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.     

Isotype specific immune responses in murine experimental toxocariasis

In this work, a murine experimental model of toxocariasis has been developed in BALB/c, C57BL/10 and C3H murine strains orally inoculated with 4,000 Toxocara canis embryonated eggs, in order to investigate the isotype-specific immune responses against excretory-secretory antigens from larvae. T. canis specific IgG+M, IgM, IgG, IgA, IgG1, IgG2a and IgG3 were tested by ELISA. The dynamics of the specific immunoglobulins (IgG+IgM) production showed a contrasting profile regarding the murine strain. Conversely to the results obtained with the IgM isotype, the IgG antibody class showed similar patterns to those obtained with IgG+IgM antibodies, only in the case of the BALB/c strain, being different and much higher than the obtained with IgG+IgM antibodies, when the C3H murine strain was used. The antibodies IgG+IgM tested in BALB/c and C57BL/10 were both of the IgM and IgG isotypes. Conversely, in the C3H strain only IgG specific antibody levels were detected. The IgG1 subclass responses showed a similar profile in the three murine strains studied, with high values in BALB/c, as in the case of the IgG responses.     

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.     

Generation of monoclonal antibodies to Macaca mulatta (rhesus) IgA

One IgG1 and five IgM murine monoclonal antibodies (mAb) specific for rhesus (Rh) IgA were generated. These mAbs bound to Rh IgA but not IgG or IgM when tested by enzyme-linked immunosorbent assay. Immunoblotting revealed that the mAbs reacted with the alpha heavy chain of Rh but not human IgA. The IgG1 anti-Rh IgA mAb detected IgA-producing cells in sections of monkey gut examined by immunofluorescent staining. These mAbs should be useful for characterizing IgA responses in the Rh monkey.     

High efficiency cross-reactive monoclonal antibody production by oral immunization with recombinant norwalk virus-like particles

Monoclonal antibodies (MAbs) are important for in-depth antigenic characterization and diagnosis of infections with human caliciviruses that cause almost all outbreaks of nonbacterial gastroenteritis. We compared different routes of immunization with nonreplicating virus-like particles (VLPs) from recombinant Norwalk virus (rNV) and recombinant Mexico virus (rMX) administered to BALB/c mice to determine the efficiency of hybridoma production. Oral immunization with VLPs without adjuvant resulted in high yields of MAb-secreting hybridomas (90%) to these VLPs of IgG (61%), IgM (29%) and IgA (10%) isotypes. Fusions with mesenteric lymph node lymphocytes yielded MAbs of various subclasses including IgG2a, IgG3, IgM and IgA. These results suggest that an immunization route that mimics the natural route of viral infection pathway may facilitate MAb technology by increasing the yields of antibody secreting hybridoma cells.     

Subpopulations of antibodies to phosphocholine in human serum

We investigated the heterogeneity of anti-phosphocholine (PC) antibodies present in human serum taken from individuals before and after immunization with a multivalent pneumococcal vaccine. The fine specificity of IgM, IgG, and IgA anti-PC antibodies was determined in an ELISA by using phosphocholine or p-nitrophenyl phosphocholine (NPPC) to inhibit binding of antibody to PC-histone. We identified two populations specific for PC that differed in their binding properties. One population is inhibited by NPPC much better than by PC and is most evident in IgG antibodies. The second population has similar avidity for PC and NPPC and is consistently associated with the IgM and IgA isotypes as well as with IgG. The IgG antibodies in both populations were predominantly of the IgG2 subclass. Both populations were found in serum samples taken before immunization with pneumococcal vaccine, suggesting that they had been stimulated through prior environmental contacts with PC-containing antigens. Previously, we found populations with similar fine specificity patterns in the murine response to PC. The two murine antibody populations have been shown to derive from different immunoglobulin variable region genes. The presence of comparable antibody populations in the human suggests the possibility that these two fine specificity families have been conserved in evolution.     

Local and systemic antibody response in infants after oral administration of inactivated enteropathogenicE. coli serotype O111 and O55

In ten infants divided into two groups (up to one month of age and at 2–7 months of age) the dynamics and formation of different antibody isotypes produced locally in the intenstine and in serum after orally administered inactivated enteropathogenicE. coli strains O111 and O55 was followed during 30 d after the first and booster dose by using an indirect immunofluorescence method. Infants up to one month of age produced antibodies of IgM isotype in stool together with the IgA isotype after the first and booster dose of the vaccine against both antigens. Serum IgG antibody increased after 2 d following the first and second dose of antigens and remained higher during 5 d. The infants aged 2–7 months expressed predominantly the IgA isotype response in stool after the first and booster dose of antigens. The serum immunoglobulin levels did not change after oral antigen administration.     

Intraclonal diversification in immunoglobulin isotype secretion: an analysis of switch probabilities

The production of all immunoglobulin isotypes except IgD was studied in a large number of single lipopolysaccharide (LPS)-reactive B cell clones. The majority, but not all, of the IgM-producing clones were found to secrete one or more other isotypes. IgG3 and IgG2b were most frequently found while IgA secretion was extremely rare. Many clones produced all four IgG subclasses and the statistical analysis of the data indicates, with a high degree of significance, that single clonal precursors give rise to progenies producing multiple isotypes. By assuming that intraclonal diversification follows the C-gene order in chromosome 12, the absolute switch probabilities of normal, unprimed LPS-reactive B cells can be calculated. The multi-potentiality of C-gene expression was further analyzed at the single cell level: a sizeable fraction of all activated B cells express two different IgG isotypes in the membrane-bound form, indicating consecutive switch events. In contrast, the majority of IgE and IgA secreting cells appear to switch directly from IgM. These results might reflect the functional relevance of S-region homologies in the control of C-gene expression.     

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.     

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.     

Genetic parameters for natural antibody isotype titers in milk of Dutch Holstein‐Friesians

The objective of the present study was to estimate genetic parameters for natural antibody isotypes immunoglobulin (Ig) A, IgG1 and IgM titers binding the bacterial antigens lipopolysaccharide, peptidoglycan and the model antigen keyhole limpet hemocyanin in Dutch Holstein‐Friesian cows (n = 1695). Further, this study included total natural antibody titers binding the antigens mentioned above, making no isotype distinction, as well as total natural antibody titers and natural antibody isotypes IgA, IgG1 and IgM binding lipoteichoic acid. The study showed that natural antibody isotype titers are heritable, ranging from 0.06 to 0.55, and that these heritabilities were generally higher than heritabilities for total natural antibody titers. Genetic correlations, the combinations of total natural antibody titers and natural antibody isotype titers, were nearly all positive and ranged from ?0.23 to 0.99. Strong genetic correlations were found between IgA and IgM. Genetic correlations were substantially weaker when they involved an IgG1 titer, indicating that IgA and IgM have a common genetic basis, but that the genetic basis for IgG1 differs from that for IgA or IgM. Results from this study indicate that natural antibody isotype titers show the potential for effective genetic selection. Further, natural antibody isotypes may provide a better characterization of different elements of the immune response or immune competence. As such, natural antibody isotypes may enable more effective decisions when breeding programs start to include innate immune parameters.     

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.     

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.     

Subclass restriction of murine antibodies: V. The IgG plaque-forming cell response to thymus-independent and thymus-dependent antigens in athymic and euthymic mice

Antigens differ in their abilities to stimulate antibodies of various isotypes. Many thymus-independent (TI) polysaccharide antigens stimulate largely IgG3 and IgM antibodies while thymus-dependent (TD) protein antigens stimulate predominantly IgG1 and smaller amounts of other isotypes. Here we determine whether thymus dependence or independence is a property of antigens which is expressed equally by all isotypes. To do this nu/+ and nu/nu mice were immunized with several TI and TD antigens and antibody responses of IgM and the four IgG subclasses measured. We found that, within the conditions of these experiments, all IgG isotypes were influenced equally by the presence or absence of T lymphocytes. Second, in agreement with J. L. Press (J. Immunol.126, 1234, 1981), we propose a division of TD antigens into two types based upon the ability to stimulate responses in the CBA/N mouse.     

Anaphylactic properties of mouse monoclonal IgG2a antibodies

Mouse monoclonal antibodies (10 hybridoma antibodies specific for soluble antigens, 8 hybridoma antibodies specific for H-2 $\text{K}\text{D}$ antigens, and 9 myeloma immunoglobulins, among which 5 had a known specificity) of the IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM isotypes were studied for their ability to induce mouse mast cell degranulation in vitro, in the presence of specific antigen or after heat aggregation. Monoclonal IgG1 antibodies, as well as IgG2b, IgG3, IgA, and IgM behaved as polyclonal antibodies of corresponding classes: all IgG1 induced mast cell degranulation with typical characteristics of IgG-mediated anaphylactic reactions, whereas IgG2b, IgG3, IgA, and IgM did not. By contrast, 2 hybridoma IgG2a and 3 myeloma IgG2a induced intense mast cell degranulation that could not be explained by a contamination with IgG1 or IgG1-IgG2a hybrid molecules. IgG2a-mediated reactions were observed in four different situations: soluble antigen-hybridoma IgG2a complexes, specific H-2 antigen-bearing mast cells challenged with hybridoma IgG2a anti-H-2, heat-aggregated myeloma IgG2a, and soluble antigen-myeloma IgG2a complexes. The conclusion was reached that mouse mast cells could be activated by mouse monoclonal IgG2a antibodies through a noncytotoxic, complement-independent mechanism involving mast cell Fcγ receptors.     

A modified hybridoma technique for production of monoclonal antibodies having desired isotypes

In the present study, we describe a modified hybridoma technique for production of monoclonal antibodies (mAbs) having a desired isotype. Mice were immunized with the antigen of interest. After having reached a high antibody titer, cells expressing IgM or IgG molecules were isolated from spleen cells of the immunized mice using a Magnetic Cell Sorting System. The isolated cells were fused with myeloma cells using the conventional fusion protocol. With the isolated IgM⁺ spleen cells, more than 75% (85 ± 7%; means ± SD) were IgM producing cells and a large number of IgM mAbs specific to the protein of interest were obtained. With the isolated IgG⁺ spleen cells, 41 ± 40% of the generated hybridomas produced IgG antibody and no IgM producing hybridoma was generated. A large number of IgG mAbs specific to the protein of interest could be produced. The results indicate that the generated hybridomas produce corresponding antibody isotypes as expressed on the surface of their starting cells. The technique that we have developed will be very useful for production of desired mAbs having a specific isotype.     

Genetic and Non-Genetic Inheritance of Natural Antibodies Binding Keyhole Limpet Hemocyanin in a Purebred Layer Chicken Line

Natural antibodies (NAb) are defined as antibodies present in individuals without known antigenic challenge. Levels of NAb binding keyhole limpet hemocyanin (KLH) in chickens were earlier shown to be heritable, and to be associated with survival. Selective breeding may thus provide a strategy to improve natural disease resistance. We phenotyped 3,689 white purebred laying chickens for KLH binding NAb of different isotypes around 16 weeks of age. Heritabilities of 0.12 for the titers of total antibodies (IgT), 0.14 for IgM, 0.10 for IgA, and 0.07 for IgG were estimated. We also estimated high, positive genetic, and moderate to high, positive phenotypic correlations of IgT, IgM, IgA, and IgG, suggesting that selective breeding for NAb can be done on all antibody isotypes simultaneously. In addition, a relatively substantial non-genetic maternal environmental effect of 0.06 was detected for IgM, which may reflect a transgenerational effect. This suggests that not only the genes of the mother, but also the maternal environment affects the immune system of the offspring. Breaking strength and early eggshell whiteness of the mother’s eggs were predictive for IgM levels in the offspring, and partly explained the observed maternal environmental effects. The present results confirm that NAb are heritable, however maternal effects should be taken into account.     

Anti-dsDNA Antibody Isotypes in Systemic Lupus Erythematosus: IgA in Addition to IgG Anti-dsDNA Help to Identify Glomerulonephritis and Active Disease

Objectives

To evaluate the role of serum IgG, IgM and IgA anti-dsDNA antibody isotypes in the diagnosis of systemic lupus erythematosus (SLE), and their association with clinical features and disease activity, in a large cohort of SLE patients.

Methods

Sera of 200 SLE patients (mean age 34±10.3 years; 26 male and 174 female; median duration of disease 115 months, range 7–378), and of 206 controls, including 19 Sjögren''s syndrome, 27 rheumatoid arthritis, 26 psoriatic arthritis, 15 idiopathic inflammatory myopathies (IIM), 13 systemic sclerosis, 49 infectious diseases and 57 healthy subjects, were tested for anti-dsDNA IgG, IgM and IgA isotypes.

Results

Selecting a cutoff corresponding to 95% specificity, the sensitivity of IgG, IgM and IgA anti-dsDNA antibodies in SLE was 55%, 30% and 49%, respectively; 12.5%, 1% and 7.5% of SLE patients had positive IgG, IgM or IgA isotype alone, respectively. SLE patients with glomerulonephritis showed higher levels of IgA anti-dsDNA (p = 0.0002), anti-dsDNA IgG/IgM (p = 0.001) and IgA/IgM (p<0.0001) ratios than patients without renal disease. No significant associations have been found between anti-dsDNA isotypes and other clinical features. IgA anti-dsDNA (p = 0.01) (but not IgG or IgM) and IgG/IgM ratio (p = 0.005) were significantly higher in patients with more active disease (ECLAM score >4).

Conclusions

The detection of IgA anti-dsDNA autoantibodies seems to improve our ability to diagnose SLE and to define lupus nephritis phenotype and active disease. By contrast, IgM anti-dsDNA antibodies might be protective for renal involvement. These data support the hypothesis that anti-dsDNA antibody class clustering may help to refine SLE diagnosis and prognosis.