An immunological enhancement phenomenon in experimental brucella infection of the chicks.

The passive transfer of chicken anti-brucella immune globulins promote the survival of Brucella abortus in the spleen of chickens infected with this bacteria. This enhancing effect was reduced significantly when the immune globulins were absorbed with heat-killed brucella cells to remove all the anti-brucella agglutinins and immune adherence antibodies. Therefore, it is most probable that the enhancing effect is related to the presence of brucella antibodies.     

Determination of brucella immunoglobulin G agglutinating antibody titer with dithiothreitol.

The routine brucella agglutination test measures both immunoglobulin M (IgM) and IgG brucella antibody titers; however, only an elevated IgG titer is significant for differentiating active from inactive disease in patients with symptoms lasting 3 or more weeks. The IgG antibody titer can be determined by treating the serum wih 2-mercaptoethanol to inactivate the IgM brucella antibodies while leaving the IgG brucella antibodies intact. Dithiothreitol, which also inactivates IgM, was compared with 2-mercaptoethanol for the determination of IgG brucella agglutination titers. The dithiothreitol and 2-mercaptoethanol test results agreed within +/- 1 dilution step in 103 of 105 serum specimens tested, for a 98.1% rate of agreement. The results indicate that dithiothreitol can be used in place of 2-mercaptoethanol for determining IgG brucella agglutination titers. Dithiothreitol does not have the offensive odor or the irritant properties of 2-mercaptoethanol.     

Immunoradiometric assay for examination and quantitation of Brucella abortus-specific antibodies reactive with the antigen(s) used in the indirect hemolysis test

An immunoradiometric assay was designed to quantitate antibodies which bind to Brucella abortus antigens adsorbed to bovine erythrocytes. This allowed examination of antibodies specific for B. abortus antigens detectable in the indirect hemolysis test for bovine brucellosis. Assay parameters were optimized for measuring antigen-specific immunoglobulin G1 (IgG1), IgG2, and IgM antibodies. The immunoradiometric assay allowed examination of binding interactions which occur during the indirect hemolysis test. Affinity-purified antibovine IgG1, IgG2, and IgM were used to detect specific bovine antibodies of these classes (and subclasses). The binding of the anti-immunoglobulins was linear as a function of immunoglobulin concentration. However, the binding of bovine antibodies of the different classes and subclasses to B. abortus antigen was nonlinear. Since B. abortus-specific antibodies of all classes and subclasses were present in the "standard serum" during the immunoradiometric assays, it is possible that the non-linearity was due to competition between antibodies for antigenic sites. IgG2 and IgM antibodies specific for B. abortus antigen(s) appeared to be capable of binding independently to antigen(s). However, the binding efficiencies of IgG1 antibodies changed as the ratio of antigenic sites to antibodies was increased.     

Specificity and prevalence of natural bovine antimannan antibodies.

Immune responses to the carbohydrate components of microorganisms, mediated both by antibodies and by lectins, are an important part of host defense. In the present experiments, the specificity and presence of natural bovine antibodies against mannan, a common fungal antigen, were examined by enzyme-linked immunosorbent assay (ELISA), using Saccharomyces cerevisiae mannan as an antigen. The results showed that all serum samples from animals of three age groups (newborn, calf, and adult) tested contained antimannan antibodies, and the titer of these antibodies increased significantly in adults. However, titers among individual adult cattle differed widely. Inhibition assays showed that yeast mannan was the strongest inhibitor. D-Mannose exhibited only a minor inhibitory effect at high concentrations. This suggests that most of these antibodies recognize an oligosaccharide-based epitope(s) different from those recognized by lectins. Cattle possess three serum C-type lectins (collectins) capable of recognizing mannan in a calcium-dependent manner. Addition of EDTA to the reaction did not reduce antibody binding, suggesting that the binding of these antibodies to mannan was not affected by the presence of collectin. The antibodies purified from either calf or adult serum by mannan-Sepharose affinity chromatography consisted of mainly immunoglobulin G (IgG) and a smaller amount of IgM. IgG1 was shown to be the dominant antimannan IgG isotype by isotype-specific ELISA. Together, these results demonstrate the production of natural antimannan antibodies in cattle in an age-dependent manner. These antibodies might be involved in defending the host against mannan-containing pathogens as a specific line of defense in conjunction with the innate response by lectins.     

Subclass distribution of rubella virus-specific immunoglobulin G

An enzyme-linked immunosorbent assay was used to study the subclass distribution of rubella virus-specific immunoglobulin G (IgG) in 97 serum samples from healthy donors and from patients with recent or remote rubella infections. Plastic beads coated with rubella antigen were incubated with test serum and then with monoclonal antibodies to the four human subclass of IgG. Rubella virus-specific IgG1 was present in all serum samples containing rubella virus-specific IgG antibodies. Rubella virus-specific IgG2 was present in 1 of 35 samples from healthy donors that also contained specific IgG1. Rubella virus-specific IgG3 was found in serum samples from patients with recent rubella infections but had disappeared by 6 months after the onset of symptoms. Rubella virus-specific IgG4 was found in low amounts in 7 of 35 samples from healthy immune donors. Of 20 serum samples that were negative by other serological techniques, 8 gave absorbances above cutoff levels in the assays for rubella virus-specific total IgG and IgG1. In 1 of 20 serum samples, the assays for total IgG and IgG2 were positive. High absorbance in the assay for rubella virus-specific IgG4 was found in one serum. This serum was negative in all other assays for rubella virus-specific antibodies.     

Amplification of the enzyme-linked immunosorbent assay for measuring allergen-specific IgE and IgG antibody

The amplified enzyme-linked immunosorbent assay (a-ELISA) has been successfully applied to the measurement of allergen-specific IgE and IgG antibodies. Data presented indicate a high positive correlation among measurements of IgE-specific antibodies to ragweed antigen E using the a-ELISA, RAST and radiometric assay of Zeiss. Similarly, a high correlation between allergen-specific IgG antibodies measured by the a-ELISA and Farr assay was also observed. Data presented indicate that the measurement of serum IgE antibodies in the linear region of the ELISA titration plot is unaffected by competition with IgG antibodies. The assay is sensitive, reliable, and without risk to laboratory personnel. It is capable of measuring both IgE and IgG antibodies in patients using the same assay system.     

A biotin-avidin-amplified inhibition enzyme immunoassay for detection of CMV antibodies in human serum

In this inhibition immunoassay undiluted serum reacts in solution with crude cellular CMV antigen in wells of microtestplates coated with hyperimmune CMV-reactive monkey IgG. CMV antibodies in the serum under test block (completely or partial) the fixation of antigen to the capture layer. Unblocked antigenic activity is in subsequent steps measured by the use of biotinylated CMV-reactive monkey IgG and peroxidase-conjugated avidin. The assay was evaluated in comparison with the CF test and was found superior both in terms of qualitative and quantitative detection of CMV antibodies. The results were uninfluenced by the presence in the sera of rheumatoid factor or autoantibodies (antinuclear antibodies). A characteristic feature of this inhibition immunoassay was the absence of equivocal results as demonstrated by analysis of 500 donor sera which were classified in two distinct separate groups: reactive and nonreactive. The assay is simple and reproducible and provides for a good reagent economy. Crude antigen can be used without sacrifice of specificity. Antigen from one Roux bottle proved sufficient for 25,000 duplicate tests.     

A radioimmunoassay for serum and gingival crevicular fluid antibodies to a purified protein of Streptococcus mutans.

A solid-phase radioimmunoassay was developed to measure serum IgG antibodies to a purified protein antigen I/II prepared from Streptococcus mutans. The assay was specific to this antigen and significant binding of 125I-radiolabelled antiserum was found only in sera from rhesus monkeys immunized with the antigen I/II but not in sham-immunized monkeys or those immunized with streptococcal antigen III. A very significant correlation was found in serum IgG antibodies tested by the radioimmunoassay and an immunofluorescent technique (r = 0.88, P less than 0.001). The sensitivity of the double-layer radioimmunoassay was increased 10 times by the addition of a third antibody layer and this enabled gingival crevicular fluid antibodies to be measured. Comparison of paired samples of serum and crevicular fluid revealed a very significant correlation between IgG antibodies to streptococcal antigen I/II in the the two fluids (P less than 0.001). These findings suggest that serum antibodies can reach the tooth surface via gingival crevicular fluid.     

Solid-phase radioimmunoassay of herpes simplex virus IgG and IgM antibodies.

A solid-phase radioimmunoassay for detection of herpes simplex virus-specific IgG and IgM antibodies in human serum specimens is presented. Virus antigen is adsorbed on polystyrene balls and antibodies which attach to the antigen are detected by 125I-labeled antihuman-gamma or antihuman-mu immunoglobulins. A total of 76 specimens have been tested. The appearance of virus-specific IgG and IgM antibodies in primary herpetic infections was readily demonstrated. When serum samples from patients with past exposure to herpes simplex virus were tested, endpoint titers of virus-specific IgG antibodies were found to be 8 to 2048 times higher than titers determined by a complement fixation test. Apparent cross reactivity with varicella-zoster virus was observed in the present radioimmunoassay.     

Solid-phase radioimmunoassay of serum IgG, IgM, and IgA antibodies to cytomegalovirus

A radioimmunoassay (RIA) using polystyrene beads as the solid phase for cytomegalovirus (CMV) antigen and iodinated immunosorbent purified anti-human IgG, IgM, and IgA as indicator antibodies was developed for the detection of immunoglobulin class-specific antibodies to CMV. An antigen prepared from extracellular virus was essential for reliable results, and a preparation ultracentrifuged and sonicated twice was better than a crude antigen. The optimal antigen gave low cpm values with a negative reference serum, resulting in cpm ratios of 10 or higher between early convalescent phase serum and negative reference serum. Of six patients with an increase in CMV CF titres, all six had an increase in RIA IgG titres, four had an increase in IgA titres, and all had IgM antibodies. The IgG titres were high, up to 1/64,000. In a group of 17 infants negative in CMV CF test, 14 had CMV IgG antibodies in RIA test, indicating mainly low levels of maternal antibodies. In six of seven patients with CMV isolations from urine specimens, an increase in IgG or IgA titres or the presence of IgM antibodies was found, and only one of these patients had an increase in CMV CF titre. The specificity of the developed CMV RIA test was further demonstrated by detecting no significant increase in RIA titres in serum specimens of patients with primary herpes simplex infection, chickenpox, herpes zoster, or infectious mononucleosis.     

Anti-inflammatory activity of human IgA antibodies and their Fab alpha fragments: inhibition of IgG-mediated complement activation

The interaction of human IgA antibodies with the classical pathway of complement activation was investigated in a homologous human system, by means of two IgA1 and three IgG1 myeloma proteins having antibody activity against a defined antigen, staphylococcal alpha-toxin. In a solid-phase antigen-dependent C3b-binding ELISA system, the monoclonal IgG antibodies were previously shown to activate the classical complement pathway synergistically, resembling polyclonal IgG antibodies, whereas IgA antibodies were unable to activate complement by either pathway. In the present study, IgA antibodies were found to inhibit significantly the activation of complement initiated by antigen-bound polyclonal or mixed monoclonal IgG antibodies, in relation to the amount of IgA antibodies applied and bound to antigen. IgA1 myeloma proteins devoid of antigen-binding activity were without effect. Inhibition was independent of the ability of the IgA antibodies to compete against the IgG antibodies in binding to antigen, and was demonstrable with physiological concentrations of antibodies. Similar results were obtained with polyclonal serum IgA having antigen-binding activity. However, the binding of C1q to antigen-complexed IgG was inhibited only by a monoclonal IgA antibody that could compete against one of the three monoclonal IgG antibodies that bound C1q synergistically. This observation implied that at least two mechanisms were involved in the inhibition of C3b fixation. Fab alpha fragments of monoclonal IgA antibodies, obtained by cleavage with IgA1 protease from Haemophilus influenzae type b, were found to have a similar inhibitory effect on C3b fixation to the intact IgA1 antibodies. This observation supports the hypothesis that IgA1 proteases contribute to the invasive pathogenicity of certain mucosal bacteria, by cleaving secretory IgA1 antibodies to antigen-binding Fab alpha fragments, which are not only defective in mucosal defense properties, but which also protect the organisms from other immune effector systems, such as complement activation.     

肠道病毒71型与脊髓灰质炎病毒抗体交叉反应性研究

通过临床血清样本研究及实验动物验证,探讨肠道病毒71型(EV71)与脊髓灰质炎病毒(PV)之间的抗体交叉反应性。以酶联免疫吸附试验(ELISA)分别检测健康儿童血清中IgG型抗体与EV71及PV的反应性;用原核表达与亲和纯化等方法分别获得EV71和PV的衣壳蛋白VP1及非结构蛋白3C;将PV-VP1、EV71-VP1、EV71灭活疫苗及PV灭活疫苗(IPV)分别经皮下免疫BALB/c小鼠制得免疫血清,以蛋白免疫印迹实验分别研究PV-VP1或EV71-VP1小鼠阳性血清与EV71-VP1和PV-VP1的反应情况,进一步以竞争ELISA试验分别研究EV71-VP1对PV-VP1与其特异性免疫血清特异结合的影响。微量中和试验研究PV阳性抗血清对EV71的体外中和效应。结果:已接种PV疫苗但未曾感染EV71的健康儿童的IgG抗体与EV71有较高的反应性,且针对EV71及PV两种病毒的IgG抗体的相对含量成正相关;蛋白免疫印迹实验显示PV-VP1与EV71-VP1免疫的小鼠抗血清中存在交叉抗体,竞争ELISA试验进一步表明EV71-VP1蛋白能明显抑制PV-VP1与其特异性免疫血清抗体的结合,反之亦然。但中和实验揭示PV阳性血清在体外不能中和EV71病毒。PV之间存在大量交叉反应性抗体,但PV免疫后产生的与EV71交叉反应的抗体是非中和性质抗体。非中和抗体可能通过免疫调理效应在EV71病毒的致病的过程中发挥重要作用。     

Evaluation of a dipstick ELISA and a rapid immunochromatographic test for diagnosis of Dengue virus infection

Here we report standardization of a dipstick enzyme-linked immunosorbent assay (ELISA, Dipstick ELISA) and its comparative evaluation with a commercial Rapid PanBio Immunochromatographic test (IC test) for detection of Dengue (DEN) virus-specific IgM and IgG antibodies in patient sera. Among crude and purified viral antigens prepared from mouse brains or cell cultures, a DEN virus type 2 antigen purified from cell cultures by sucrose density gradient centrifugation was found superior in terms of the signal/ noise (S/N) ratio in the assay system. The sensitivity of detection of the virus by specific IgM antibody was improved by removal of IgG from patient sera prior to testing. The evaluation of the Dipstick ELISA by use of 156 serum samples revealed an overall accordance of 96% and 93% with the IC test in detection of IgM antibodies to DEN viruses (IgM antibodies) and IgG antibodies to DEN viruses (IgG antibodies), respectively. The sensitivity of the Dipstick ELISA and the IC test with reference to the mu-capture ELISA was 83% and 87%, respectively, with a specificity of 98% in both cases. The sensitivity of the Dipstick ELISA with reference to the IC test in detecting IgM and IgG antibodies was 84% and 94%, respectively, and the specificity of the Dipstick ELISA was 98% and 92%, respectively.     

Monoclonal antibodies to stages of Trypanosoma cruzi: characterization and use for antigen detection.

Monoclonal antibodies to the amastigote and epimastigote stages of Trypanosoma cruzi were produced and characterized by immunoglobulin class and subclass. Of the 17 monoclonal antibodies, 14 were of the immunoglobulin M (IgM) class and 2 were of the IgG2 and 1 was of the IgG1 subclass of IgG. Five of the monoclonal antibodies recognized the antigens of amastigotes only, two recognized the antigens of epimastigotes only, and ten recognized an antigen(s) common to both stages of T. cruzi. By using an immunofluorescence test with monoclonal antibodies, it was possible to visually localize amastigote- or epimastigote-specific antigens and the antigens common to both. Antigens specific for epimastigotes were noted on the flagellum or in spots over the entire body of the parasite. The antigens common to both amastigotes and epimastigotes were on one of the extremities of the amastigotes and on the region of the flagellar pouch of the epimastigotes. Four of the monoclonal antibodies were capable of detecting T. cruzi antigen in serum from mice infected with the parasite and in the supernatant of infected cell cultures, suggesting that monoclonal antibodies may be useful for antigen isolation and diagnostic methods.     

The effect of 7S and 19S antibodies on the primary response to Salmonella typhi antigen

The effect of IgM and IgG antibodies on the primary response to Salmonella typhi antigen has been studied. IgG antibodies given before antigenic challenge or combined with antigen in antibody excess profoundly suppressed the formation of agglutinins in rabbits and humans. IgM antibodies appeared to have little or no inhibitory effect when given as complexes in antibody excess or when infused in relatively small amounts. Larger amounts do inhibit agglutinin formation in rabbits. It is suggested that the antibodies exert their inhibitory effect by combining with antigen and removing it as a stimulant for antibody formation.     

Detection of immunoglobulin G antibodies to cytomegalovirus antigens by antibody capture enzyme-linked immunosorbent assay

An antibody capture enzyme-linked immunosorbent assay (ELISA) that uses horseradish-peroxidase-labeled antigen for the detection of immunoglobulin G (IgG) antibodies to cytomegalovirus (CMV) is described. A microtiter plate was coated with anti-human IgG and consecutively incubated with serum specimens, enzyme-labeled CMV antigen made from CMV-infected cell nuclei, and substrate. The CMV IgG antibody content was determined spectrophotometrically and expressed as absorbance. Furthermore, to reveal any nonspecific reactions, all sera were tested against an enzyme-labeled control antigen made from uninfected cell nuclei. The problem with nonspecific reactions was small and was circumvented by the addition of unlabeled control antigen to the conjugates. For epidemiological studies the test was not as sensitive as other serological tests. On the other hand, the IgG antibody capture ELISA was highly sensitive for detecting the serological antibody response in patients with primary and recurrent CMV infections. Thus, one positive serum remained positive at a serum dilution of 1:10(7). The specificity of the test was shown by a blocking experiment and by testing 126 complement fixation-positive sera, of which 97% were positive. There was a rather good correlation between the complement fixation test and the IgG antibody capture ELISA (rs = 0.79, P less than 0.001). The test is especially useful when tests for CMV antibodies of the IgM, IgA, and IgE classes are run by similar antibody capture ELISAs, since the same procedure and conjugate are used.     

Analysis of false negative results in the immunoassay for anti-acetylcholine receptor antibodies in myasthenia gravis

Possible causes for the failure of immunoassays to detect anti-acetylcholine receptor activity in serum from confirmed myasthenia gravis (MG) patients were investigated. A more sensitive assay, using Protein A to trap immune complexes (ARIA), was applied to 65 MG sera which were negative in the usual assay and to 42 normal human sera. Normal and negative MG sera had antibody (Ab) activity in the same range (50-70 pM). Titers present in 70% of normal sera appeared to be specific antireceptor antibodies as defined by tests for antigen specificity. Thus, higher sensitivity assays did not improve discrimination of MG from normals. In a second group of 108 MG sera studied, 48 were negative by the usual assay criteria in a rat acetylcholine receptor immunoassay. Further detailed analysis of this negative group showed that 3/48 had IgG3 antibody not detectable in the test, 14/48 had Ab's recognizing human receptor determinants exclusively, 29/48 had toxin blocking Ab's not determined by immunoassays, and 6/48 were negative in all tests. The results indicate that the exclusive occurrence of toxin-blocking antibodies in MG subjects is a major factor contributing to false negatives in the ARIA test. Estimates of the amount of Ab's with this functionality indicated that they are present in very much smaller amounts than other classes of anti-receptor Ab's. Degree of blocking activity in patient serum showed a fair correlation with severity of disease. Thus, blocking antibodies appear capable of causing all degrees of disease severity in the absence of other types of antireceptor Ab's. The development of a sensitive and quantitative in vitro assay for blocking antibodies combined with the usual immunoassay would be a major improvement for a MG diagnostic test, with greater than 94% positivity predicted.     

Active release of bound antibody by Streptococcus mutans.

Previous studies have shown that Streptococcus mutants is capable of releasing many surface protein antigens, particularly antigen P1. Antigen P1 is immunodominant and has been implicated in adherence of S. mutants to the acquired pellicles. The purpose of this study is to investigate the significance of release of this antigen by the cells. S. mutants NG8 (serotype c) was incubated with an anti-P1 rabbit immunoglobulin G (IgG) or a human colostral IgA which contains natural anti-P1 activity. Results indicated that the bound antibodies were released by the cells in a pH- and time-dependent manner. The optimal pH for release was between 6 and 8, and the release rate reached a plateau in 1 h at 37 degrees C. The release of bound antibodies was considered an active process, since heat-killed cells remained capable of antibody binding but failed to release the antibodies. The release was also dependent on the age of the culture, with early-exponential-phase cells releasing the maximum amount of bound IgG. The released IgG was isolated by polyethylene glycol precipitation and protein A-Sepharose column chromatography and found to be associated with antigen P1, indicating that the antibodies were released together with the antigen in the form of immune complexes. The binding of S. mutans by secretory IgA (SIgA) inhibited the adherence of the cells to salivary agglutinin-coated hydroxylapatite. However, when the SIgA-coated S. mutans was allowed to release the bound antibodies, the inhibitory effect of SIgA on adherence was abrogated. These results suggest that S. mutans is capable of shedding surface-bound antibodies in the form of antibody-antigen immune complexes. Such an action may be a strategy employed by the cells to counter the neutralizing effect of naturally occurring antibodies in the oral cavity.     

Comparative immunoglobulin G antibody profiles between mother and child (CGMC test) for early diagnosis of congenital toxoplasmosis

Early diagnosis of congenital toxoplasmosis is rendered difficult when specific immunoglobulin M (IgM) and/or IgA antibodies are absent in the blood of the newborn infant. Since maternal IgG antibodies can cross the placenta, determination of IgG antibodies in newborn infants has hitherto not been used routinely for the diagnosis of congenital infection. The aim of this study was to assess the diagnostic usefulness of an immunoblot assay which compares the early IgG profiles between the mother and her child (comparative IgG profile between mother and child; CGMC test) directed against a total cell lysate of Toxoplasma gondii tachyzoites. Serum samples from 97 newborn infants at risk of toxoplasma infection were obtained from umbilical cord blood at birth or postnatally until 3 months of life and were directly compared with serum samples from the respective mothers. Congenital toxoplasmosis was diagnosed only when IgG-reactive protein bands that were present in any newborn serum samples were absent in the corresponding maternal serum sample. Congenital infection was defined by conventional serological assays when IgM and/or IgA antibodies were present in newborn infant blood or when IgG titers rose within the first 12 months or were persistently stable for more than 8 months. Using these criteria, congenital infection was definitely confirmed in 11 cases. Three additional cases were diagnosed based on indicative data. The CGMC test, which was performed without knowledge of the results of conventional serologal assays, had sensitivity and specificity of 82.4 and 93.0%, respectively, and positive and negative predictive values of 73.7 and 95.7%, respectively. When true positives and true negatives were considered, the comparative IgG profile had a ratio of 90.9% true results. The CGMC test thus is useful as an additional assay for the rapid diagnosis of congenital toxoplasmosis when paired serum samples from mother and child are available.     

Ursodeoxycholic acid treatment lowers the serum level of antibodies against pyruvate dehydrogenase and influences their inhibitory capacity for the enzyme complex in patients with primary biliary cirrhosis

A two-year randomized, double-blind, placebo-controlled clinical trial used paired serum samples from 122 patients with primary biliary cirrhosis to compare the effect of ursodeoxycholic acid and colchicine on their immune parameters. IgG antibodies to pyruvate dehydrogenase, the major autoantigen in primary biliary cirrhosis, were determined by enzyme-linked immunosorbent assay and immunoblot; enzyme inhibition assay against pyruvate dehydrogenase was used to test the changes of the functional reactivity of the serum autoantibodies. Treatment with ursodeoxycholic acid decreased both the level of IgG antibodies to pyruvate dehydrogenase (P<0.01) and the inhibitory titer of the sera for pyruvate dehydrogenase (P<0.01). Treatment with colchicine or placebo showed no statistically significant changes in either the antibody levels or the inhibitory titers. Ursodeoxycholic acid thus alters the immune parameters of patients with primary biliary cirrhosis. The mechanism of these changes needs further investigation.Abbreviations AMA Anti-mitochondrial antibodies - PBC Primary biliary cirrhosis - OD Optical density - PDH Pyruvate dehydrogenase - UDCA Ursodeoxycholic acid