Diagnostic value of dengue virus-specific IgA and IgM serum antibody detection

The diagnostic value of dengue virus (DV)-specific immunoglobulin A (IgA) serum antibody detection, by an indirect immunofluorescence assay (IFA) was evaluated. For this study, the kinetics of DV-specific IgA serum antibodies was analysed in two experimentally immunised macaques, paired samples from 35 patients suspected of a primary or secondary DV infection, paired sera from patients with high levels of IgA specific antibodies against influenza virus (n = 15), sera from patients with other viral infections (n = 40) and healthy blood donors (n = 10), which served as controls. The presence of DV-specific IgA serum antibodies in humans and in monkeys was compared with that of DV-specific IgM demonstrated in a capture enzyme-linked immunosorbent assay (ELISA). The development of DV-specific IgA and IgM antibodies in macaques proved to be similar to that observed in humans with a DV infection. In sera obtained from suspected primary DV patients during the acute phase and convalescent phase, DV-specific IgA was detected in 1/6 (17%) and 6/6 (100%), whereas IgM was detected in 4/6 (67%) and 5/6 (83%), respectively. In sera from suspected secondary DV patients during the acute phase and convalescent phase, DV-specific IgA was detected in 18/29 (62%) and 28/29 (97%), whereas IgM was detected in 20/29 (69%) and 28/29 (97%), respectively. The control group consisted of five paired serum samples from yellow fever vaccinated individuals and a patient with acute tick-borne encephalitis, 15 paired serum samples from patients with high levels of IgA antibodies specific for influenza virus and 40 serum samples from patients with specific IgM antibodies against other viruses. Ten serum samples from healthy blood donors were included. Among the control serum samples, in one patient, both DV-specific IgA and IgM antibodies were present, and in three sera DV-specific IgM antibodies could be demonstrated. These data suggest that detection of DV-specific IgA serum antibodies by IFA may have additional value for the diagnosis of DV infection.     

Study of the immunological effectiveness of the enteral vaccination of volunteers with detergent Sonne dysentery monovaccine]

The authors evaluated the immunological results of enteral and subcutaneous injection of detergent Sonne dysentery vaccine to volunteers (changes in homo- and heterologous antibodies in the blood and saliva recorded by means of common indirect hemagglutination test and that modified according to Coombs' test, and IgG, IgM, and IgA concentration recording. Both immunization methods induced specific antibody formation: enteral vaccination caused chiefly the accumulation of secretory antibodies of IgA class, and subcutaneous -- of serum antibodies belonging to IgG class. The supposed mechanisms determining the capacity of enteral administration of the antigen to provoke an immune response different from that caused by its subcutaneous injection are discussed.     

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 mucosal and serum antibodies specific for the capsular polysaccharide of Haemophilus influenzae type b by enzyme-linked immunosorbent assay

A sensitive enzyme-linked immunosorbent assay (ELISA) with rough-surfaced glass beads used as the solid phase was developed for detection of IgG, IgM, and IgA antibodies to Haemophilus influenzae type b capsular polysaccharide (HITB-CP). A successful method for indirect coating of glass bead surfaces with HITB-CP, and parameters affecting the specificity and sensitivity of the assay are described. This ELISA system proved to be 100 times as sensitive as the standard indirect fluorescent-antibody assay. The assay was applied to the measurement of antibodies to HITB-CP in serum and nasal secretions and proved to be a useful tool in the evaluation of immunological response to HITB infection.     

Natural and immune human antibodies reactive with antigens of virulent Neisseria gonorrhoeae: immunoglobulins G, M, And A

Natural and immune human antibodies reactive with heat-labile and heat-stable antigens of virulent Neisseria gonorrhoeae were studied by use of an indirect fluorescent-antibody (IFA) procedure. The immunoglobulin class of the reactive antibodies was identified by using fluorescein-conjugated antisera specific for human IgG, IgA, or IgM in the IFA procedure. The effects of heat and mercaptoethanol on IFA reactivities were also studied. It appeared that antibodies of the IgG, IgM, and IgA classes present in the sera of both infected persons (immune antibodies) and normal persons with no history of gonococcal infection (natural antibodies) react with heat-stable somatic antigens. Immune IgG antibodies, however, were distinguishable from natural IgG antibodies by their ability to recognize heat-labile surface antigens. The distinction between natural and immune IgM antibodies was less obvious. IgM antibodies from both infected and normal persons appeared to react with heat-labile antigens. Some, but not all, infected persons had immune IgA antibodies to heat-labile as well as to heat-stable antigens. Treatment of sera with mercaptoethanol had no effect on IgG antibodies. The IFA activity of IgM antibodies was decreased, but not abolished. The effects of mercaptoethanol on IgA antibodies were variable. Some sera showed a decrease in IgA titer, and others showed an increase in IgA activity to certain antigens. Immune IgG antibodies were more resistant to heating than were natural IgG antibodies. Natural and immune IgM antibodies appeared equally sensitive to heating. IgA activity, on the other hand, was increased by heating sera at 60 C, but was decreased at higher temperatures. Thus, it appears that natural and immune human IgG antibodies to N. gonorrhoeae may be distinguished by their interactions with heat-labile antigens and by their resistance to heating.     

Normal human sera contain antibodies directed at Fab of IgA

Serum samples from 26 normal volunteers were evaluated by isotype-specific ELISA for the presence of IgG and IgM antibodies directed at IgA. Although there were wide variations in antibody levels, anti-IgA antibodies of both isotypes were found in all individuals tested. The anti-IgA activity was detected against a variety of polymeric and monomeric IgA1 and IgA2 myeloma proteins containing both kappa and lambda light chains. By using Fab and Fc fragments generated by incubation of an IgA1 myeloma protein with IgA1 protease, it was shown that the anti-IgA activity was specific for the Fab portion of the IgA molecule. It was also demonstrated that the serum of two individuals contained both IgG and IgM activity directed at autologous affinity-purified IgA. IgM antibody levels against both whole IgA and Fab of IgA were significantly higher than IgG antibody levels. Cells producing anti-IgA antibodies of both isotypes were detected in lipopolysaccharide-stimulated human spleen.     

Composition of immunoglobulins in brucellosis patients using DEAE-cellulose column chromatography.

The composition of immunoglobulins in patients with brucellosis was studied. The method of ion-exchange chromatography on DEAE-cellulose columns was used to define more precisely the physico-chemical character of cysteine-resistant antibodies. The study of IgM, IgA and IgG fractions obtained from the patients sera showed the IgG fraction to possess the greatest serological activity in the agglutination reaction, in the passive haemagglutination reaction and in Coomb's test. Specific antibodies in the remaining 2 fractions (IgA and IgM) were found only in single patients in low titres, mainly in Coomb's test (incomplete antibodies). The study of IgM, IgA and IgG serum fractions before and after cysteine treatment revealed cysteine-resistant antibodies to be usually IgG globulins. The presence of specific IgG antibodies in the sera of patients was found to correlate with active clinical manifestations of brucellosis.     

Competition between Serum IgG,IgM, and IgA Anti-Glycan Antibodies

Anti-glycan antibodies are an abundant subpopulation of serum antibodies with critical functions in many immune processes. Changes in the levels of these antibodies can occur with the onset of disease, exposure to pathogens, or vaccination. As a result, there has been significant interest in exploiting anti-glycan antibodies as biomarkers for many diseases. Serum contains a mixture of anti-glycan antibodies that can recognize the same antigen, and competition for binding can potentially influence the detection of antibody subpopulations that are more relevant to disease processes. The most abundant antibody isotypes in serum are IgG, IgM, and IgA, but little is known regarding how these different isotypes compete for the same glycan antigen. In this study, we developed a multiplexed glycan microarray assay and applied it to evaluate how different isotypes of anti-glycan antibodies (IgA, IgG, and IgM) compete for printed glycan antigens. While IgG and IgA antibodies typically outcompete IgM for peptide or protein antigens, we found that IgM outcompete IgG and IgA for many glycan antigens. To illustrate the importance of this effect, we provide evidence that IgM competition can account for the unexpected observation that IgG of certain antigen specificities appear to be preferentially transported from mothers to fetuses. We demonstrate that IgM in maternal sera compete with IgG resulting in lower than expected IgG signals. Since cord blood contains very low levels of IgM, competition only affects maternal IgG signals, making it appear as though certain IgG antibodies are higher in cord blood than matched maternal blood. Taken together, the results highlight the importance of competition for studies involving anti-glycan antibodies.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. I. Occurrence of antibodies to enterobacterial common antigen (ECA)

The antibodies against the Enterobacterial Common Antigen (ECA) were detected using the ELISA in 293 serum samples collected from 185 persons suspected for yersiniosis, as well as 115 serum samples from healthy individuals (blood donors). The presence of IgA antibody in diagnostically significant titres for ECA were detected by ELISA in 3.5%, IgG in 13.0%, and IgM in 5.2% of blood donors. Statistical analysis showed that the frequency of detecting antibodies for ECA among the patients with yersiniosis was significantly higher (p < 0.05) in relation to the blood donors. Most frequently the elevated antibody levels were detected among patients with reactive arthritis (IgA 29.2%, IgG 35.4%, IgM 16.7%) while the most infrequent among patients with abdominal pain in acute phase of yersiniosis (IgA 14.9%, IgG 25.3%, IgM 19.5%). The level of antibodies for ECA, together with age increased reaching its peak, on the average, among individuals aged 41 - 60 years. In majority of the individuals studied antibodies of the IgG class reached the level much higher in relation to those of the IgA and IgM classes. The obtained results showed that the detection of antibodies to ECA may be useful in serodiagnosis of Yersinia infections.     

Natural antibodies and immune antibodies of human ABO blood group system

Sera of normal and isoimmunized persons genotype OO and A₁O were fractionated to separate the three main immunoglobulin components IgG, IgM and IgA from each other. The anti-B activity was measured in every fraction and individual serum. The results indicate that the natural anti-B antibodies are of IgM molecular type, contrasting with the immune anti-B antibodies which are IgM, IgG and eventually IgA. The restriction to the IgM class and other data previously reported are discussed in relation to the origin of the natural antibodies of the human ABO blood group system.     

Immunofluorescence analysis of IgA binding by human mononuclear cells in blood and lymphoid tissue

The nature of IgA-binding cells and their tissue distribution was examined by an indirect immunofluorescence assay with the use of IgA1 and IgA2 paraproteins and fluorochrome- or biotin-labeled F(ab')2 fragments of idiotype-specific antibodies. The frequency of IgA-binding mononuclear cells was approximately 13% in blood and spleen samples but less than 1% in tonsil samples. IgA binding could be visualized by flow immunocytometry on monocyte/macrophages, but not on T and B cells. IgA polymers were bound better than IgA dimers and monomers. Nonhomologous IgA myelomas of both IgA1 and IgA2 subclasses inhibited the IgA-binding to monocytes, whereas aggregated normal serum IgG, IgM paraproteins, and an IgG myeloma did not. IgA binding was relatively insensitive to changes in temperature or cation concentration. IgA-binding monocytes were found in IgA-deficient patients at the same frequency as in normal individuals. The results indicate that monocytes constitutively express class-specific binding sites for both IgA1 and IgA2 molecules.     

The determination in saliva of IgA antibodies to Shigella ribosomes for the diagnosis of dysentery

The levels of antiribosomal antibodies to Shigella ribosomes in serum and saliva samples from 38 dysentery patients (15 S. sonnei cases and 23 S. flexneri cases), 14 patients with salmonellosis and 136 healthy adults were determined in ELISA with ribosomes from S. sonnei R-mutant used as solid-phase antigen. High levels of "normal" antiribosomal IgA, IgG and IgM antibodies were revealed in the sera of healthy persons while the level of salivary IgA antibodies was very low. In dysentery infection no increase in the levels of serum IgG and IgM antibodies and only a slight increase in the level of IgA antibodies were revealed. Local immune response was manifested by the early (on days 2-4 from the onset of infection) and significant augmentation (12- to 16-fold) of salivary antiribosomal IgA antibodies. An increase in the level of these antibodies was registered in 95-100% of dysentery patients but not in patients with salmonellosis, which made it possible to recommend the method for diagnosing shigellosis. Immune response to Shigella ribosomal antigens, in contrast to the response induced by Shigella O-antigen, is almost exclusively local.     

The localization of the antibody response in milk or bile depends on the nature of the antigen

Immunization in the Peyer's patches of rats with horse spleen ferritin or Escherichia coli 06 carrying type 1 pili resulted in an IgA antibody response detected in milk and bile and an IgG and IgM antibody response in serum, milk, and bile. The IgA antibody response to type 1 pili was as a mean 5.0-fold higher in milk than in bile. In contrast IgA antibody activity to 06 LPS was as a mean 6.3-fold higher in bile than in milk. The IgA antibodies to ferritin were randomly distributed between milk and bile. The IgG and IgM antibody activity to all three antigens studied were higher in the milk than in the bile. The secretory antibody response could be transferred from immunized rats to unimmunized rats with mesenteric lymph node cells (MLN) taken from donor rats 4 days after immunization in the Peyer's patches. IgA antibodies to pili and ferritin appeared solely in the milk of the recipients, whereas IgA antibodies to the 06 LPS only appeared in the bile. The ratios serum:milk and serum:bile for the IgG and IgM antibodies indicated an antigen-specific direction of homing with local production of these two isotypes primarily in the mammary gland. Antibody-forming cells of the IgA class could not be detected in the MLN on the day the cells were transferred. It is concluded that the difference seen in antibody distribution between milk and bile is not due to dissemination of antigen, but instead a result of different homing or expansion at the mucosal-glandular site dependent on the antigen specificity of the migrating cells.     

Dynamics of the level of antibodies to protein antigens of the Meningococcus serogroup A in the serum of patients with meningococcal infection

Two characteristics were determined in the sera of patients by means of radioimmunoassay (RIA) techniques: the RIA titer and the characteristic indicating the presence of highly avid antibodies in the serum. This investigation revealed that in the sera of patients with meningococcal infection of moderate severity the values of the RIA titer of IgM, IgG and IgA, as well as the values indicating the levels of highly avid IgM, IgG and IgA, increased as the infection progressed. In patients with the severe form of meningococcal infection the values of the RIA titer of IgA and IgA increased, while the RIA titer of IgG remained practically unchanged. No dynamic changes in the characteristics indicating the presence of highly avid antibodies in the serum were observed.     

Role of Salmonella and Yersinia in the pathogenesis of spondyloarthropathies and rheumatoid arthritis. II. Antibodies to Salmonella and Yersinia in sera and synovial fluids

IgA, IgG and IgM antibodies against Yersinia Yop proteins, Yersinia LPS and Salmonella LPS from different serogroups were determined by enzyme-linked immunosorbent assay (ELISA) in a 885 serum samples and 92 synovial fluids. The control group consisted of 200 healthy blood donors. Compared with control subjects, patients with arthritis showed significantly increased titres of antibodies against Yersinia Yop, Yersinia LPS and Salmonella LPS appropriately in 21.7%, 44.0% and 56.0% serum samples. The prevalence of positive antibody levels was highest in Yersinia serogroup O3 and Salmonella serogroup B and D antibodies. The IgA titres were found to be much higher in adults than in children and youngsters but IgM titres consequently decreased with age. Investigation of synovial fluids obtained from patients with arthritis showed that Yersinia and Salmonella antibodies in synovial fluid mirror those in serum by concentration, by specificity and by distribution in classes.     

Characterization of anti-Cryptosporidium IgA antibodies in sera from immunocompetent individuals and HIV-infected patients.

Human antibody response to Cryptosporidium parvum has been previously shown as involving immunoglobulin (Ig)M and IgG isotypes. The interest in anti-cryptosporidial IgA antibody response has been recently stimulated by studies on the therapeutic effects of secretory IgA antibodies to Cryptosporidium in animal models and in patients. In the present study, isotypes of serum anti-Cryptosporidium antibodies have been characterized in donors of the following categories: (a) healthy adults, (b) healthy children, (c) immunocompetent children with transient cryptosporidial diarrhea, (d) HIV-infected patients without clinical and parasitological evidence of Cryptosporidium infection and (e) AIDS patients with cryptosporidial diarrhea. Antibodies were detected using C. parvum oocysts purified by density gradient centrifugation from bovine faeces. The IgA antibodies were revealed using alpha-chain specific antibodies. Indirect immunofluorescence analysis with oocysts was used as control. Although high levels of serum antibodies of the IgA class were detected in some donors in the group of healthy adults, elevated values were consistently found in HIV-infected patients. Higher values were found in HIV patients with clinical cryptosporidiosis. The presence of a secretory component in serum IgA antibodies in these patients has been documented. Data indicate that IgA serum antibodies are produced as well as IgM and IgG antibodies upon contact with the parasite, and suggest that elevated IgA serum antibodies to Cryptosporidium are not associated with protection in HIV patients.     

IgA and IgM anti-Naegleria fowleri antibodies in human serum and saliva

Antibodies from IgA and IgM classes that recognize Naegleria fowleri (Nf) proteins were detected by the ELISA assay in serum and saliva from three groups of people: (i) subjects with upper respiratory tract infections (URTI) living in the parasite-endemic area, (ii) healthy persons from the same area, and (iii) healthy persons from a parasite-nonendemic area. In serum and in saliva the titers of IgA antibodies to Naegleria fowleri in the group of patients with URTI was significantly higher than that of the healthy group in the parasite-endemic area; also the titers of IgM antibodies in serum were significantly higher in patients. On the contrary, in saliva the antibodies were higher in healthy people from the parasite-endemic area. In all cases the subjects from the parasite-nonendemic area had lower antibody titers in serum and saliva.     

Standardization of ELISA IgM and IgA for immunodiagnosis of human trichinosis

An ELISA test for trichinosis using as antigen a larvae soluble fraction from Trichinella spiralis was carried out for the detection of IgM and IgA specific antibodies in 45 serum samples from patients confirmed or suspected to have trichinosis by strong clinical and epidemiological evidences. All the patients had positive serology detected by precipitin test, bentonite floculation test, indirect hemagglutination test and ELISA IgG test. The cut-off value was determined using two criteria. Criterion A was determined in each plate, using three positive controls and two negative ones; the average of the negative controls and the weakest positive control, multiplied by a 1.2 factor was, considered the cut-off value. Criterion B was determined using the average plus three standard deviations from 64 apparently healthy persons serum samples. In both cases, three serum dilutions (1:10, 1:100 and 1:500) were used. The sensitivity of ELISA IgM was 100.0, 93.3 and 82.2% using serum dilutions of 1:10, 1:100 and 1:500 respectively (criterion A) and 100.0, 97.8 and 95.6% for the same dilutions (criterion B), whereas the values for ELISA IgA were: 100.0, 91.1 and 86.7% (criterion A) and 100.0, 100.0 and 91.1% (criterion B). In order to find out the specificity of ELISA IgM and ELISA IgA, additional 118 serum samples from individuals with other parasitoses, such as cysticercosis (18) hydatidosis (39), fascioliasis (12), toxocariasis (30), Chagas' disease (12) and individuals with non-specific eosinophilia (7), were also tested. ELISA IgM presented a specificity of 92.3, 93.4 and 97.3% (criterion A) and 96.2, 97.8 and 97.8% (criterion B) whereas the results for ELISA IgA were 97.8, 98.9 and 99.4% (criterion A) and 98.4% for the 1:10 and 1:100 dilutions and 100.0% for the 1:500 dilution (criterion B). The positive predictive values of ELISA IgM were 76.3, 77.8 and 88.1% (criterion A) and 86.5, 91.7 and 91.5% (criterion B) whereas the negative ones were 100.0, 98.3 and 95.7% (criterion A) and 100.0, 99.4 and 98.9% (criterion B). The positive predictive values of ELISA IgA were 91.8, 95.3 and 97.5% (criterion A) and 93.8, 93.8 and 100.0% (criterion B) whereas the negatives ones were: 100.0, 97.8 and 96.8% (criterion A) and 100.0, 100.0 and 97.8% (criterion B). The use of ELISA IgM and ELISA IgA in the immunodiagnosis of trichinosis is discussed.     

Humoral immune response in patients with bronchopulmanory infections to immunotherapy

The content of IgA, IgM, IgG and the level of specific antibodies in the blood serum of patients with bronchopulmonary diseases after a course of immunotherapy with polycomponent vaccine B[symbol: see text]-4 was studied. A rise in the concentration of IgM due to the synthesis of specific antibodies to Proteus vulgaris, Escherichia coli and Klebsiella pneumoniae was found to occur. The examination of sick children revealed that a high proportion of them (54%) showed a pronounced decrease in the level of IgA. The use of the preparation made it possible to enhance the level of IgA.     

Humoral reaction to Bordetella pertussis antigens: pertussis toxin, filamentous hemagglutinin and lipopolysaccharide in children with clinical symptoms of whooping cough. II. Occurence and level of B. pertussis antigens in children with suspected whooping cough

The aim of this study was to determine and evaluate IgG, IgM and IgA levels to pertussis toxin (PT), filamentous hemagglutinin (FHA) and endotoxin (LPS) of B. pertussis in children with clinical symptoms of whooping cough. The serum samples obtained from 265 children (age range: 2 months-16 years) suspected of pertussis were examined by indirect haemagglutination (IH) and ELISA tests. Higher antibody level was most frequently observed in IgA class to PT, FHA and LPS in 45.3%, 35.1% and 66% of pertussis patients sera respectively. The least positive results were obtained in IgM class to PT and FHA (in 9.8% and 2.6% of children sera respectively) but in the case of LPS applied as the antigen in ELISA, higher IgM level was determined in 46.8% of pertussis patients sera. The four times increase of antibody level to LPS determined by IH was observed in 86.7% of children suspected of pertussis. Humoral response to B. pertussis infection is mainly connected with higher IgA level to PT, FHA, LPS and IgM to LPS in children with clinical symptoms of whooping cough.