and their molecular weights determined. Two kinds of L chains of molecular weight 25,000 and 27,000 were revealed by SDS-polyacrylamide gel electrophoresis. In addition two Ig-like proteins consisting of heavy chains only, of 19S H-type and with similar molecular weight, were detected in Xenopus serum ans isolated. These proteins share common antigenic determinants with Xenopus 19S Ig heavy chains and are devoid of light chain determinants.     

Studies on Xenopus laevis immunoglobulins

The anuran amphibian Xenopus laevis has been shown to produce two classes of antibodies to HGG, BSA and Hc. These antibodies were characterized by gel filtration on Sephadex G-200 as `19S'' and `7S'' immunoglobulins. In the course of immunization, antibody activity could be initially detected in the `19S'' immunoglobulin fraction, followed by the appearance of the activity in the `7S'' immunoglobulin fraction at a later stage of immunization. A switch-over from `19S'' to `7S'' activity was not observed. Both immunoglobulins were composed of heavy and light polypeptide chains. The `19S'' protein had heavy chains with a molecular weight of 74,500, similar to human μ-chain (73,900). The `7S'' protein differed from human IgG in respect to the molecular weight of its heavy chain which was shown to be 64,500. Light chains of both immunoglobulins of Xenopus were found to have a molecular weight of 26,700, similar to human immunoglobulin light chains (25,000).