Class IgG,IgM and IgA antibodies againstStaphylococcus aureus antigens in human serum and saliva

Using the ELISA method antibodies against the sonicate, teichoic acid (TA) and exoproducts ofStaphylococcus aureus were determined in sera and saliva of healthy individuals. Main serum antibodies against all the antigens used were shown to be class IgG antibodies. However, antigens of the sonicate stimulated significantly even the systemic IgA response. In the saliva class IgA antibodies predominated, but IgG antibody levels against TA and exoproducts approached the level of IgA antibodies. Levels of IgM antibodies against all antigens tested were low in both the serum and saliva which corresponds with the anamnestic type of response. On the basis of these results one may assume that not only IgG, but also IgA antibodies are important in the systemic immunity against staphylococcal infection and in the immunity of mucous membranes; besides IgA, even class IgG antibodies play an important role.     

The low-molecular protein of the cell wall in streptococci group A devoid of serological type specificity]

The scheme for the isolation and purification of low-molecular cell-wall protein without type specificity, including the extraction of the cell walls of group A streptococci, type M 29, with 1% solution of Triton X-100, the separation of the extract by ion-exchange chromatography in DEAE-trisacryl M with the subsequent two-stage gel filtration in superfine Sephadex G-50, is described. The isolated protein had a molecular weight of 4,000 daltons and contained no admixtures of group-specific polysaccharide A, phosphorus, nucleic acids and Fc receptors and interacted with antisera to group A streptococcal cells of heterologous type M in the enzyme immunoassay (EIA). Purified protein was characterized by a high content of glycine. The antigenic determinants of immobilized protein, recognized by antibodies in EIA, were sensitive to the action of trypsin and resistant to the action of pepsin, papain, pronase E and sodium periodate.     

Protective efficacy of IgM monoclonal antibodies in experimental group B streptococcal infection is a function of antibody avidity

We have produced and characterized six mAb directed against group B streptococci (GBS). All antibodies are IgM. We have previously shown that some of these antibodies are highly protective in the treatment of experimental infections in neonatal rats, whereas others do not appear to have any protective efficacy. Using an ELISA, we demonstrate the specificity of both protective and nonprotective antibodies. Two antibodies, binding different epitopes, are directed against antigenic structures present on all GBS; two are specific for type III carbohydrate determinants; one binds to a protein Ag present on all type I and II GBS; and one appears to bind to type Ia GBS only. Quantitative absorption assays provide evidence that the difference between protective antibodies and nonprotective antibodies is the avidity that the antibody demonstrates for the epitope recognized on the surface of the bacteria; 10 to 15 times as much protective antibody binds to GBS as does nonprotective antibody. Direct binding experiments with radiolabeled antibody confirm this conclusion.     

Resistance of normal serum IgA and secretory IgA to bacterial IgA proteases: evidence for the presence of enzyme-neutralizing antibodies in both serum and secretory IgA, and also in serum IgG

Normal serum IgA and secretory IgA (sIgA) of subclass IgA1 were isolated from pooled human serum and milk, respectively. They were tested for their susceptibility to bacterial IgA proteases from Haemophilus influenzae, Streptococcus pneumoniae, Neisseria gonorrhoeae, and Neisseria meningitidis that cleave IgA of only the IgA1 subclass. They were also tested for susceptibility to a novel IgA-protease from Clostridium ramosum that cleaves IgA of the IgA1 as well as the IgA2 subclass of the A2m(1) allotype. Both normal serum IgA1 and sIgA1 exhibited resistance to most IgA proteases. The one exception was the IgA protease from C. ramosum which readily cleaved both the serum IgA1 and sIgA1 into Fab and Fc fragments. Secretory component (SC) had nothing to do with the resistance of these IgAs. The resistance of these IgAs to most of the IgA proteases was found to be due to their enzyme-neutralizing antibody activity, since the Fab but not the Fc fragment of sIgA1 showed enzyme-inhibitory activity against these IgA proteases. Similar enzyme-neutralizing antibody activity was found in the pepsin-digested normal serum IgG-(Fab')2 fragment. These results indicate that the induction of the enzyme-neutralizing antibodies against the bacterial IgA proteases took place not only in mucosal sIgA but also in serum IgA and IgG. No enzyme-neutralizing antibody activity against the novel IgA-protease of C. ramosum was detected in any immunoglobulin preparations used in the present study or in the serum of a patient who carries the IgA protease-producing strain of C. ramosum in his feces.     

Changes in virulence, M protein and IgG Fc receptor activity in a type 12 group A streptococcal strain during mouse passages

A type 12 group A strain (1800) was passaged serially through mice 25 times. The ability to servive in normal human blood dropped from a growth index of 52 after the first passage to 1 after four passages. After 14 passages the growth index increased again and stabilized above 30. The virulence for mice increased from a LD100 of 10(8) colony forming units (CFU) to 10-100 CFU after 7 passages and then remained constant. The Mqw antigen disappeared after 4 passages as tested by immunodiffusion, electroimmunoassay and indirect bactericidal tests. Three antisera, raised in rabbits against strains originally belonging to types M3, M12 and M46 but devoid of type antigens after mouse passages showed high bactericidal indices against the 1800 strain after 14 or more passages on mice. Anti-type M1 serum was also found bactericidal for the passaged strains. The IgG Fc-receptor activity of the strain isolated after each mouse passage was tested in hemagglutination experiments with human red blood cells coated with "incomplete" anti-Rh and hot hydrochloric acid extracts of the strains. The capacity to agglutinate "Ripley"-coated cells increased gradually during the first 12 passages and subsequently the titres of the extracts stabilized between 1:160 and 1:320. The HUN coat, useful for detection of the G3m (5) maraker gave titraes increasing with the number of passages while the titres for IgG1 coats kept at 1:4 or below. On background of these results, the possible role of the IgG Fc-receptor as a virulence factor is discussed.     

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.     

T15 group A streptococcal Fc receptor binds to the same location on IgG as staphylococcal protein A and IgG rheumatoid factors

Previous work has shown that IgG rheumatoid factors (RF) bind to the C gamma 2-C gamma 3 interface region of human IgG in the same area that binds staphylococcal protein A (SPA). Group A, C, and G strains of Streptococci possess Fc receptors that bind to IgG but not to fragments containing only the C gamma 2 or C gamma 3 domains. This work describes the binding site location on human IgG for the binding of the isolated Fc receptor from the T15 strain of a Group A streptococcus and its relationship to the site that binds SPA and the IgG RF. The isolated T15 Fc receptor (T15) with a molecular mass of 29.5 kD inhibited the binding of IgG RF to IgG. The binding of T15 itself to IgG was strongly inhibited by SPA (42.0 kD) and its monovalent fragment D (7 kD). Human IgG fragments consisting of the C gamma 3 domains did not inhibit the binding of T15 to IgG, whereas those with both domains were effective inhibitors. T15 did not bind to rabbit IgG fragments consisting of either the C gamma 2 or C gamma 3 domains, but did bind to those with both domains. An IgG3 myeloma protein was a poor inhibitor and has been shown to bind poorly to the IgG RF. Most IgG3 myeloma proteins did not bind to SPA. The substitution of Arg and Phe for His 435 and Tyr 436 is responsible for the poor binding of IgG3 to SPA and to the IgG RF. Chemical modification of His or Tyr on IgG reduced its ability to inhibit the binding of T15 to IgG. Reversal of the chemical modifications with hydroxylamine resulted in near complete restoration of inhibitory capacity. This information, collectively, coupled with the known positions in space of the His and Tyr residues in the C gamma 2-C gamma 3 interface region, verified that both His 435 and Tyr 436, and possibly His 310 and 433, are involved. These residues are also involved in binding SPA and the IgG RF. These data therefore indicate that the T15 Group A Streptococcal Fc receptor binds to the same location on the Fc of IgG as SPA and the IgG RF. The biologic relevance of these similarities between bacterial cell wall Fc receptors and IgG RF are not yet apparent, but suggest that RF could bear the internal image of these bacterial structures.     

Monoclonal antibodies to streptococcal group A carbohydrate. II. The VK1GAC light chain is preferentially associated with serum IgG3

A kappa-light chain variable region (V kappa) dominantly employed in the serum antibody response of A/J mice to streptococcal group A carbohydrate (GAC) has been termed VK1GAC. Examination of in vitro recombinants between the isolated heavy and light chains of VK1GAC+ and VK1GAC-anti-GAC hybridomas and non-GAC-binding myeloma proteins indicated that two antisera (anti-Id5 and anti-Id20) recognized the VK1GAC light chain when it was free in solution or paired with several heterologous heavy chains. Screening of a panel of A/J anti-GAC monoclonal antibodies with these antisera showed almost complete concordance between Id5 and Id20 expression and the presence of VK1GAC light chain as detected by its unique isoelectric focusing spectrotype. These antisera were used to examine serum expression of the VK1GAC light chain in normal and hyperimmune serum of A/J mice. Normal A/J serum contained from 20 to 100 micrograms Id5/ml serum, whereas only 1 to 10 micrograms Id20/ml serum was detected. The levels of both VK1GAC idiotypes increased dramatically 10- to 20-fold after hyperimmunization of mice with group A vaccine. When serum IgG from normal and immune mice was fractionated into the IgG subclasses (IgG1, IgG2a, and IgG3), it was found that the VK1GAC light chain does not pair randomly with heavy chains of the IgG subclasses, but rather is associated preferentially with heavy chains of the IgG3 subclass whether or not it is associated with antibodies to GAC. These results suggest that the heavy chain pairing exhibited by this VK product may not be random.     

Causal analysis of the variability of IgA, IgG, and IgM immunoglobulin levels

The interindividual variability of IgA, IgG, and IgM immunoglobulin levels was studied using path analysis in a northeastern Brazilian sample (nuclear families) to determine the genetic and/or environmental causes of their variation. The path analysis model decomposes the phenotype into genetic causes (autosomal and X-chromosome-linked genes) and environmental causes. A significant familial aggregation, mainly resulting from autosomal components, was detected for the 3 immunoglobulin levels. The values of genetic heritability were h2 = 0.410 +/- 0.030 for IgA, h2 = 0.617 +/- 0.020 for IgG, and h2 = 0.540 +/- 0.023 for IgM, and the values for environmental-cultural heritability were c2 = 0.085 +/- 0.034 for IgA, c2 = 0.084 +/- 0.027 for IgG, and c2 = 0.023 + 0.023 for IgM. Our results did not show a heritable component resulting from X-chromosome-linked genes on IgM levels, as suggested by some studies (Wood et al. 1969; Grundbacher 1972; Purtilo and Sullivan 1979). Some additional results were that (1) age and IgA concentration were positively correlated, with IgA level increasing gradually from childhood to adulthood (p < 0.001); (2) sex and the age X sex interaction act on IgG concentration (p < 0.01); (3) age and IgM concentration are correlated (with children presenting lower levels than adults, especially in males, p < 0.01); and (4) a significant association exists between sex and IgM level (with females presenting higher levels than males, p < 0.001).     

Detection of autoreactive antibodies to serum IgG and IgA in amoebic liver abscess cases.

Assessment of autoreactive antibodies in response to healthy human serum IgA and IgG was performed by indirect haemagglutination assay on serum samples from 81 amoebic liver abscess cases for IgA and 70 for IgG. Appropriate controls were taken simultaneously. IgA, IgG were isolated and purified from a healthy human serum through Sephadex G-200 and protein A CL 4B sepharose chromatography. These immunoglobulins were used for the detection of its own antibodies in amoebic liver abscess cases. This revealed that 43.20% and 48.50% of the cases were positive for IgA and IgG respectively, where as only 19.35% and 28.30% of the controls were in positive category (IgA and IgG respectively). The mean titres with standard deviation of the autoreactive antibodies to serum IgA both in ALA cases and controls shows a highly significant difference between tests and controls (P less than 0.001). Similarly the mean titres with standard deviation both in ALA and controls for the serum IgG differed significantly (P less than 0.001). This suggests the presence of autoreactive antibodies against serum IgA and IgG in amoebic liver abscess cases.