Oral immunization with Streptococcus mutants in rhesus monkeys and the development of immune response and dental caries.

The oral route of immunization with Streptococcus mutants was compared with the subcutaneous route in rhesus monkeys. Significant levels of serum IgG, IgM and IgA antibodies in Strep. mutans were elicited only in monkeys immunized subcutaneously. Similarly, the skin delayed hypersensitivity reaction to Strep. mutans was elicited only in the subcutaneously immunized monkeys. Oral immunization induced a modest increase in salivary IgA antibodies to Strep. mutans, though a slight increase in IgA antibodies was also found in the saliva of all other groups of immunized and control monkeys. A small though not significant reduction in dental caries was found in the monkeys immunized orally, whereas subcutaneous immunization with Strep. mutans consistently elicited a significant reduction in caries. Oral feeding of Strep. mutans failed to induce tolerance to a subsequent subcutaneous challenge by the same organism. Furthermore, sequential subcutaneous followed by oral immunization had little effect on the titre of salivary or serum antibodies.     

The use of monoclonal antibodies in (reverse) passive haemagglutination tests for herpes simplex virus antigens and antibodies

Three monoclonal antibodies against herpes simplex virus type 2 have been tested for their suitability as reagents in reverse passive haemagglutination. Two of these antibodies with specificity for virus glycoprotein D, when linked to red blood cells, were able to capture antigens without being agglutinated, but addition of immune serum subsequently led to agglutination. Haemagglutination using these monoclonal antibody-linked, antigen-captured red cells was readily applicable to testing human sera for antibodies to herpes simplex virus and the titres obtained correlated with those from virus plaque neutralisation tests. The procedure has been termed "Specific Antigen Capture Passive Haemagglutination." A further monoclonal antibody with specificity for the major DNA-binding protein of type 2 herpes virus-infected cells (a nonstructural protein) showed conventional reverse passive haemagglutination when linked to red blood cells and was specific for type 2 herpes simplex virus. The nature and potential uses of these simple reverse passive haemagglutination procedures using monoclonal antibody reagents are discussed.     

Bacterial and strain specificities in opsonization, phagocytosis and killing of Streptococcus mutans.

Opsonization of Streptococcus mutans, followed by phagocytosis and killing by polymorphonuclear leucocytes has been postulated as an effector mechanism in protection against dental caries. Opsonization was studied by using sera from monkeys immunized with killed Strep. mutans (sero-type c) and compared with sera from sham-immunized monkeys. Antibodies to Strep. mutans (sero-type c) induced maximal phagocytosis and killing of serotypes c and e, and this was significantly greater than with serotypes a and d; there was no significant phagocytosis or killing of serotype b. There was little or no opsonization with Actinomyces viscosus, Lactobacillus casei, Strep, sanguis and Strep. salivarius. The exception was Strep. CHT which showed significant phagocytosis and killing. The results suggest that immunization with the serotype c strain of Strep. mutans might offer protection against four of the five common serotypes of this organism.     

Detection of protein A-like substances on haemolytic streptococci prior to use in mixed reverse passive antiglobulin haemagglutination (MRPAH).

Before mixed reverse passive antiglobulin haemagglutination tests (MRPAH) can be used to measure the class of bacterial antibodies, the bacteria have to be shown to be free of Protein A or Protein A-like substances on their surfaces. Two basic procedures have been examined: haemagglutination of red cells coated with immunoglobulin by the bacteria, and the MRPAH reaction itself to reveal absorption of purified gamma Fc by the bacterial suspension. The use of a purified gamma Fc component has proved successful in providing a sensitive test for the detection of Protein A-like substances on the surface of bacterial. In addition to both the Cowan and Wood strains of Staph, aureus, strains of haemolytic streptococci of groups A, C and G had Protein A-like substances on their surfaces. In contrast, strains of group B and group D, as well as Strep. milleri, had no detectable Protein A-like activity.     

Detection and quantitation of rotavirus using monoclonal antibody coupled red blood cells: comparison with ELISA

A total of 125 faecal extracts from infants were tested by reverse passive haemagglutination (RPH) using red cells coated with a monoclonal antibody against the major group-specific rotavirus antigen (VP 6). Results were compared with those obtained using a rabbit anti-rotavirus capture, guinea pig anti-rotavirus detector-based ELISA. The specificity of the assay was confirmed by use of 'normal' immunoglobulin coupled red cells and by inhibition with rabbit antiserum. The antibody-coated red cells could be stabilised by treatment with glutaraldehyde and subsequent freeze-drying with no detectable loss of activity even after storage at 45 degrees C for 4 wk. Good correlation was obtained between RPH and ELISA. Purified bovine rotavirus could be detected by RPH down to approximately 10(5) particles in a 25 microliters vol. Similar results were obtained with polyclonal antibody coupled cells and an ELISA using monoclonal antibody. Experiments using subgroup-specific monoclonal antibodies indicated the feasibility of rapid subgroup determination.     

Human serum antibody response against Streptococcus mutans antigens.

Antigens from Streptococcus mutans were examined to identify specific polypeptides that may have stimulated antibody responses and possibly play some role in caries immunity. A group of 10 adult human subjects was screened for serum antibodies reactive with antigens from S. mutans. Extracellular and cellular protein preparations from S. mutans LM7 (Bratthall serotype e) and V403 (biotype c) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western electrophoretic transfer and immunoblotting analysis. Antibodies reactive with polypeptides ranging from 34 to 400 kilodaltons in apparent molecular mass were detected by these means. Radioimmunoassay competition experiments revealed that the cellular and extracellular antigens did not compete with each other for serum antibodies. Preabsorption of sera with extracellular proteins from other oral streptococcal species prior to immunoblotting indicated that the antigens unique to S. mutans have molecular masses greater than 100 kilodaltons, and each individual produced antibodies against different antigens of high molecular mass. Examination of sera from young children also indicated heterogeneous responses against S. mutans LM7 antigens.     

A modified passive-haemagglutination technique for the detection of cytomegalovirus and herpes simplex virus antibodies: application in virus-specific IgM diagnosis

Cytomegalovirus (CMV) and herpes simplex virus (HSV) antibodies were detected by a modified passive haemagglutination (PHA) technique. The main features of this modification are the use of a simpler method for the removal of nonspecific sheep agglutinins in the sera, the deployment of commercially available CMV and HSV antigens, and a different sucrose density gradient (SDG) system for the separation of IgM from IgG. The modified procedure proved to be a reliable, specific, and sensitive technique in detecting antibodies to CMV and HSV in both whole serum and in the separated IgM and IgG fractions. It was as reliable as the complement-fixation (CF) test when applied in seroepidemiological studies and in the detection of antibodies in cord serum. Preliminary data are provided which suggest that the combination of SDG and PHA may prove to be a more reliable system for the detection of exclusion of CMV-specific IgM than an enzyme-linked immunosorbent assay (ELISA).     

Serum glucosyltransferase-inhibiting antibodies and dental caries in rhesus monkeys immunized against Streptococcus mutans.

Serum antibodies to glucosyltransferase (GTF) of Streptococcus mutans serotype c were assayed sequentially by means of an enzyme inhibition radio-assay in twenty-six Rhesus monkeys immunized with S. mutans. Pre-immune and control sera had a GTP-enhancing effect which was shown also by albumin and non-immune immunoglobulin fractions. GTF-inhibitory activity was found in IgG fractions from some immune sera and could be absorbed by S. mutans cells possessing cell-bound GTF. Inhibitory antibodies to GTF developed in the sera of four monkeys immunized with hydroxylapatite extract of culture supernatant (HACS), and in four out of fifteen monkeys immunized with S. mutans cells, but in none of the seven sham-immunized control animals. The monkeys immunized with HACS showed no reduction in caries. A correlation has been demonstrated between protection against caries and the early development of serum IgG antibodies to antigens present in HACS but there was no consistent association between protection against caries and GTF-inhibitory antibodies. The results also suggest the possibility that other antibodies, possibly present in the IgM or IgA fractions and having an enhancing effect on GTF, may increase the incidence of caries.     

Redirecting the humoral immune response against Streptococcus mutans antigen P1 with monoclonal antibodies

The adhesin P1 of Streptococcus mutans has been studied as an anticaries vaccine antigen. An anti-P1 monoclonal antibody (MAb) bound to S. mutans prior to mucosal immunization of mice was shown previously to alter the amount, specificity, isotype, and biological activity of anti-P1 antibodies. The present study was undertaken to screen this and four additional anti-P1 MAbs for immunomodulatory activity when complexed with S. mutans and administered by a systemic route and to evaluate sera from immunized mice for the ability to inhibit adherence of S. mutans to immobilized human salivary agglutinin. All five MAbs tested influenced murine anti-P1 serum antibody responses in terms of subclass distribution and/or specificity. The effects varied depending on which MAb was used and its coating concentration. Two MAbs promoted a more effective, and two others a less effective, adherence inhibition response. An inverse relationship was observed between the ability of the MAbs themselves to inhibit adherence and the ability of antibodies elicited following immunization with immune complexes to inhibit adherence. Statistically significant correlations were demonstrated between the levels of anti-P1 serum immunoglobulin G2a (IgG2a) and IgG2b, but not of IgG1 or IgG3, and the ability of sera from immunized animals to inhibit bacterial adherence. These results indicate that multiple anti-P1 MAbs can mediate changes in the immune response and that certain alterations are potentially more biologically relevant than others. Immunomodulation by anti-P1 MAbs represents a useful strategy to improve the beneficial immune response against S. mutans.     

Detection of Antisperm Antibodies on the Surface of Motile Spermatozoa. Comparison of the Immunobead Binding Technique (IBT) and the Mixed Antiglobulin Reaction (MAR)

ABSTRACT: Investigators testing for antisperm antibodies have recently focused on tests that detect the Ig classes of the sperm-bound antibodies. The aim of this study was to compare the sensitivity of two of these tests, viz. the immunobead binding technique (IBT) and the mixed antiglobulin reaction (MAR). Twenty-one male or female sera were tested for IgG and IgA antisperm antibodies with the IBT and the MAR. The sera were selected on the basis of the IBT results, and the MAR was carried out without knowledge of these results. For IgG antisperm antibodies, there was a highly significant correlation between the two tests (P = 0.0043), whereas, for IgA antisperm antibodies, the correlation was poor (P = 0.2951), because the IBT revealed a positive reaction for IgA in sera in which no such antibodies could be detected by the MAR.     

Preparation of anti-polysaccharide antiserum using liposomes as immunological adjuvant

Bovine serum albumin (BSA) was coupled to dextran by controlled periodate oxidation followed by sodium borohydride reduction. The conjugate was entrapped in negatively charged, multilamellar phosphatidylcholine (lecithin) liposomes in which the polysaccharide remained surface-exposed, at least partially. Liposome-entrapped conjugate elicited in rabbits an appreciable anti-dextran response when compared with the sera raised in saline or in Freund's adjuvant. The anti-dextran antibodies belonged to both the IgM and IgG classes. The precipitin reaction of dextran with the antiserum raised in liposomes was determined.     

The use of monoclonal antibodies to differentiate isolates of herpes simplex types 1 and 2 by neutralisation and reverse passive haemagglutination tests

Monoclonal antibodies specific for herpes simplex type 1 or type 2 were used in reverse passive haemagglutination tests or infectivity neutralisation tests to serotype 100 isolates of herpes simplex virus (HSV). All isolates were independently serotyped by measuring their sensitivity to bromovinyl deoxyuridine. Reverse passive haemagglutination tests with type-specific antibodies directed against the HSV glycoprotein D and major DNA binding protein gave results in perfect agreement with the results of drug-sensitivity measurement. A single isolate behaved anomalously in the neutralisation test with a type 1-specific antibody directed against glycoprotein A/B. Restriction-enzyme analysis of virus DNA suggests that this isolate contains a variant glycoprotein A/B. The two methods used for serotyping proved very sensitive, giving adequate results with samples containing as little as 100 plaque forming units (pfu) of HSV. The reverse passive haemagglutination test has the additional advantages of speed and simplicity.     

Anti-IgG antibodies in rheumatic diseases cross-react with Streptococcus mutans SR antigen.

We have previously shown that SR protein, a S. mutans major cell wall protein, as well as the recombinant protein SR (rSR) share common epitopes with human IgG. Since this antigenic mimicry could play a role in the induction of anti IgG, we have examined, in k-ELISA, the presence of antibodies reacting with S. mutans SR proteins and S. mutans whole cells in sera from 36 patients with rheumatic diseases. The majority of the 36 sera showed a high reactivity with rSR when compared with control sera. Eight highly positive sera were further purified on rSR and human IgG sorbents and tested against both rSR and IgG in ELISA and Western blotting. The affinity-purified antibodies reacted strongly with rSR, IgG and IgG Fab fragments but failed to react with IgG Fc fragment. In Western blotting the addition of unlabelled IgG abolished the reactivity of affinity-purified biotinylated antibodies with all antigens, confirming the existence of a common epitope shared by rSR and human IgG heavy chain. We show the existence in rheumatic diseases of high titres of anti-human IgG antibodies cross-reactive with S. mutans SR proteins. Those antibodies are principally IgG and react with the Fd part of the Fab fragment. We can hypothesize from the above data that this antigenic mimicry existing between S. mutans SR-related antigens and human IgG could play a role in the synthesis of at least a part of the anti-IgG antibodies present in rheumatic diseases sera.     

Enzyme-linked immunosorbent assay (ELISA) for detecting antisperm antibodies in mice with testicular autoimmunity

We developed an ELISA for measuring antisperm antibodies in the mouse by using serum samples obtained from mice immunized with murine testicular antigens in complete Freund's adjuvant (CFA) as well as from mice rendered vasectomized. Sperm antigens used were syngeneic epididymal spermatozoa and two types of soluble, murine testicular antigens prepared in our laboratory. This study deals with a) the sequential changes of antisperm antibody levels following immunization; b) determination of immunoglobulin classes of these antibodies; c) a correlation between the absorbance values and the endpoint titers of antisperm antibodies; and d) comparison of endpoint titers of antisperm antibodies detected by ELISA with those by immunoperoxidase staining method in immune and nonimmune sera. It is suggested that serum dilution as high as 1/800 or more is required for detecting antibody titers of immune sera, because nonimmune mouse sera reveal a definite, although low, level of absorbance value at a serum dilution of 1/400 or less.     

Auto-Antibodies to Tumour Necrosis Factor a in Healthy Humans and Patients with Inflammatory Diseases and Gram-Negative Bacterial Infections

A semi-quantitative immunoblotting method was developed to screen for serum auto-antibodies against tumour necrosis factor alpha (TNF alpha). Forty nitrocellulose strips containing identical amounts of human recombinant TNF alpha (rTNF alpha) were prepared for each set-up, and the anti-TNF alpha antibody immunoreactivities were scored according to the density of the resulting colour reaction. A significant number of sera from apparently healthy donors contained detectable auto-antibodies to TNF alpha (40%), while the strongest reaction was observed in 8%. A higher prevalence of anti-TNF alpha antibodies was found in sera from patients with Gram-negative bacterial septicaemia (66%), cystic fibrosis with chronic Pseudomonas aeruginosa lung infection (72%), and various rheumatic diseases (61%). The antibodies in sera from these patients belonged primarily to the IgG and IgM classes, the latter exhibiting the strongest response. Longitudinally collected serum samples from patients in septic endotoxin shock revealed that the anti-TNF alpha antibodies were induced initially during septicaemia, reaching maximum reactivities within the first week and returning to low or undetectable levels on days 9-20.     

A micro enzyme-linked immunosorbent assay for insulin antibodies in serum

A solid-phase micro enzyme-linked immunosorbent assay for the measurement of insulin antibodies in serum is described and its performance compared with that of an established radiobinding assay. Interassay precision in the ELISA was 10% or less at widely spaced points on the dilution curves for human, porcine and bovine insulins. Specificity was demonstrated by substituting purified human gamma-globulin for the test serum and glucagon for the insulin. The influence on ELISA of endogenous insulin in the test serum was examined by measuring antibody binding before and after extraction of the insulin. The correlation between results from extracted and unextracted sera was 0.96 and the fit ideal: y = 1.00x + 0.38%. The correlation between the results of measuring insulin antibody in 256 diabetic sera by the 2 assays was r = 0.74, P less than 0.001 (human insulin) and r = 0.71, P less than 0.001 (porcine insulin). ELISA is cheap and simple to perform. We believe it may prove to be a practical alternative to radioassay in both the routine detection and investigative research of insulin antibodies.     

Antigens which detect IgE antibodies in workers sensitive to toluene diisocyanate

Two antigens, both containing tolyl groups, were compared for ability to detect IgE antibodies in workers hypersensitive to toluene diisocyanate. One antigen, formed by reaction of p-tolyl isocyanate with human serum albumin, detected antibodies in each of ten hypersensitive workers. A second tolyl antigen, formed by reaction of p-toluoyl chloride and human serum albumin, and therefore lacking isocyanate linkages, detected antibodies in seven of the ten workers. In RAST assays, sera from toluene diisocyanate-sensitive workers demonstrated higher binding to human serum albumin-coated control discs than did sera from non-sensitive workers. The significance of this binding in calculating tolyl-reactive antibody titres is discussed.     

A method to determine significant levels of immunoglobulin G to Aspergillus fumigatus antigens in an ELISA system and a comparison with counterimmunoelectrophoresis and double diffusion techniques

A method is described which assesses results obtained from an ELISA system for the determination of human serum levels of IgG class antibodies to Aspergillus fumigatus. The method is used to discriminate positive from negative samples, and significant antibody activity may be reported to the clinician, relative to a reference positive control serum monitored simultaneously under the same test conditions. Antibody content is expressed as the absorbance of a certain dilution of serum. Duplicate samples were analysed at a single serum dilution and their absorbtion values obtained from a semi-automated ELISA microplate reader. These were entered into a computer programmed to convert the data into units on a logarithmic scale. In parallel experiments, ELISA results were compared with those obtained by the techniques of counterimmunoelectrophoresis and double diffusion which measure precipitating antibody of all classes. A relatively good degree of correlation between tests was found only among sera with a high level of antibody.     

A method to determine significant levels of immunoglobulin G to Aspergillus fumigatus antigens in an ELISA system and a comparison with counterimmunoelectrophoresis and double diffusion techniques

A method is described which assesses results obtained from an ELISA system for the determination of human serum levels of IgG class antibodies to Aspergillus fumigatus. The method is used to discriminate positive from negative samples, and significant antibody activity may be reported to the clinician, relative to a reference positive control serum monitored simultaneously under the same test conditions. Antibody content is expressed as the absorbance of a certain dilution of serum. Duplicate samples were analysed at a single serum dilution and their absorbtion values obtained from a semi-automated ELISA microplate reader. These were entered into a computer programmed to convert the data into units on a logarithmic scale. In parallel experiments, ELISA results were compared with those obtained by the techniques of counterimmunoelectrophoresis and double diffusion which measure precipitating antibody of all classes. A relatively good degree of correlation between tests was found only among sera with a high level of antibody.     

The influence of anti-fibronectin antibodies on interactions involving extracellular matrix components and cells: a possible pathogenic mechanism.

Antibodies, directed to the 30-kD collagen binding domain (CBD) of fibronectin (Fn), have been previously demonstrated in sera from patients with systemic lupus erythematosus (SLE), and we now investigate the possible pathogenic effects of these antibodies on collagen-Fn and cell-Fn interactions. The binding of type 1 collagen to Fn was demonstrated by ELISA, and could be specifically inhibited by the preincubation of solid-phase immobilized Fn with anti-Fn antibodies from SLE sera. By using indirect immunofluorescent staining, anti-Fn antibody containing SLE sera but not normal human serum (NHS) reduced the deposition of newly synthesized collagen and Fn on living human skin fibroblasts. We also found that sera from SLE patients containing anti-Fn antibodies significantly reduced thyroid cell attachment to Fn immobilized on plastic compared with NHS. These effects were shown to be due to the presence of anti-Fn antibodies in these sera, as SLE sera depleted of anti-Fn antibodies did not reduce the deposition of collagen or Fn on cultured fibroblasts, nor did they inhibit cell attachment.