Transfer of antirotaviral antibodies from mothers to their infants

Levels of rotavirus-specific immunoglobulin G (IgG), IgA, IgM, and secretory immunoglobulin in maternal and cord serum, colostrum and milk, and infants' stools were measured by enzyme-linked immunosorbent assay in 92 mothers and their infants. Although antirotaviral IgG, IgA, and secretory immunoglobulin were present in most maternal sera, only IgG crossed the placenta. All samples of colostrum and milk tested contained antirotaviral secretory immunoglobulin and IgA except those of two women in whom IgA deficiency was subsequently described. Specific IgM and IgG were also detected in many colostral samples. Antirotaviral IgA was detected in many colostral samples. Antirotaviral IgA was detected in stools of breast-fed but not bottle-fed neonates. Apparently the human infant receives rotaviral antibodies both transplacentally and via maternal colostrum and milk.     

Evaluation of antibodies reactive with Campylobacter jejuni in Egyptian diarrhea patients.

Serum and stool samples were collected from 128 individuals: 96 diarrhea patients and 32 apparently healthy controls. Stool specimens were cultured for enteric bacterial pathogens, while sera were screened by enzyme-linked immunosorbent assay for Campylobacter jejuni-reactive antibodies. Of 28 diarrhea patients who demonstrated C. jejuni-reactive antibodies (titers, > 100), 14 were culture positive for this organism. The 32 healthy controls showed significantly lower antibody titers (P < 0.05) with the exception of 10 subjects who were culture positive for C. jejuni and had reactive immunoglobulin M (IgM) (6 subjects) and IgG (7 subjects). IgA was not detected in those 10 individuals (asymptomatic). Avidity was expressed as the thiocyanate ion concentration required to inhibit 50% of the bound antibodies. The avidity was higher in symptomatic patients than asymptomatic healthy controls. IgG was less avid (0.92 M) compared to IgM (0.1 M) and IgA (1.1 M), with no correlation between antibody titer and avidity. However, the thiocyanate ion concentration required for the complete inhibition of IgG (5 M)-bound antibodies was higher than that of IgA (2 M) and IgM (3 M). This study also shows that C. jejuni antibodies were variably cross-reactive with Escherichia coli, Shigella flexneri, Shigella sonnei, and Neisseria meningitidis in addition to Campylobacter coli and Campylobacter rectus.     

Measurement of antibody-producing capacity in man. V. Immunoglobulin classes of antibodies to flagellin

The immunoglobulin classes of antibody to flagellin were determined in twenty-eight persons injected with flagellin from Salmonella adelaide. Sera were examined from non-immunized persons and from persons given primary and secondary immunization. Results obtained by titration before and after treatment of whole serum with 2-mercaptoethanol (ME) were compared with those obtained after fractionation of serum by gel filtration on Sephadex G-200 and by radio-immunoelectrophoresis (RIEP). The ME procedure, whilst not fully reliable for determining class of specific antibody in individual sera, is suitable for routine analyses of many sera. After gel filtration, ME-sensitive antibodies were mostly contained in the IgM protein peak whereas ME-resistant antibodies were mainly associated with the IgG protein peak. Using RIEP, specific antigen binding to the IgM, IgG and IgA precipitin lines was demonstrated. Binding of flagellin to the IgM precipitin line correlated with the presence in whole serum of ME-sensitive antibody and binding to the IgG line with ME-resistant antibody. Antigen binding to the IgA line did not correlate with results from ME-sensitivity studies using whole sera.Natural antibody in serum before immunization was entirely IgM. The antibody after primary immunization was mainly IgM but varying amounts of IgG were also produced; a change to the synthesis of entirely IgG antibody during the primary response was rare. The antibody after secondary immunization was mostly IgG; there was no evidence of increased IgM synthesis. IgA antibodies to flagellin were detected during both primary and secondary responses in about half of the subjects tested, but were not quantified.     

The Immunoglobulin Class of anti-spermatozoal antibodies in Serum

ABSTRACT: The aim of this study was to determine the immunoglobulin class of circulating antisperm antibody using a technique called the indirect immunobead test (IBT). In the IBT sperm bound antibodies are detected using polyacrylamide beads coated with rabbit antihuman immunoglobulin classes IgG, IgA, and IgM. Of the 20 infertile men with serum immobilizins, 100% were found to be positive for sperm-bound IgG, 50% positive for IgA, and 0% positive for IgM, using the IBT. Similarly, 20 infertile females with serum immobilizins showed 95% positivity for IgG, 60% for IgA, and 15% for IgM. Thus there was a good correspondence between the presence of serum immobilizins as determined by the sperm immobilization test (SIT) and the IBT. This study provides data that indicates that IgG and IgA are the two major immunoglobulin classes of sperm antibody in male and female immune sera as detected by a simple, sensitive immunological technique, the serum IBT.     

Multiple-antigen slide test for detection of immunoglobulin M antibodies in newborn and infant sera by immunofluorescence.

A microimmunofluorescence test was evaluated for use in measuring immunoglobulin M (IgM) antibodies in infant sera to five of the agents implicated in congenital and neonatal disease. Pen point dots of Toxoplasma gondii, cytomegalovirus, rubella virus, herpes simplex virus, and chlamydial cell culture antigens were applied to each circle of eight-circle printed slides. These multiple-antigen slides greatly facilitated the screening of 607 sera from infants and 117 sera from mothers for the presence of IgM antibody to these agents. Forty sera could be examined microscopically in approximately 30 min. All sera reacting with one or more antigens were tested for rheumatoid factor by the latex method, absorbed with glutaraldehyde-cross-linked human IgG, and retested for the presence of IgM antibody. IgM antibody to cytomegalovirus was demonstrated in sera from four newborns, but IgM antibody to rubella virus could not be detected until 21 days after birth, although rubella virus was isolated from sera from five younger infants. This may indicate that rubella IgM levels in many congenitally infected newborns are too low to be measured by the immunofluorescence method. Five percent of the sera from infants in this study possessed demonstrable IgM antibody to one of the antigens.     

HC-IgA antibodies of different specificities are normally present in serum: quantitation by ELISA and relationship to the major Ig classes.

Enzyme-linked immunosorbent assays (ELISA) were developed for direct measurement of protein HC-IgA complexes (HC-IgA) in serum with antibody specificity for rabbit IgG (rheumatoid factor (RF) activity), lipopolysaccharide from Yersinia enterocolitica serotype O:3 (Y3) and tetanus toxoid (TT). About 80% of patients with rheumatoid arthritis had increased concentrations of HC-IgA-RF. The values were correlated with the concentrations of IgA-RF and IgM-RF. HC-IgA anti-Y3 was measured in 45 sera with anti-Y3 antibodies of IgM, IgG and IgA class. The HC-IgA anti-Y3 levels were correlated with those of anti-Y3 of IgG and IgM class, but not of IgA class. For HC-IgA anti-Y3, the closest correlation was that with the specific IgM antibody concentration, rs = 0.63 (p less than 0.001). In 25 normal sera, significant correlations were observed between HC-IgA anti-TT and specific antibodies of IgG and IgA class, but not of IgM class. In 107 sera containing IgA M-components, the total concentration of HC-IgA correlated poorly with both protein HC and with IgA concentrations. It was concluded that specific HC-IgA antibodies are normal constituents of serum, and that their concentrations are not directly related to the serum content of specific IgA antibodies.     

Human-isotype-specific enzyme immunoassay for antibodies to pneumococcal polysaccharides.

A simple enzyme immunoassay has been developed to allow the quantitation of the human response to immunization with pneumococcal polysaccharide. The assay uses the 14-valent vaccine (Pneumovax) as a convenient antigen to adsorb to the solid-phase microdilution plate wells and commercially available isotype-specific antibody conjugates. The results have been expressed as arbitrary pneumococcal polysaccharide antibody units by reading off a standard curve constructed by using heterogeneous pooled serum. All nonimmunized subjects tested had immunoglobulin G (IgG) antibodies present in serum. All six control subjects who were immunized with Pneumovax demonstrated an IgG response, and the majority responded with a rise in IgA- and IgM-specific antibody concentrations at a mean of 6 weeks postimmunization. Five out of six cord sera tested contained IgG antibodies only, which were present in concentrations similar to those seen in adults, whereas in 6- to 12-month-old infants only low levels of IgG and IgM and no IgA antibodies were detected. Serum taken from 10 hypogammaglobulinemic patients immediately prior to infusion of immunoglobulin showed low to negative IgG antibody concentrations, and no IgA or IgM antibody was present.     

Antiglobulins and cryoglobulins in rabbits producing homogeneous streptococcal antibodies

After immunization with group B or group C streptococcal vaccines, antiglobulins and cryoglobulins were found in the sera of rabbits producing homogeneous antibodies. The cryoglobulins contained antiglobulin activity and homogeneous streptococcal antibodies. The antiglobulins reacted with both rabbit and human IgG. Precipitating antibody activity to rabbit IgG lacking sialic acid was identified in one cryoglobulin.

A rabbit that produced two homogeneous antibodies to group B streptococci produced a third homogeneous immunoglobulin without anti-streptococcal antibody activity after deliberate immunization with rabbit IgG. The appearance of the new homogeneous immunoglobulin coincided with re-stimulation of antiglobulin formation. The possibility is discussed that antiglobulin antibodies may be responsible for the clonal proliferation of plasma cells that leads to homogeneous immunoglobulin production.

     

Isotype-specific enzyme immunoassay for influenza antibody with monoclonal antibodies to human immunoglobulins

Mouse monoclonal antibodies specific for human immunoglobulin isotypes were investigated for use in an isotype-specific enzyme immunoassay for detection of antibody to influenza type A hemagglutinin (H1 and H3). The monoclonal antibody reagents were compared with isotype-specific, hyperimmune rabbit antisera from the National Institutes of Health. Endpoint titers for immunoglobulin G (IgG) obtained with the two reagents were within fourfold of each other 84% of the time (79 of 84) and within eightfold of each other 95% of the time (89 of 94). Regression analysis of the data gave a multiple correlation coefficient (r2) of 0.77 and a Spearman rank value of 0.83 (P less than 0.001). For IgA reagents, endpoint titers agreed within fourfold 77% of the time (88 of 114) and within eightfold 92% of the time (105 of 114). The r2 was 0.73, and Spearman rank was 0.83 (P less than 0.001). IgM antibody was detected in only 17 of 114 sera by either monoclonal or polyclonal reagents. Of these sera, 14 (82%) gave titers with the two reagents that were within fourfold of each other. A similar number of fourfold titer rises were detected with each reagent in paired sera showing hemagglutination inhibition titer rises. Monoclonal antibody reagents detected 27 IgA, 29 IgG, and 6 IgM rises, while polyclonal antisera detected 26 IgA, 31 IgG, and 7 IgM rises. These results show that monoclonal antibodies specific for human immunoglobulin isotypes are suitable as reagents for diagnostic assays. The advantages of monoclonal antibodies are their high degree of specificity and the ability to be standardized and produced in unlimited quantities. Moreover, the availability of immunoglobulin subclass- and allotype-specific monoclonal antibodies will enable a more detailed analysis of the antibody response to influenza as well as other infectious agents.     

The enzyme-linked immunosorbent assay (ELISA) for determination of IgG and IgM antibodies after infection with mumps virus

Under routine laboratory conditions ELISA was tested for suitability for serological demonstration of specific antibodies of the immunoglobulin classes G and M against mumps virus. Sera from patients with known clinical and virological data were used. The results of ELISA were compared with those of CFT. 45 paired sera were tested in ELISA IgG, 87 first sera in ELISA IgM. Both tests were highly sensitive, antibodies were detected earlier and with higher titers than with the CFT. The ELISA IgM is particularly suitable for early diagnosis of mumps infection with the first serum. In addition 23 paired sera from patients with acute parainfluenza virus infection were examined for cross-reacting antibodies. Low anti-mumps IgG antibody titers were found in some sera. These findings reduced the mumps specificity of the IgG test. In five serum samples from one patient — obtained before, during, and after an infection with mumps — the course of IgG and IgM antibodies could be demonstrated. Advantages and limitations of ELISA IgG and IgM are summarized.Behringwerke AG, Marburg, West Germany     

Immunoglobulin class of Brucella antibodies in human sera

Non-agglutinating Brucella antibodies in fifteen cases of chronic brucellosis were found to be immunoglobulin classes G and A. The use of specific antiglobulin reagents in the indirect antiglobulin test allows an easy analysis. Direct agglutination by IgM but not by IgG Brucella antibody is analogous to the situation that occurs in the Rh-D antigen—antibody system.     

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.     

Solid-phase enzyme immunoassay for determination of antibodies to cytomegalovirus.

A solid-phase enzyme immunoassay for the determination of immunoglobulin H (IgG) and IgM antibodies to cytomegalovirus is described. The enzyme immunoassay gave reliable and consistent results which were in concordance with those obtained by the complement fixation test and the indirect immunofluorescence test. Antibodies to herpes simplex and varicella-zoster viruses did not interfere in the enzyme immunoassay for cytomegalovirus IgM antibodies. In a few patients with IgM antibodies to Epstein-Barr virus, cytomegalovirus IgM antibodies were also detected. False-positive cytomegalovirus IgM antibody results were observed in sera containing both the rheumatoid factor and cytomegalovirus IgG antibodies. This rheumatoid factor interference was overcome by the absorption of sera with polymerized human gamma globulin. The absorption did not affect true cytomegalovirus IgM antibody titers. Also described is a simple enzyme immunoassay that makes possible a more sensitive detection of the rheumatoid factor than the latex agglutination test.     

Demonstration of antimyelin antibodies by immunofluorescence in Guillain-Barré syndrome

IgG, IgA and IgM antibodies reacting with myelin sheaths were found in the sera but not in the cerebrospinal fluid of four out of six patients with Guillain-Barré syndrome. These antibodies had organ specificity for myelin of the central and peripheral nervous systems but did not demonstrate species specificity. Similar antibodies, but in much lower titres, were found infrequently in the sera of patients with liver disease and rarely in normal individuals. The organ specificity and the close correlation between antibody titres and disease activity suggest the possibility that these antibodies may play a role in the pathogenesis of Guillain-Barré syndrome. The detection of antimyelin antibodies by immunofluorescence may provide a relatively simple ancillary laboratory aid in the diagnosis of the neurological entity.     

Monoclonal antibody-based capture enzyme immunoassays for specific serum immunoglobulin G (IgG), IgA, and IgM antibodies to respiratory syncytial virus

Monoclonal antibodies (MAbs) to the fusion protein (F), attachment protein (G), and nucleoprotein (N) of respiratory syncytial (RS) virus were evaluated for use as detector antibodies in immunoglobulin G (IgG), IgA, and IgM capture enzyme immunoassays. MAb assays were tested against assays using polyclonal antibodies (PAbs) with serum specimens from patients with and without evidence of recent RS virus infection. Assays developed with N MAbs were comparable to or better than PAb assays for detecting specific IgG and IgM antibodies but were somewhat less sensitive for IgA. F MAb assays were less sensitive for IgG and IgM antibodies but identified specific IgA in some specimens negative by N MAb assay. G MAb assays were insensitive for IgG and IgM antibodies but did detect about 50% of the IgA antibodies identified by the PAb assay. The basis for the low sensitivity of the G MAb assays is unclear, since many of these specimens were positive for IgG antibodies to G by Western immunoblot. The sensitivity of MAb assays varied with patient age: N MAb assays detected specific antibody responses to RS virus in all immunoglobulin classes in both adults and infants less than 1 year of age, F MAb assays detected specific IgG responses in adults and IgA responses in both adults and infants, and G MAb assays only detected IgA responses in adults. A mixture of N and F MAbs was complementary overall, identifying 54 of 55 (IgG), 51 of 52 (IgA), and 16 of 17 (IgM) serum specimens positive by PAb assay. These MAb assays were also specific with specimens tested from persons without a history of recent RS virus infection. The availability of these MAb-based assays offers other laboratories the opportunity to have long-term, standardized reagents and tests for serological diagnosis of RS virus infection.     

Class-specific antibody determination against Aspergillus fumigatus by means of the enzyme-linked immunosorbent assay. III. Comparative study: IgG, IgA, IgM ELISA titers, precipitating antibodies and IgE binding after fractionation of the antigen

IgG, IgA and IgM ELISA antibody titers against Aspergillus fumigatus were elevated in sera of patients with aspergilloma and allergic bronchopulmonary aspergillosis (ABPA), showing higher titers for the IgG antibodies compared with the IgA and IgM antibodies. No differences were found between titers of identical antibody classes in the two groups of sera. IgG and IgA ELISA titers were highly specific whereas IgM ELISA showed more unspecific binding of IgM antibodies. Antibodies, as measured by ELISA, studied after fractionation of the antigen into fractions of decreasing molecular weight, showed a preferential binding by the high molecular weight fractions. Precipitating antibodies studied in patient sera did not always correspond with the IgG ELISA titers. IgE antibody binding was observed in all fractions from Sephadex G-100 fractionated components; maximum binding was found with fractions of 28,000-60,000 daltons. The low molecular weight fractions (18,000-less than 5,000 daltons) showed less IgE binding but the quantity of this fraction was higher. The discrepancies noted between the IgG and IgA ELISA titers and the binding of IgM or IgE antibodies indicate that antigenic components may in part differ in the binding of antibody classes.     

Comparative analysis of antibodies to Francisella tularensis antigens during the acute phase of tularemia and eight years later.

Approximately 8 years after treatment for tularemia, 14 of 22 (63.6%) individuals tested still had a positive microagglutination test for Francisella tularensis antibodies. An enzyme-linked immunosorbent assay for anti-F. tularensis outer membrane antibodies was positive for 55% (immunoglobulin A [IgA]), 95% (IgG), and 27% (IgM) of the late-phase sera, but with antibody levels significantly reduced from those in the acute-phase sera. IgG and IgA antibody levels in the late-phase sera showed significant correlation with levels in the acute-phase sera. The IgG/IgM ratio calculation discriminated between acute-phase and persistent antibodies for most sera, but Western blot (immunoblot) patterns did not. Immunoblotting indicated that the F. tularensis lipopolysaccharide is a major target for antibodies in both groups of sera. Our results substantiate the need for caution in the interpretation of positive serological test results for tularemia, which could result from disease occurring years earlier.     

Differentiation of cytomegalovirus antigens by their reactivity with various classes of human antibodies in the indirect fluorescent antibody test

Human sera containing immunoglobulin G (IgG), IgA, and IgM antibodies were tested by immunofluorescence for reactivity with cytomegalovirus-infected cell cultures and with early antigens of cytomegalovirus produced by treating the infected cultures with either bromodeoxyuridine or cytosine arabinoside. IgG antibody but not IgM antibody reacted with early antigens produced in bromodeoxyuridine-treated infected cultures. This observation on a small sample of sera suggested that a positive IgM reaction with an infected, nontreated culture and a negative reaction with a bromodeoxyuridine-treated infected culture may indicate a positive specific IgM reaction for cytomegalovirus, even in the presence of IgM rhematoid factor. The hyothesis requires further testing. The different classes of antibody did not all react or did not react to the same extent with early antigens produced in infected cells blocked with cytosine arabinoside or bromodeoxyuridine. This observation indicates that different antigens were being produced as a result of the two treatments.     

Biological activity of serum antibodies to a nonacylated form of lipoprotein D of Haemophilus influenzae.

Protein D, a surface-exposed 42-kDa membrane lipoprotein, is well conserved among both type b and nontypeable Haemophilus influenzae strains, and it is considered a vaccine against H. influenzae infections. Here, we report the large-scale purification of a nonacylated form of protein D (PDm) from the periplasmic space of Escherichia coli overexpressing PDm. Screening of human sera for levels of antibodies to PDm demonstrated that the immunoglobulin G (IgG) antibody level is above background levels in infants less than 6 months of age. Following a drop to background values in the age group 6 months to 1 year, IgG antibody levels start to increase, together with IgA antibody levels, after 1 year of age. The first appearance of serum IgM antibodies is in 6-month- to 1-year-old infants whose IgG antibody levels have dropped to the postnatal background level. Affinity-purified antibodies from humans and from PDm-immunized rats detected epitopes of protein D which are normally exposed on the bacterial surface. Affinity-isolated human anti-PDm antibodies eluted in acidic buffer were not bactericidal against H. influenzae. Loss of bactericidal activity may occur in this buffer, as was demonstrated in pooled human sera with high bactericidal activity after incubation in the same buffer. Hyperimmunization of rats with PDm induced high levels of serum IgG and IgA antibodies against PDm and significant bactericidal activity against homologous and heterologous H. influenzae strains.     

Antibody response in the parotid fluid and serum of Irus monkeys (Macaca fascicularis) after local immunization with Streptococcus mutans.

The antibody response of Macaca fascicularis in parotid saliva and serum to local immunization by two routes with Streptococcus mutans was studied and compared over 1 year. Antibodies were titrated and classified by indirect immunofluorescent staining using specific antiglobulin conjugates. Antiglucosyltransferase activity was assayed by an enzyme inhibition test. Animals were immunized first by injecting formalin-killed bacterial cells and cell products subcutaneously into the vicinity of the four major salivary glands. The monkeys were next immunized by retrograde instillation of antigen into the parotid duct. Extensive subcutaneous local immunization gave a serum response only. After parotid duct immunization, high titers of immunoglobulin A (IgA) antibody, along with traces of immunoglobulin G (IgG) immunoglobulin M (IgM) antibody, appeared in the parotid saliva, and in the serum high titers of IgG antibody were present along with lower titers of IgA and IgM. IgA antibodies in parotid fluid were shown by double immunofluorescent staining to be associated with antigenic determinants which cross-reacted with an antiserum directed to human secretory component. Titers in parotid fluids and sera fell sharply when immunization was stopped. This response pattern was reproducible. High concentrations of antibody capable of inhibiting glucosyltransferase prepared from S. mutans were found in the sera, but relatively little was detected in the parotid fluids. Extensive immunization via the parotid duct resulted in transient functional impairment of the gland, as evidenced by diminished salivary flow rates. We conclude that parotid ductal immunization can be an effective method for stimulating a salivary secretory IgA antibacterial antibody response.