Double-blind analysis of the relation between adult periodontitis and systemic host response to suspected periodontal pathogens.

By using a sensitive enzyme-linked immunosorbent assay, 200 randomly selected sera from Red Cross blood donors were screened for immunoglobulin G (IgG), IgA, and IgM levels against Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Bacteroides intermedius. A subgroup of 79 blood donors was clinically examined for type and extent of periodontal destruction, and serological and clinical data were subjected in all possible dual combinations to correlation analyses. The results revealed that the majority of the blood donors suffered from moderate to severe adult periodontitis, often coupled with severe gingival inflammation. No cases of localized juvenile periodontitis or rapidly progressive periodontitis were observed. The extent of periodontal destruction proved to be significantly correlated only to the IgG response levels against B. gingivalis. Corresponding correlation tests assessing the relationships of loss of attachment, bone loss, pocket depth, and papillary bleeding index with the IgG responses to A. actinomycetemcomitans were of marginal significance, while the IgG responses to B. intermedius revealed no relationship to the periodontal health status. The specific IgM responses proved to be unrelated to the clinical parameters, but interestingly, they were found to be highly correlated with each other. Specific IgA levels were frequently too low for enzyme-linked immunosorbent assay testing and, therefore, had to be exempted from statistical analyses. Assessments of the serotype specificity of strongly elevated IgG responses to A. actinomycetemcomitans disclosed no evidence for an association of a particular serotype-specific IgG response with the occurrence of adult periodontal destruction. In contrast to results of earlier studies, a number of sera were found to contain strongly elevated IgG levels against two or even all three serotypes. Although derived by an alternative approach, the reported results largely corroborate earlier observations linking only the occurrence of elevated anti-B. gingivalis IgG responses to the presence of marked periodontal lesions in adults.     

Diagnosis of influenza A virus infections by detection of specific immunoglobulins M, A, and G in serum.

The value of immunoglobulin M (IgM) detection in the early diagnosis of influenza A was examined in a prospective study during an outbreak of influenza A/Philippines/2/82 (H3N2) virus infection in February and March 1986. The IgM response was investigated in sera from 64 adults with influenzalike symptoms; we found a fourfold rise in titer or high titers (greater than or equal to 80) of influenza A virus antibodies by the complement fixation test. The IgM response, analyzed by the hemadsorption immunosorbent technique, was compared with the IgG and IgA responses analyzed by an indirect enzyme-linked immunosorbent assay and the hemadsorption immunosorbent technique, respectively. Antigen detection in nasopharyngeal secretions by immunofluorescence was performed for all patients on admission to hospital. Specific IgM was detected in 86% (55 of 64) of the patients with influenza A. In sera from 36% (18 of 64) of the patients it was detected already on admission. Influenza A virus antigen was detected in nasopharyngeal cells by immunofluorescence on admission in 53% (34 of 64) of the patients. A combination of immunofluorescence and IgM results gave a significantly higher diagnostic rate, 69% (P less than 0.01), on admission than did each of the two tests separately. An IgA serum antibody response was seen in 76% (48 of 64) of the cases but did not contribute to any increase in the diagnostic rate. IgM detection by the hemadsorption immunosorbent technique was found to be a valuable supplement for the diagnosis of influenza A in an early phase of the disease.     

Purification of immunoglobulins G by protein A/G affinity membrane chromatography

An affinity membrane grafted with protein A/G or protein A was characterized for human and mouse immunoglobulins G purification. Breakthrough curves up to ligand saturation were measured and used to study the effects of flow velocities, feed solution concentrations and protein A/G versus protein A membranes. Increased flow-rate did not decrease the amount of IgG bound to the membranes. Increased feed solution concentration allowed more IgG to bind prior to breakthrough. Kinetic parameters for immunoglobulins G sorption to immobilized protein A were measured in batch experiments. The static binding capacity was determined to be 6.6 mg ml(-1) membrane volume. Finally, this affinity membrane was used to purify IgG from cell culture supernatant. The electrophoresis of the purified IgG fractions did not show any contaminant.     

Serum and cerebrospinal fluid immunoglobulins M, A, and G in Japanese encephalitis.

A comparison was made of virus-specific immunoglobulin M (IgM), IgA, and IgG detected by capture or indirect enzyme immunoassay in serum and cerebrospinal fluid of patients with Japanese encephalitis. The IgM capture enzyme immunoassay was more sensitive than assays for other isotypes of viral antibody; IgM was detected in 75% of specimens collected less than or equal to 4 days after the onset of illness. Specific IgA was detected in both serum and cerebrospinal fluid; however, IgA levels were significantly lower than IgM levels.     

Degradation of human immunoglobulins G and M and complement factors C3 and C5 by black-pigmented Bacteroides

Strains of Bacteroides, Capnocytophaga and Fusobacterium were examined by immunological methods for their ability to degrade the human serum proteins IgG, IgM, C3 and C5. The proteolytic activity of the strains was measured in terms of the breakdown of serum into trichloroacetic acid-soluble material. Only black-pigmented Bacteroides strains showed proteolytic activity. Strains of B. gingivalis degraded IgG, IgM, C3 and C5, strains of B. intermedius IgG and C3, strains of B. endodontalis C3 and IgG and a strain of B. loeschei degraded only IgG. These findings are discussed in relation to the pathogenicity of the black-pigmented Bacteroides.     

Detection of antirotavirus immunoglobulins A, G, and M in swine colostrum, milk, and feces by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was developed to allow direct detection of class-specific antirotavirus antibodies. In colostrum and in milk, antirotavirus antibodies were found in the three immunoglobulin classes. Antirotavirus immunoglobulins G and M were predominant in colostrum, whereas antirotavirus immunoglobulin A was predominant in milk and feces.     

Measurement of immunoglobulins G, A, and M levels in B-lymphocytes culture

Nephelometric immunoassays were developed for human IgG, IgA, and IgM quantitation in B-lymphocytes culture media. They allowed measurement of immunoglobulin (Ig) levels over a broad range of concentrations with good accuracy and precision. The kinetics of Ig production in B-lymphocyte cultures was followed and the mean amount of each Ig was determined in six different samples after three days of culture. The nephelometric immunoassays reported here could be used to study, in vitro, the influence of various molecules (inhibitory or amplifying effect) on B-lymphocytes' functional capacities.     

Experimental hypersensitivity pneumonitis: serum immunoglobulins G1, G2, M, A and E in Micropolysopra faeni sensitized and desensitized calves

Weekly evaluation of serum antibody activity in IgA, IgM, IgG1 and IgG2 as well as serum IgE levels indicates highly significant reduction in IgE and elevation of IgA in calves experimentally desensitized with Micropolyspora faeni soluble antigen. Serum IgG2, IgG1 and IgM levels were also elevated. Results are discussed in the light of the possible significance of reagin-mediated reactions in initiation of hypersensitivity pneumonitis.     

Immunoblot analysis of Toxoplasma gondii antigens by human immunoglobulins G, M, and A antibodies at different stages of infection.

The Toxoplasma gondii antigenic components eliciting the immunoglobulin G (IgG), IgM, and IgA antibody responses were studied by using follow-up sera from a laboratory worker who developed an acute glandular toxoplasmosis after an accidental infection with the protozoa. IgG toxoplasma antibodies reacted with multiple components over a wide molecular weight range from 6,000 to 150,000. In contrast, IgM toxoplasma antibodies reacted predominantly with polypeptides of 6, 25, and 35 kilodaltons, which might be useful in new diagnostic procedures. The general pattern of antigenic components in the IgA toxoplasma antibody response closely resembled that in the IgM response, even though some characteristic features were constantly observed. The possibility that the restricted IgM and IgA antibody responses relate to the pathogenetic events in human toxoplasmosis is considered.     

Detection of immunoglobulins G and M to rubella virus by time-resolved immunofluorometry.

We describe new methods for the detection of immunoglobulin G (IgG) and IgM rubella-specific antibodies in serum. The IgG assay was based on a solid-phase rubella antigen immobilization approach, and the IgM assay was based on the IgM capture assay principle. Both assays used biotinylated antibodies (anti-human IgG and antirubella monoclonal antibody, respectively). The tracer system was based on streptavidin labeled with a fluorescent europium chelate. The final measurements were done by using time-resolved fluorescence. Both assays were thoroughly evaluated with clinical samples and compared successfully with established techniques. We anticipate that these assays are suitable for routine clinical use.     

Differential binding properties of protein A and protein G for dog immunoglobulins.

We studied the binding of dog immunoglobulins G, A, M and E to protein A and protein G. Passive cutaneous anaphylaxis (PCA) testing was used for the measurement of dog IgE and enzyme-linked immunosorbent assays (ELISA) were used for the measurements of dog IgG, IgA and IgM. Protein A from lyophilized cells of Staphylococcus aureus bound 97% of IgE, 98% of IgG, 81% of IgA, and 97% of IgM. Protein A-Sepharose CL-4B bound 87% of IgE, 100% of IgG and IgA, and 98% of IgM. In a stepwise elution with varying pH, a small amount of IgE was eluted at pH 5 and pH 6 and all the remaining Igs were eluted at pH 3 from the protein A column. In contrast to protein A, dog IgE was not bound to Protein G-Sepharose, while 100% of IgG, 95% of IgA, and 44% of IgM were bound to Protein G-Sepharose.     

Antiviral activity of colostrum and serum immunoglobulins A and G

Enteric virus-specific IgA and IgG present in paired human sera and colostrums were measured by the enzyme-linked immunosorbent assay (ELISA). Virus-specific IgA was present in all colostrums, but virus-specific IgG could not be detected. The reverse was true when sera were assayed. Most of these colostrums also neutralized either polio virus or reovirus, as did IgA, which was separated from a pool of colostrums by exclusion chromatography. No correlation could be made between levels of neutralizing and ELISA antibody titers in colostrums.     

Role of immunoglobulins G1 and G2 in anaphylactic shock in the guinea pig

Heating serum from actively sensitised guinea pigs did not remove its ability to sensitise recipient animals in vivo and parenchymal lung strips in vitro to anaphylaxis. Thermoresistant antibodies should thus account for the transferable sensitising effect, which persists for at least 9 days. IgG1 and IgG2, contained in the serum, were separated by affinity chromatography to determine the importance and the participation of these subclasses in passive anaphylactic shock. IgG1, present in smaller amounts than IgG2, was more effective in sensitising isolated lung strips. The intravenous administration of ovalbumin to guinea pigs, which had been injected with 0.8 mg/kg of IgG1 or 2 mg/kg of IgG2 9 days beforehand, induced an intense bronchoconstriction with leucopenia and moderate thrombopenia, suggesting an as yet undescribed role for IgG2 in passive tissue sensitisation. The use of mepyramine, an antagonist of the histamine H1 receptor, WEB 2086, an antagonist of platelet-activating factor, and nordihydroguaiaretic acid, a dual inhibitor of cyclooxygenase and lipooxygenase, alone or associated, demonstrated that the anaphylactic contraction of lung strips from guinea pigs sensitised by IgG1 is mediated by histamine and arachidonate derivatives, whereas that of lung strips from guinea pigs sensitised with IgG2 is mostly mediated by histamine. In addition, the association of the three potential antagonists slightly reduced the anaphylactic contraction of lung strips provided by guinea pigs sensitised by serum. Our results, using a sensitisation procedure considered until now to involve exclusively IgE antibodies, indicate that IgG1 and IgG2 are in fact the essential antibodies for passive anaphylactic shock in the guinea pig.     

Contribution of immunoglobulins M and G, complement, and properdin to the intracellular killing of Escherichia coli by polymorphonuclear leukocytes.

The effect of immunoglobulins and complement (C) on phagocytosis and intracellular killing of Escherichia coli was studied in vitro. The incubation system consisted of monolayers of human polymorphonuclear leukocytes and C-resistant, [3H]thymidine-labeled E. coli C source was human serum deprived of immunoglobulins and properdin by immunoabsorption. In the absence of C, only immunoglobulin G-coated bacteria were phagocytosed, whereas immunoglobulin M lacked opsonic activity. In the presence of C, phagocytosis was enhanced; however, immunoglobulin M was now more efficient than immunoglobulin G. Intracellular killing was notably augmented when C was activated by immunoglobulin G- or immunoglobulin M-coated bacteria; in contrast, the alternative activation of C by properdin had no effect on phagocytosis or intracellular killing. These results demonstrate the importance of immunoglobulins together with C not only for phagocytosis but also for efficient intracellular killing.     

Multiple protein extract microarray for profiling human food-specific immunoglobulins A, M, G and E

Existing food immunoglobulin (Ig) tests require large volumes of serum, are limited to one immunoglobulin class, are not amenable to high throughput analysis and only give a limited picture of the immunological response to food antigens. Conversely a new generation of Component Resolved Diagnostic systems using pure proteins is highly specific and totally dependent on the availability of the protein in its recombinant or natural origin form. Here we demonstrate a proof-of-concept of a microarray test based on protein extracts of food components. Our approach relies on innovations on three different fronts: the novelty of using arrayed food samples sequentially extracted with detergent and chaotropic agents, the ability to measure four different Ig classes simultaneously and the ability to analyse the generated data via a suitable bioinformatics/statistical analysis interface. This approach combines high numerical power of microarrays with automation, high throughput analysis and enables detailed investigation of the Ig profiles to food antigens. The prototype shown contains extracts of approximately 350 food ingredients that cover most of the food products found in the UK. Here we showed that the use of a sequential extraction technique to solubilise and then denature food samples has its benefits in the assessment of variations in antigenicity when tested with human sera. A patient dependent degree of class specificity was observed with human sera (IgG specificity correlates well with IgA>IgM>IgE). Besides generating a simultaneous profile for IgA, IgM, IgG and IgE the array system has shown good discrimination between challenge responders in atopic and non-atopic individuals. Poly- and mono-specific IgE responders were easily identified. The mathematical modelling of specific IgE content showed good correlations when compared with established IgE antibody testing assay (UniCAP). Although in its proof-of-principle stages, the immune profiling technique described here has the potential to provide unique insights into exposure/sensitization and establish relationships between specific immunoglobulin classes and subclasses against food protein antigens. In further developments, the immune profiling technique could also be extended to other related areas such as parasite and bacterial gut infection. Full analyses of large longitudinal and retrospective clinical trials are on going to determine the positive and negative predictive values of the technique.     

Participation of normal human immunoglobulins M, G, and A in opsonophagocytosis and intracellular killing of Bacteroides fragilis and Bacteroides thetaiotaomicron by human polymorphonuclear leukocytes.

Restoration of the ability of hypogammaglobulinemic serum to support opsonophagocytosis and intracellular killing of Bacteroides fragilis and Bacteroides thetaiotaomicron by human polymorphonuclear leukocytes was achieved by supplementation with normal human immunoglobulin M, but not with normal human immunoglobulin G. Polymorphonuclear leukocyte bactericidal activity in the presence of immunoglobulin A-deficient human serum was equivalent to that observed in the presence of normal human serum.     

Selective reactivity of antibodies to human immunoglobulins G, M, and A with rubella virus proteins

Proteins of purified rubella virus were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with human sera and immunoglobulin class heavy-chain-specific peroxidase conjugates. The levels of rubella antibodies in these sera were predetermined by the radial hemolysis test, the density gradient centrifugation method for immunoglobulin M (IgM) antibodies, and IgG-, IgM-, and IgA-specific enzyme immunoassays. In immunoblotting, rubella-specific IgG antibodies reacted with both envelope glycoproteins (E1 and E2) and the capsid protein (C). In contrast, rubella IgM antibodies reacted predominantly with E1, whereas the specific reactivity of IgA antibodies was directed mainly to the capsid protein. Purified IgM rheumatoid factor added to IgG-positive, IgM-negative serum did not give false-positive reactivity in the immunoblotting test as it did in solid-phase enzyme immunoassays. The immunoglobulin class-specific reactivities with the different viral proteins are expected to have diagnostic applications.     

Serological diagnosis of Toxoplasma gondii infections by rapid separation of serum immunoglobulins M and G with CM Bio-Gel A.

A simple and rapid method has been developed for the separation of serum immunoglobulin M (IgM) and IgG. CM Bio-Gel A chromatography was used in the technique, which resulted in an IgM-rich fraction containing 31% of the original serum IgM and less than 2% of the serum IgG. The procedure was used to detect masked IgM antibodies in patients suspected of having Toxoplasma gondii infections.