Ultracentrifugal studies of sera from cattle vaccinated or naturally infected with Brucella abortus

In conformity with the findings of previous investigators, it was shown by density gradient ultracentrifugation that the antibodies in sera collected from calves shortly after vaccination with Brucella abortus, strain 19, were entirely or mainly rapidly-sedimenting. These macroglobulin (19S or IgM) antibodies showed complement-fixing as well as agglutinative activity with Br. abortus antigen. In later bleedings from the same vaccinated calves, antibodies with an intermediate sedimentation rate, (IgG), were present, as well as IgM. Sera from 15 of 22 non-vaccinated, relatively recent field cases of brucellosis appeared to contain only the IgG class of antibodies. In one herd, however, two cows with IgM only and five with both IgM and IgA were found; all seven of these cattle had been serologically negative before their introduction into this known infected herd a few months earlier. The agglutinative activity of sera from four cases of brucellosis of long standing and from eight cows, 4 to 13 years of age, that had been vaccinated as calves, was confined to the IgG fraction.     

The Rose Bengal Test in human brucellosis: a neglected test for the diagnosis of a neglected disease

Brucellosis is a highly contagious zoonosis affecting livestock and human beings. The human disease lacks pathognomonic symptoms and laboratory tests are essential for its diagnosis. However, most tests are difficult to implement in the areas and countries were brucellosis is endemic. Here, we compared the simple and cheap Rose Bengal Test (RBT) with serum agglutination, Coombs, competitive ELISA, Brucellacapt, lateral flow immunochromatography for IgM and IgG detection and immunoprecipitation with Brucella proteins. We tested 208 sera from patients with brucellosis proved by bacteriological isolation, 20 contacts with no brucellosis, and 1559 sera of persons with no recent contact or brucellosis symptoms. RBT was highly sensitive in acute and long evolution brucellosis cases and this related to its ability to detect IgM, IgG and IgA, to the absence of prozones, and to the agglutinating activity of blocking IgA at the pH of the test. RBT was also highly specific in the sera of persons with no contact with Brucella. No test in this study outperformed RBT, and none was fully satisfactory in distinguishing contacts from infected patients. When modified to test serum dilutions, a diagnostic titer >4 in RBT resulted in 87.4% sensitivity (infected patients) and 100% specificity (contacts). We discuss the limitations of serological tests in the diagnosis of human brucellosis, particularly in the more chronic forms, and conclude that simplicity and affordability of RBT make it close to the ideal test for small and understaffed hospitals and laboratories.     

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.     

Characterization of immunoglobulin classes of human antibodies to cryptococcus neoformans

Immunoglobulin G (IgG), immunoglobulin M (IgM) and immunoglobulin A (IgA) levels were determined by radial immunodiffusion techniques in sera from 11 patients with cryptococcosis. Most specimens showed increased levels of IgM. Studies with fluorescein-labeled monospecific antihuman IgG and IgM, however, indicated that IgG was the immunoglobulin reactive in the indirect fluorescent antibody (IFA) test. In addition, cross-reacting sera from mycotic infections other than cryptococcosis were also shown to contain IFA antibodies of the IgG class. Sera treated with 2-mercaptoethanol continued to react in both the IFA test and the tube agglutination test. No correlation could be established between IgG and IgM concentrations and serological reactivity in the sera evaluated in this study.     

Study of interaction between IgG and IgM antibodies against rubella virus by the immunofluorescence method.

In immunoglobulin fractions or after elimination of IgG by absorption the immunofluorescence test for rubella IgM antibodies is more sensitive than in whole serum. Blocking of IgM activity by IgG antibodies was eliminated when the time of incubation of the serum with virus antigen was prolonged. After prolonged incubation higher titres of rubella antibodies were also obtained in the IgM immunoglobulin fractions. Protein A in Staphylococcus aureus suspension effectively absorbs antibodies of IgG class. The IgM antibody titres in absorbed sera of patients infected with rubella were in some cases 2 to 4 times higher than in unabsorbed sera.     

Evaluation of IgG, IgM, and IgA antibodies to mycoplasma penetrans detected by ELISA and immunoblot in HIV-1-infected and STD patients, in São Paulo, Prazil

The aim of the present study was to determine the frequency of IgG, IgA, and IgM antibodies to Mycoplasma penetrans in HIV-1-infected patients and in patients with sexually transmitted diseases. We tested serum samples from 106 HIV-1-positive patients and 110 individuals with clinical symptoms of urethritis. ELISA and the immunoblot test were performed using M. penetrans lipid associated membrane proteins as antigen. By ELISA, we found a higher frequency (P < 0.05) of IgG against M. penetrans in HIV-1-infected and STD patients (25.5 and 17.3%) than in controls (1.2%), as well as a higher frequency of IgA (P < 0.05) (15.1 and 17.3% compared to 1.2%). For IgM, no differences were observed (P >/= 0.05) (3.8, 9.1, and 5. 8%, respectively). When the frequencies of IgG, IgM, and IgA antibodies of the HIV-1-infected patients were compared taking into account the CD4/CD8 cell ratios < 0.3 and >/= 0.3, no significant differences were observed between the two groups (13.3, 10, and 20%, compared to 20, 0, and 5%, respectively) (P > 0.05), possibly due to the low number of samples on which we could perform T-cell counts (53/106). The M. penetrans peptide of 38 kDa, considered immunodominant, was recognized in immunoblot by 51.8% of positive sera by ELISA for IgG, 50.0% for IgM, and 75% for IgA in the AIDS patients group, and by 47.4, 60.0, and 75.0%, respectively, in the sexually transmitted disease group. Cross-reactions in immunoblot for IgG were observed in sera from individuals infected with Mycoplasma pneumoniae and Mycoplasma hominis, and cross-reactions in immunoblot for IgA were observed in sera from individuals infected with M. hominis; all of them were ELISA negative to M. penetrans.     

Enzyme-linked immunosorbent assay (ELISA) for IgM, IgG and IgA class antibodies against Candida albicans antigens: development and comparison with other methods

An extract of Candida albicans was used as an antigen on microtitre plates in the enzyme-linked immunosorbent assay (ELISA) to measure IgM, IgG and IgA class antibodies in the sera of hospitalized patients. It was found that of these patient sera that reacted positively in Ouchterlony immunodiffusion (ID) when undiluted, 58% were also positive in the ELISA against the same antigen preparation. However, all the sera with an ID titre of 1:2 or higher were ELISA-positive, demonstrating especially IgG and IgA. Of the sera positive by counterimmunoelectrophoresis against somatic and metabolic antigens of C. albicans, 86% were positive by ELISA. Reactions in precipitin-negative sera, if they occurred, usually demonstrated IgM or IgA. The sera with high passive haemagglutination or indirect immunofluorescence titres against surface antigens of C. albicans were positive in the IgG and IgA assays, while approximately one third were positive in the IgM assay.     

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.     

Comparative Seroepidemiologic Analysis of <Emphasis Type="Italic">Chlamydophila Pneumoniae</Emphasis> Infection using Microimmunofluorescence,Enzyme Immunoassay and Neutralization Test: Implications for Serodiagnosis

A seroepidemiologic study using the microimmunofluorescence (MIF) technique was conducted to determine the prevalence of Chlamydophila pneumoniae IgG antibodies among 205 healthy Singapore university undergraduates using the MRL Diagnostics MIF test kit. The overall seroprevalence was 35.1% with significantly higher seropositivity rates among males than females (48.2 vs. 18.7%, P < 0.001). A comparative study using the Labsystems MIF test kit was conducted on sera from 192 students. Using the MRL MIF test as the reference, the sensitivity and specificity of Labsystems MIF test were 92.6 and 87.9%, respectively. A total of 78 samples comprising 15 MIF-negative and 63 MIF-positive samples were also tested for complement-independent neutralizing antibodies in vitro. All the 78 samples and 11 additional MIF-negative samples were also tested for IgM, IgG and IgA against C. pneumoniae by enzyme immunoassay (EIA) using the Labsystems EIA test kit. None of these 89 samples were seropositive for IgM. The percentages of IgG and IgA seropositivity increased with increasing grades of MIF-positivity. Among the IgG seropositive samples, 69.1% were also positive for IgA, suggesting that a high proportion of infected individuals also had IgA antibodies denoting chronicity. Neutralizing antibodies were detected in 22.2% of MIF-positive sera, but only in 6.7% of MIF-negative sera. 26.4 and 34.2% of samples which were IgG and IgA seropositive respectively also exhibited neutralizing activity. The percentages of MIF-positive sera with neutralizing activity increased with the grade of MIF positivity, i.e. 0% (1+), 7.1% (2+), 18.8% (3+), and 63.6% (4+). High-grade MIF positivity (particularly with MRL MIF kits) may represent a useful serologic marker of predictive value for neutralizing activity.     

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.     

Comparative study of cell-immunoenzymatic methods for the estimation of IgG and IgM anti-brucella antibodies in the diagnosis of human brucellosis

Two methods which employ whole cells are described and compared for the detection of human IgG and IgM anti-brucella antibodies. Dot ELISA and ELISA were shown to be suitable for a screening diagnosis of human brucellosis. Titres of antibodies obtained by dot ELISA showed 100% coincidence for IgG and 97% for IgM, compared with agglutination and complement fixation tests; when ELISA was used 11 % positive sera were not detected. The comparison of these two methods with the conventional serological test kit indicated that both dot ELISA and ELISA were sensitive, reproducible and specific for the quantification of IgG and IgM anti-brucella antibodies.     

Isotype concentrations of human antibodies to group A meningococcal polysaccharide

Antibodies to meningococcal group A polysaccharide (MenA) in the sera of 34 vaccinated adults were quantitated by an isotype-resolving solid-phase RIA (IgA, IgM, IgG1, IgG2, IgG3, and IgG4). All individuals had antibodies before vaccination. The geometric mean concentration was 2.9 micrograms/ml. Two weeks after vaccination the mean antibody concentration had trebled. Average proportions of the three isotypes were then as follows: IgA 15%, IgM 48%, IgG 37%. No differences were found between individuals who had been immunized with the polysaccharide 7 to 8 yr earlier and "primary responders." The subclass composition of IgG antibodies was determined in the 24 postvaccination samples with a definite IgG response (greater than 2-fold increase). IgG1 was the predominant subclass in antibodies of some sera and IgG2 in others, but the average proportions of both subclasses were nearly the same. IgG3 and IgG4 were only found in occasional sera, but when present, each subclass accounted for up to 6%. Although the ratio of kappa and lambda chains could not be determined, there was evidence to suggest that it was higher in anti-MenA antibodies than in antibodies to protein antigens.     

Comparative study of cell-immunoenzymatic methods for the estimation of IgG and IgM anti-Brucella antibodies in the diagnosis of human brucellosis

Two methods which employ whole cells are described and compared for the detection of human IgG and IgM anti-brucella antibodies. Dot ELISA and ELISA were shown to be suitable for a screening diagnosis of human brucellosis. Titres of antibodies obtained by dot ELISA showed 100% coincidence for IgG and 97% for IgM, compared with agglutination and complement fixation tests; when ELISA was used 11% positive sera were not detected. The comparison of these two methods with the conventional serological test kit indicated that both dot ELISA and ELISA were sensitive, reproducible and specific for the quantification of IgG and IgM antibrucella antibodies.     

A Hospital-Based Serological Survey of Cryptosporidiosis in the Republic of Korea

The seroprevalence of cryptosporidiosis was examined using patients'' sera collected from hospitals located in 4 different areas of the Republic of Korea. ELISA was used to measure antibody titers against Cryptosporidium parvum antigens from a total of 2,394 serum samples, which were collected randomly from patients in local hospitals; 1) Chungbuk National University Hospital, 2) Konkuk University Hospital, 3) local hospitals in Chuncheon, Gangwon-do (province), 4) Jeonnam National University Hospital, from 2002 through 2003. Of the 2,394 samples assayed, 34%, 26%, and 56% were positive for C. parvum-specific IgG, IgM, and IgA antibodies, respectively. Positive IgG titers were most common in sera from Jeonnam National University Hospital, Gwangju, Jeollanam-do, and positive IgM titers were most common in sera from Chungbuk National University Hospital, Cheongju, Chuncheongbuk-do. The seropositivity was positively correlated with age for both the IgG and IgA antibodies but was negatively correlated with age for the IgM antibodies. Western blotting revealed that 92%, 83%, and 77% of sera positive for IgG, IgM, and IgA ELISA reacted with 27-kDa antigens, respectively. These results suggested that infection with Cryptosporidium in hospital patients occurs more commonly than previously reported in the Republic of Korea.     

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.     

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.     

Echinococcus multilocularis: Immunoglobulin and antibody response in C57BL6J mice

Each of 50 male C57BL/6J mice was infected intraperitoneally with 50 cysts of Echinococcus multilocularis. At 2, 4, 6, 8, and 14 weeks after infection, 10 mice were sacrificed, their larval cyst masses weighed, and their sera collected. Each serum sample from uninfected control and infected mice was adsorbed twice with two batches of E. multilocularis antigen conjugated to Sepharose beads. The concentrations of IgG1, IgG2a, IgG2b, IgM, and IgA in unadsorbed and IgG1, IgG2b, and IgM in adsorbed sera were quantified by the radial immunodiffusion technique. Hydatid mice produced increasingly large amounts of IgG1 and IgM; small measurable increases of IgG2b and no significant increases of IgG2a and IgA were observed during the course of infection. During the rapid growth phase of the cysts (6 to 14 weeks) IgG1 antibodies were found to range from 86 to 93% and IgM antibodies from 17 to 33% of the total IgG1 and IgM. However, the actual protein concentrations of IgM antibodies (761 and 1215 mg/dl) were higher than the sum of the protein concentrations of IgG1 and IgG2b antibodies (411 and 779 mg/dl). The significance of the relative concentrations of IgM, IgG1, IgG2a, and IgG2b antibodies is discussed with reference to their effectiveness in antibody-dependent cellular cytotoxicity and complement-mediated lysis in the control of alveolar hydatid disease.     

Specificities of human heterophile Hanganutziu and Deicher (HD) antibodies to glycosphingolipids and a glycoprotein

The specificities of five heterophile Hanganutziu and Deicher (HD) antibody-containing sera from four different cancer patients and one other diseased patients were compared. Three glycosphingolipids and one glycoprotein antigens and their chemically modified derivatives were used. The antibodies of all whole sera showed similar specificities. IgG and IgM antibody fractions of each serum were separated. Although antibodies of the same class showed similar specificities, differences were detected between the specificities of IgG and IgM. IgG antibody specificities were dependent on the hydrophobic (ceramide) group while IgM antibodies were directed more to the terminal sialic acid moiety of the glycosphingolipid antigens. The results suggested that a similar population of IgG-producing lymphocytes is stimulated in patients. Due to the similarities in specificities of HD antibodies, the results of this study will facilitate the future isolation of either IgG or IgM antibody-producing lymphocyte(s) from a patient with HD antibodies and the establishment of a monoclonal antibody through hybridization with a human myeloma cell line.     

Antibody and immune complexes induce tissue factor production by human endothelial cells

Patients with systemic lupus erythematosus (SLE) have an increased incidence of arterial and venous thromboses. The mechanism by which thromboses develop in these patients is unknown. We had previously observed that the sera of patients with SLE contain antibodies and immune complexes that can bind to endothelial cells. Because endothelial cells can synthesize tissue factor, a potent activator of coagulation, we studied the effect of IgG complexes and sera from patients with SLE on the production of tissue factor by these cells. Human umbilical venous endothelial cells incubated with heat-aggregated IgG (HA-IgG) (0.5 to 4.0 mg) elaborate procoagulant activity in a dose-dependent manner. All procoagulant activity was found in the particulate cell fraction, and none was secreted into the medium. Maximum expression of procoagulant activity required 6 to 8 hr, and its production was totally inhibited by the addition of cyclohexamide or actinomycin D. The presence of gel-filtered platelets augmented production of procoagulant activity by endothelial cells stimulated by HA-IgG. Endothelial cell procoagulant activity was not inactivated by diisofluoropropylphosphate, required the presence of Factor VII for its expression, and was neutralized by a specific anti-tissue factor antibody. Endothelial cells incubated with sera from 14 of 16 patients with SLE produced increased amounts of tissue factor compared with 21 normal sera (p less than 0.025). Fractions of two SLE sera containing monomeric IgG, IgA, or IgM, as well as fractions containing IgG complexes, each stimulated endothelial cells to produce more tissue factor than similar fractions prepared from two normal sera. These studies demonstrate that endothelial cells will produce the procoagulant tissue factor after exposure to anti-endothelial cell antibodies or IgG-containing immune complexes. The production of tissue factor by endothelial cells at sites of immune vascular injury may play a role in the development of thromboses in patients with SLE.