Immunoblot analysis of serum IgG, IgA and IgE responses against larval excretory-secretory antigens of Anisakis simplex in patients with gastric anisakiasis

To increase our understanding of the immune response to Anisakis infection, antigen specific IgG, IgA and IgE responses were identified using an immunoblot technique after polyacrylamide gel electrophoresis of excretory-secretory products from the larval stage of Anisakis simplex. Nine sera were drawn from proven cases of gastric anisakiasis within 3 days after symptoms had developed. The molecular weight of the major antigenic bands were distributed between 50 kDa and 120 kDa of the antigens. In nine cases of gastric anisakiasis, three of them were positive for IgG response, five for IgE, and six for IgA, respectively. None of control sera recognized the antigenic bands in IgA and IgE responses. In contrast, two controls had IgG antibodies against 1-2 proteins in the 65-95 kDa region. The antigenicity of the excretory-secretory products was lost following treatment by 0.2% trypsin, but not by 0.2 M periodic acid. Based on the results of reactivity to lectins, antigenic bands of the ES products possessed mucin type glycoconjugate residues in their protein portion. This indicates that the humoral responses of IgA and IgE antibodies to the larval ES antigens are a more reliable index of infection than that of the IgG response.     

Evaluation by ELISA of anisakis simplex larval antigen purified by affinity chromatography

In order to improve the specificity and sensitivity of the techniques for the human anisakidosis diagnosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG specific antibodies were isolated by means of protein A-Sepharose CL-4B beads columns. IgG anti-A. simplex and -A. suum were coupled to CNBr-activated Sepharose 4B. For the purification of the larval A. simplex antigens, these were loaded into the anti-A. simplex column and bound antigens eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex specific proteins were loaded into the anti-A. suum column. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis were carried out. Further, we studied the different responses by ELISA to the different antigenic preparations of A. simplex used, observing their capability of discriminating among the different antisera raised in rabbits (anti-A. simplex, anti-A. suum, anti-T. canis). The discriminatory capability with the anti-T. canis antisera was good using the larval A. simplex crude extract (CE) antigen. When larval A. simplex CE antigen was loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with A. simplex CE antigen, its capability for discriminate between A. simplex and A. suum was improved, increasing in the case of T. canis. The best results were obtained using larval A. simplex CE antigen loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with adult A. suum CE antigen. When we compared the different serum dilution and antigenic concentration, we selected the working serum dilution of (1/4)00 and 1 microg/ml of antigenic concentration.     

Ordering of antibodies, that react with cardiac muscle fiber and interstitial connective tissue antigens, in different immunoglobulin classes

Sera of patients suffering from rheumatic diseases and myocarditis were examined on the sections of human and bovine myocardial tissue by indirect immunofluorescence with the use of pure IgG antibodies or monospecific sera against IgG, IgA and IgM. It was shown that antibodies reacting with different myofibers and interstitial connective tissue of the heart belong to the main immunoglobulin classes (IgG, IgA and IgM). There was a significant predominance of IgG antibodies as shown by the frequency of their detection and by the titer height. The predominance of antibodies to certain classes of immunoglobulins did not correlate with a specific disease entity. The frequency of detecting antibodies to a certain immunoglobulin class was in good agreement with the time of the disease onset. Moreover, the frequency of positive reactions due to IgG, IgA, and IgM antibodies correlated with the level of the appropriate immunoglobulins in the test sera.     

Detection of HIV-Gag p24-Specific Antibodies in Sera and Saliva of HIV-1-Infected Adults and in Sera of Infants Born to HIV-1-Infected Mothers

Secretory immunoglobulin A (IgA) is known to play an important role in the mucosal defense against a variety of pathogens. Although the role of IgA antibodies during sexual transmission of HIV is not clear, HIV-specific IgA antibodies have been detected in various mucosal secretions of HIV-infected individuals. Using a monoclonal antibody against human IgA, we established an ELISA system to detect anti-HIV p24 IgA antibodies in sera and saliva. We have analyzed the levels of anti-HIV p24 IgG and IgA antibodies in sera and saliva of 107 and 119 adults, respectively, with HIV infection at different clinical stages, and in the sera of 13 infants born to HIV-infected mothers. The level of anti-HIV p24 IgA antibodies was lower in sera and higher in saliva as compared to that of anti-HIV p24 IgG antibodies. Where the percentage of HIV-specific serum antibody-positive cases decreased with disease progression, that of saliva antibody-positive cases increased in AIDS patients. Among the 13 infants born to HIV-infected mothers, 7 infants were HIV-p24-specific serum IgA positive. These sera were negative for anti-HIV p24 secretory IgA, suggesting that some infants develop their own immune responses against HIV infection. Thus, the detection of HIV-specific IgA antibodies, especially in saliva, could be a simple and reliable test for the diagnosis of HIV infection.     

Serodiagnostics of chlamydial infections--significance of positivity in IgA and/or IgM antibody classes only

There was followed the development of serological findings in patients with proved positivity only in classes IgA and/or IgM of chlamydial antibodies (without IgG), which can be suspected of showing "false" positivity. 184 patients were repeatedly examined for chlamydial antibodies in their sera (interval between collections up to three months) using a genus specific rELISA. Sera were also tested for the evidence of IgM antibodies against capside antigen of Epstein-Barr virus (EBV) and against cytomegalovirus (CMV) using ELISA methods. In 75 (40.8%) of patients, IgA/IgM individual positivities were demonstrated even during the following sample test(s). In 28 (15.2%) of them, IgG evidence preceded and in 29 (15.7%) other patients positive seroconversion followed in this class. In 13 (7.1%) patients, IgG antibodies disappeared and subsequently reappeared. Only in 39 (21.2%) of these probands, antibodies IgA/IgM were not demonstrated at another examination. Active EBV, resp. CMV infection was proved in 24 (13.0%), resp. in 18 (9.8%) of patients. It is concluded that the evidence of positivities only in classes IgA and/or IgM mostly signal the onset of a primary infection (reinfection) or an active infection in patients with IgG production failures respectively. In these cases, a "false" positivity can be supposed to occur only in a minor extent.     

Seric and secretory antibodies reactive to α, β and γ intimins of Escherichia coli in healthy Brazilian adults

Intimin is essential for attaching and effacing lesions by pathogens such as enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC), and the antigenic polymorphism of intimin determines distinct subtypes. Our aim was to investigate the presence of immunoglobulin G (IgG) and IgA antibodies reactive to α, β and γ intimins in serum and colostrum from healthy Brazilian adults. We found seric IgG and secretory IgA antibodies reactive to conserved and variable regions of α, β and γ intimins and a positive correlation between the concentrations of these antibodies in both serum and colostrum that suggested cross reactivity among anti-intimin antibodies, as was confirmed by immunoblotting and absorption. The concentrations of anti-conserved region antibodies were higher than those of variable region antibodies. The presence of antibodies reactive to EHEC antigens could result from contact with EPEC or with other bacteria of the environment even though this bacterium is not frequent in Brazil, and suggests possible protection against EHEC.     

Levels of immunoglobulins of isotype IgG and IgG2 in subjects lacking formation of IgG antibodies against lipopolysaccharide of Chlamydia

In some patients with antibodies against LPS antigen of Chlamydia, specific immunoglobulins G are not present. The findings of isolated anti-LPS IgA/IgM antibodies are to be considered as possibly non-specifically or "false" positive. The aim of this study was to investigate if there is any difference in the level of total immunoglobulins of isotypes IgG and IgG2 in probands with isolated positivity anti-LPS IgA (i.e. without simultaneous presence of specific IgG; n = 34) as compared with a control group of subjects presenting positive anti-LPS IgA and IgG antibodies (n = 44). Antibodies against LPS antigen of Chlamydia were determined by ELISA method ("Chlamydien--rELISA", medac, Germany). Total immunoglobulin levels were determined by nephelometry using the following kits: "Immunoglobulin G Reagent, ARRAY Systems", Beckman Coulter, USA and "Human IgG2 Subclass Beckman ARRAY Kits", The Binding Site, UK. The measured values were related to the age-dependent laboratory standard values and the differences between the tested groups were statistically evaluated (chi(2) test). Decreased total IgG have been demonstrated in 4 (11.8 %) probands and in 5 (11.4 %) subjects of the control group; increased total IgG were found in 2 (5.9 %) probands and in 1 (2.3 %) subject of the control group. Decreased levels of total IgG2 were not determined in any proband and were found in 1 (2.3 %) subject of the control group. Increased levels of IgG2 were registered in 12 (35.3 %) probands and in 15 (34.1 %) control subjects. No statistically significant differences were found between the two groups. It can be concluded that no relationship was proved between the levels of total IgG and IgG2 and the absence of formation of specific anti-Chlamydia-LPS IgG. Further research will be necessary for the elucidation of this phenomenon (e.g. the presence of specific anti-LPS IgG in immunocomplexes).     

IgA and IgA diphtheria antitoxin responses from human tonsil lymphocytes.

Human tonsil lymphocytes were stimulated with diphtheria toxoid and then cultured in a Marbrook culture system so that antibodies could be measured in the culture supernatant. Specific antibodies were measured with excess radiolabeled antigen and antisera specific for each immunoglobulin class. Good IgG and IgA diphtheria antitoxin responses have been obtained and responding culture supernatants were shown to neutralize toxin. The relationship between antitoxin response in vitro and immunization of donors with toxoid was investigated. It was found that at least two immunizations after the age of 6 months were necessary to prime the tonsils for an in vitro antibody response. The IgG and IgA in culture supernatants were demonstrated by immunodiffusion and were measured by radioimmunoassay. By sucrose density gradient ultracentrifugation, it was shown that 40% of the IgA produced in the cultures was greater than 7S. Evidence was obtained that neither the IgA nor the specific IgA antitoxin bears secretory piece. It appears that human lymphocytes from tonsils produce polymer IgA in vitro without secretory piece.     

Species-specific monoclonal antibodies for a membrane antigen(s) in all developmental forms of Trypanosoma cruzi

Two monoclonal antibodies (designated as TCF48 and TCF87 were raised against Trypanosoma cruzi, strain Tulahuen, Both antibodies reacted with all developmental forms of several different strains of Trypanosoma cruzi. The antibodies showed no detectable cross-reactivity with other species of Trypanosomatidae, so far examined. TCF48 and TCF87 were classified as immunoglobulin subclasses IgG1 and IgG2b, respectively. Apparent molecular weight of the corresponding antigen(s) to these monoclonal antibodies was 25,000 in amastigotes and epimastigotes, and 25,000 and 24,000 in trypomastigotes, as determined by the Western immunoblotting analysis. This antigen appeared to be located at the plasma membrane and the flagellum ofT. cruzi. However, no evidence supported the localization of the epitope(s) at the external surface of the live cell. Since this antigen reacted with the sera from the chronically infected mice, these monoclonal antibodies may be useful in the study of Chagas' disease.     

Monoclonal antibodies of IgA isotype specific for lipopolysaccharide of Salmonella enteritidis: production, purification, characterization and application as serotyping reagents

Hybridomas secreting immunoglobulin A (IgA) monoclonal antibodies (MAbs) against Salmonella enteritidis lipopolysaccharide (LPS) were generated after mucosal immunization of BALB/c mice with heat killed bacteria. Antigen binding properties and specificity of the produced MAbs were studied in ELISA and immunoblotting with purified LPS. Two IgA MAbs agglutinated all Salmonella OD1 strains and all S. enteritidis clinical isolates. MAb 178H11 recognized O:9 antigen of subserogroup OD1 LPS. MAb 177E6/A9 reacted also with OD3 LPS antigen and agglutinated OD3 strains. These data suggest the existence of different O:9 antigen subspecificities, one presented in subgroup OD1 and the other common for OD1 and OD3. Thus the produced IgA MAbs prove to be useful reagents, which could differentiate OD1 and OD3 from OD2 strains.     

Jacalin interaction with human immunoglobulin A1 and bovine immunoglobulin G1: affinity constant determined by piezoelectric biosensoring

The affinity of the D-galactose-binding lectin from Artocarpus heterophyllus lectin, known as jacalin, with immonuglobulins (Igs) was determined by biofunctionalization of a piezoelectric transducer. This piezoelectric biofunctionalized transducer was used as a mass-sensitive analytical tool, allowing the real-time binding analysis of jacalin-human immunoglobulin A1 (IgA(1)) and jacalin-bovine IgG(1) interactions from which the apparent affinity constant was calculated. The strategy was centered in immobilizing jacalin on the gold electrode's surface of the piezoelectric crystal resonator using appropriate procedures based on self-assembling of 11-mercaptoundecanoic acid and 2-mercaptoethanol thiol's mixture, a particular immobilization strategy by which it was possible to avoid cross-interaction between the proteins over electrode's surface. The apparent affinity constants obtained between jacalin-human IgA(1) and jacalin-bovine IgG(1) differed by 1 order of magnitude [(8.0 ± 0.9) 10(5) vs (8.3 ± 0.1) 10(6) L mol(-1)]. On the other hand, the difference found between human IgA(1) and human IgA(2) interaction with jacalin, eight times higher for IgA(1), was attributed to the presence of O-linked glycans in the IgA(1) hinge region, which is absent in IgA(2). Specific interaction of jacalin with O-glycans, proved to be present in the human IgA(1) and hypothetically present in bovine IgG(1) structures, is discussed as responsible for the obtained affinity values.     

Secretory immunoglobulin A antibodies against the sigma1 outer capsid protein of reovirus type 1 Lang prevent infection of mouse Peyer's patches

Reovirus type 1 Lang (T1L) adheres to M cells in the follicle-associated epithelium of mouse intestine and exploits the transport activity of M cells to enter and infect the Peyer's patch mucosa. Adult mice that have previously cleared a reovirus T1L infection have virus-specific immunoglobulin G (IgG) in serum and IgA in secretions and are protected against reinfection. Our aim in this study was to determine whether secretory IgA is sufficient for protection of Peyer's patches against oral reovirus challenge and, if so, against which reovirus antigen(s) the IgA may be directed. Monoclonal antibodies (MAbs) of the IgA isotype, directed against the sigma1 protein of reovirus T1L, the viral adhesin, were produced and tested along with other, existing IgA and IgG MAbs against reovirus T1L outer capsid proteins. Anti-sigma1 IgA and IgG MAbs neutralized reovirus T1L in L cell plaque reduction assays and inhibited T1L adherence to L cells and Caco-2(BBe) intestinal epithelial cells in vitro, but MAbs against other proteins did not. Passive oral administration of anti-sigma1 IgA and IgG MAbs prevented Peyer's patch infection in adult mice, but other MAbs did not. When anti-sigma1 IgA and IgG MAbs were produced in mice from hybridoma backpack tumors, however, the IgA prevented Peyer's patch infection, but the IgG did not. The results provide evidence that neutralizing IgA antibodies specific for the sigma1 protein are protective in vitro and in vivo and that the presence of these antibodies in intestinal secretions is sufficient for protection against entry of reovirus T1L into Peyer's patches.     

Evaluation by the skin prick test of Anisakis simplex antigen purified by affinity chromatography in patients clinically diagnosed with Anisakis sensitization

Anisakis simplex crude extracts (CE) (IPI, ASAC and ALK-ABELLO), A. simplex larval antigens purified using a column of IgG anti-A. simplex (PAK) or a column of IgG anti-Ascaris suum (PAS), antigen eluted from columns of IgG anti-A. suum (EAS) and an A. suum adult CE were assayed by the skin prick test. Thirty percent of assayed patients showed a negative reaction in the Anisakis skin prick test. Of 70% positives, two patients had a weal greater than that produced by histamine with the A. simplex extract from ABELLO and IPI. The A. suum skin prick test was positive in 35% of patients, with a lower reaction than that observed with the A. simplex extract from IPI in 57% of the sera and a higher reaction in 28% of the sera. All patients with positive reactions with the crude extract also showed positive weals with the two purified antigens, PAK and PAS. All patients, except three, with a reaction to A. suum antigen, were positive to the EAS antigen. In five patients the weal size produced by PAS was greater than that observed with PAK, whereas in another six patients the contrary was observed. Only one of these six patients did not react to EAS antigen, coincident with the patient showing only a slight increase (7%) in the weal size induced by PAK vs. PAS. When the EAS antigen was tested on patients positive to both PAK and PAS, six patients presented a weal size of >30% and only three patients who were positive to PAS did not react to the EAS antigen. These three patients were also negative against the A. suum CE. Purification by affinity chromatography eliminates from the PAS antigen the proteins responsible for producing cross-reactions with Ascaris (present in the EAS antigen).     

Enterobacterial 38-kDa outer membrane protein is an age-dependent molecular marker of innate immunity and immunoglobulin deficiency as results from its reactivity with IgG and IgA antibody

In earlier studies on an animal model we observed protective properties of outer membrane proteins (OMPs) of Shigella, Hafnia, and Escherichia coli strains. In order to investigate human sera for reactivity with OMPs we subjected these proteins to immunoblotting with umbilical cord plasma and sera from children and adults. The IgG and IgA antibodies interacted primarily with a 38-kDa protein, in similar way for several enterobacterial strains, but different for Pseudomonas aeruginosa. This observation prompted us to determine the reactivity with the purified 38-kDa OMP in the sera of several groups of children. The reactivity of the protein from Shigella flexneri serotype 3a with sera in ELISA was age dependent, increasing from low reactivity in infants to the adult antibody level. The IgG and IgA antibody specific response thus revealed the normal pattern of immunity. The level of IgA and IgG antibody was significantly low in child patients with IgA and/or IgG immunoglobulin deficiencies, but was at the healthy control level in children with recurrent respiratory tract inflammation. These data correlated with total IgA and IgG levels in immunoglobulin-deficient children. The results indicate that this protein may serve as an immunodiagnostic marker, but also as an antigen carrier in vaccines.     

Assay of IgA to Shigella flexneri during shigellosis

Abstract An enzyme-linked immunosorbent assay (ELISA) to Shigella flexneri 2a whole bacterium was used to determine IgM, IgG and IgA serum titers in 50 acute-phase shigellosis patients and 37 controls, i.e., hospital patients without known recent infections. Compared to controls, the shigellosis patients displayed statistically raised average serum titers to S. flexneri in all 3 above immunoglobulin classes, most notably IgA, which displayed an average 42-fold increase. Specific IgM and IgG were 5- and 16-fold higher, respectively. All sera displayed statistically raised titers in at least one immunoglobulin class. A Widal agglutination detected a 7-fold increase in serum titers; this was comparable to the IgM ELISA. Statistical analysis showed that the intra-assay error of the ELISA varied from 5 to 14%, depending on the absorbance from which titers were calculated. A second ELISA was performed on the above shigellosis sera to determine titers to purified lipopolysaccharide (LPS): a statistical correlation was found between these and the above values for all 3 immunoglobulin classes. We conclude that the use of S. flexneri whole bacterium as an antigen in an IgA ELISA is a statistically valid and convenient parameter for monitoring shigellosis, comparable to the use of LPS as antigen, and more sensitive than IgM or IgG ELISAs or agglutinations.     

Utilization of released proteins (RPs) of Yersinia enterocolitica for serologic diagnosis of yersiniosis. III. Western-blot

The usefulness of the immunoblotting using released proteins (Yersinia outer proteins-Yop) as the antigen for the serological diagnosis of yersiniosis was estimated. The IgA, IgG, and IgM antibody responses of patients with yersiniosis and healthy blood donors were studied by western-blot prepared in our laboratory, and two commercial assays. The results indicate that antibodies of all three classes are most consistently directed against the proteins of YopD, YopM and YopE. Good correlations between the three western-blots were obtained for all proteins except the protein V-AG. Patients with yersinia-triggered reactive arthritis have IgA class antibodies against the YopD more often and for longer period than the non-arthritic patients with yersiniosis.     

CEA-containing immune complexes in sera of patients with colorectal and breast cancer — analysis of complexed immunoglobulin classes

Summary A sandwich enzyme immunoassay was developed to detect circulating immune complexes containing carcinoembryonic antigen (CEA) and immunoglobulin (Ig) G, IgA, or IgM using a nitrocellulose-bound anti-CEA antibody as the solid phase reagent. Elevated levels of CEA-containing circulating immune complexes (CEA-IC) were found in 15.4% of 117 sera from patients with colorectal cancer in a postsurgery follow-up study. Also in 24.5% of 102 sera from patients with breast cancer in different states of disease CEA-IC were found. The predominant Ig determined in CEA-IC of colorectal cancer patients was IgA, followed by IgG and IgM, whereas IgG and IgM were the most frequent Igs in CEA-IC of breast cancer patients. Elevated CEA levels were found in 12.0% of the colorectal cancer patients and in 25.4% of sera from breast cancer patients. No significance for the coincidence of elevated CEA levels and CEA-IC was recorded in all patients sera tested. In sera of patients with disease recurrence, however, both parameters were shown to be elevated (CEA 80.7% and CEA-IC 42.3%). The data presented indicate the detection of CEA-IC as an additional parameter for the identification of patients at increased risk for disease recurrence.     

Elevated urinary excretion of immunoglobulins in nonproteinuric patients with type 1 diabetes

Increased albuminuria is a hallmark of early diabetic nephropathy, whereas the role of immunoglobulins (Igs), such as IgG (its 1-4 subtypes), IgA, and IgM, different in charge and size, has not been examined in early nephropathy in the past due to lack of a sensitive and reliable method. Our study group consisted of subjects with type 1 diabetes (T1D) and normoalbuminuria (n = 78), microalbuminuria (n = 78), and of 75 healthy subjects (HS). A Luminex-based immunoassay (1,000 time more sensitive than nephelometry-based method) was validated for the urine matrix and used for the measurements of IgG1-4, IgA, and IgM in our study groups. The Luminex-based assay detected Igs in 87% of HS subjects and in 100% of T1D subjects. Recovery of known amounts of Igs added to urine was 92-118%. In the normoalbuminuria group, urinary concentrations of albumin, IgG2, IgA, and IgM were significantly higher than in HS, whereas in the microalbuminuria, further elevation of IgG2, IgG4, and IgA was the most pronounced. In all three groups, fractional excretion of Igs was at least 100 times lower than that of albumin. Fractional excretion of IgG2 was the highest among all Igs. We validated a sensitive method for measuring Igs in urine using Luminex. We found that elevated concentrations of Igs, particularly in IgG2 and IgA, is present in subjects with T1D and no proteinuria. Elevation of those particular Ig subtypes suggests a contribution of novel mechanisms in early diabetic nephropathy, different from charge and size barrier impairment.     

Western blot antibody determination in sera from patients diagnosed with Anisakis sensitization with different antigenic fractions of Anisakis simplex purified by affinity chromatography

Using Western blot techniques, the specificities of crude and purified (PAK and PAS) Anisakis simplex antigens were compared against 24 sera from patients diagnosed with Anisakis sensitization. All patients recognized a 60 kDa protein against the A. simplex crude extract, while 37.5% and 12.5% reacted with proteins of 40 and 25 kDa, respectively, when IgG was tested. In the case of IgE determination, 41.6% of sera were negative, while 12.5% and 20.8% appeared to cross-react against Toxocara canis and Ascaris suum, respectively. When the PAK antigen (A. simplex antigen purified by means of a column of IgG anti-A. simplex) was tested, immune recognition towards the 60, 40 and 25 kDa proteins increased in 83.3%, 16.7% and 4.2%, respectively, when the Ig antibodies were tested. In the case of the PAS antigen (PAK antigen purified by means of a column of IgG anti-A. suum), the reaction against the 40 and 25 kDa proteins increased to 45.8% and 25%, respectively, when Ig antibodies were used. Finally, when the EAS antigen (eluted from the anti-A. suum column after PAK purification) was tested, 83.3% of the assayed sera reacted against the 14 kDa protein, when the Ig antibodies, IgG and IgM immunoglobulins were measured. With the IgE determination, the reactions were observed in 41.7% of patients with proteins between 60 and 35 kDa against the PAS antigen. With the EAS antigen, reactive bands of 184, 84 and 14 kDa appeared. In conclusion, in the purification process of the A. simplex larval crude extract, the proteins implicated in cross-reactions with Ascaris and Toxocara were eliminated, with an important concentration of proteins responsible for the induction of specific responses.