Human serum antibody response inCampylobacter jejuni enteritis as measured by enzyme-linked immunosorbent assay

An ELISA for detection of IgG, IgA, and IgM antibody using an acid-glycine extract from Campylobacter jejuni as antigen was developed. To determine the value of this assay for the diagnosis of acute Campylobacter jejuni infections, the IgG, IgA, and IgM immune response against Campylobacter jejuni was investigated at various timepoints after infection in patients with culture-proven infection. A total of 112 sera from 46 patients and 78 sera from a control group were tested. All but one of the 46 patients with culture-proven Campylobacter jejuni enteritis developed IgG antibodies against Campylobacter jejuni. IgA and IgM ELISA both showed 97% specificity, and sensitivity of 63% and 30% respectively. IgG antibody titers generally remained at a constant level for more than 50 days, whereas IgA and IgM antibody titers declined more rapidly to normal values within 30 to 50 days after onset of clinical symptoms. Detection of Campylobacter jejuni specific IgA antibodies in a single serum sample provided the most useful assay for serological diagnosis of Campylobacter jejuni enteritis. The presence of Campylobacter jejuni specific IgM antibodies was the sole diagnostic criterion in three cases. Serological diagnosis of Campylobacter jejuni enteritis should therefore include both IgA and IgM antibody determination.     

Antibody responses to Campylobacter infections determined by an enzyme-linked immunosorbent assay: 2-year follow-up study of 210 patients

An enzyme-linked immunosorbent assay (ELISA) was adapted to measure immunoglobulin G (IgG), IgM, and IgA classes of human serum antibody to Campylobacter jejuni and Campylobacter coli. Heat-stable antigen, a combination of C. jejuni serotype O:1,44 and O:53 in the ratio 1:1, was used as a coating antigen in the ELISA test. A total of 631 sera from 210 patients with verified Campylobacter enteritis were examined at various intervals after infection, and a control group of 164 sera were tested to determine the cut-off for negative results. With a 90th percentile of specificity, IgG, IgM, and IgA showed a sensitivity of 71, 60, and 80%, respectively. By combining all three antibody classes, the sensitivity was 92% within 35 days after infection, whereas within 90 days after infection, a combined sensitivity of 90% was found (IgG 68%, IgM 52%, and IgA 76%). At follow-up of the patients, IgG antibodies were elevated 4.5 months after infection but exhibited a large degree of variation in antibody decay profiles. IgA and IgM antibodies were elevated during the acute phase of infection (up to 2 months from onset of infection). The antibody response did not depend on Campylobacter species or C. jejuni serotype, with the important exception of response to C. jejuni O:19, the serotype most frequently associated with Guillain-Barré syndrome. All of the patients infected with this serotype had higher levels of both IgM (P = 0.006) and IgA (P = 0.06) compared with other C. jejuni and C. coli serotypes.     

Identification of Campylobacter jejuni and Campylobacter coli antigens with mucosal and systemic antibodies.

The development of a rapid and specific diagnostic assay for Campylobacter infections is important in determining the etiology of acute diarrhea in humans. Studies have shown that sonicated whole bacteria or partially purified antigens cross-reacted with antibodies against other closely related bacteria. To solve the problems of specificity, we identified specific antigens of Campylobacter jejuni and Campylobacter coli for use in diagnostic assays. We investigated the responses of serum, urine, and intestinal lavage antibodies in infected (fed live bacteria) and parenterally immunized (intraperitoneal injection of sonicated whole bacteria with adjuvant) mice directed against C. jejuni or C. coli by Western blot (immunoblot) analysis. Antibody responses were examined weekly for up to 28 days. Fewer antigens were detected by urinary and intestinal lavage fluid immunoglobulin A (IgA) than serum IgG and IgM for both parenterally immunized and infected mice. Serum from parenterally immunized mice detected more antigens than that from infected mice. Two high-molecular-weight antigens (62,000 and 43,000) were predominantly detected by serum, urine, and intestinal lavage fluids of both parenterally immunized and infected mice. Serum antibodies from 28-day parenterally immunized mice detected one antigen specific to C. coli with a molecular weight of 38,000 and one antigen specific to C. jejuni with a molecular weight of 27,000. An immunodominant protein with a molecular weight of 31,000 common to both C. jejuni and C. coli was also recognized by serum antibodies from parenterally immunized mice.     

Human antibody response to Campylobacter jejuni flagellin protein and a synthetic N-terminal flagellin peptide.

We measured isotype-specific human antibodies directed against Campylobacter jejuni native flagellin and a synthetic peptide derived from the N-terminal amino acid sequence of the protein by using a microdilution enzyme-linked immunosorbent assay (ELISA). Serum samples from patients with gastrointestinal infection caused by C. jejuni (n = 20) and control samples (number from normal subjects = 20; number from patients with diarrhea other than campylobacter = 20) were tested in this assay. Serum specimens from patients with campylobacter infection showed statistically significant higher isotype-specific antiflagellin antibody titers than control samples did. Detection of immunoglobulin G (IgG) antibodies was less specific (70%) than detection of either IgA or IgM antibodies in infected patients (95%). The sensitivity of testing for any of the isotypes ranged from 64 to 100% in acute-phase serum specimens and 85 to 95% in convalescent-phase serum specimens. An ELISA with an N-terminal synthetic peptide derived from the flagellin protein as antigen was not sensitive (60%) for detecting campylobacter infection but was very specific (97.5%). In conclusion, detection of serum IgA or IgM against C. jejuni flagellin may be a useful marker of infection. Although the N-terminal synthetic peptide was antigenic in a few patients with infection and showed good specificity in the ELISA, additional amino acid sequences with better sensitivity for detecting infection need to be identified.     

Heterozygosity at the Km locus associated with humoral immunity to Campylobacter jejuni

Serum samples from 43 Caucasian subjects convalescing from acute Campylobacter jejuni infection were typed for nine Gm and two Km determinants. The sera were also used to measure IgA, IgG, and IgM classes of antibody to acid-labile surface proteins of C. jejuni by an enzyme-linked immunosorbent assay. A highly significant association (p = 0.004) was found between Km1/Km3 heterozygotes and the level of IgA antibodies. These results suggest the existence of complementary immune response genes which in the heterozygous condition permit a humoral response to C. jejuni.     

Detection of immunoglobulin M (IgM), IgA, and IgG Norwalk virus-specific antibodies by indirect enzyme-linked immunosorbent assay with baculovirus-expressed Norwalk virus capsid antigen in adult volunteers challenged with Norwalk virus.

Pre- and postexposure sera collected from 17 adult volunteers challenged with Norwalk virus as described previously (D. Y. Graham, X. Jiang, T. Tanaka, A. Opekun, P. Madore, and M. K. Estes, J. Infect. Dis. 170:34-43, 1994) were examined for Norwalk virus-specific immunoglobulin M (IgM), IgA, and IgG by indirect enzyme-linked immunosorbent assays with recombinant Norwalk virus antigen bound to the solid phase. Sixteen of the 17 volunteers had evidence of past infection, all presenting with preexisting IgG antibody of high avidity; only one volunteer had no evidence of previous infection. Virus infection was detected in 14 of the 16 volunteers with evidence of past infection, and 9 of the infected volunteers had symptomatic illness. A significant rise in both virus-specific IgA and IgG titers was detected after challenge in all of the volunteers who became ill. Five of the asymptomatic volunteers who were infected had rising titers of virus-specific IgG, but only two of the five had a concomitant rise in their virus-specific IgA antibody titers. Antibody rises were detectable in eight of nine ill volunteers 8 to 11 days after challenge but in the asymptomatic volunteers only after more than 15 days had elapsed. Virus-specific IgM was detected after challenge in all 14 infected volunteers. Between symptomatic and asymptomatic volunteers there were no significant differences in titers of virus-specific IgG and IgA in serum before challenge; however, there were significantly higher titers in symptomatic volunteers between 8 and > 90 days after challenge for virus-specific IgG and 8 and 24 days after challenge for virus-specific IgA.     

Campylobacter etiology in human gastroenteritis demonstrated by antibodies to acid extract antigen

Campylobacter antibodies of the immunoglobulin G (IgG), IgM, and IgA classes were determined by enzyme immunoassay with acid glycine extract antigen in patients and controls involved in two Campylobacter outbreaks and in 266 unselected patients with acute enteritis. The assay showed a specificity of 99% for each immunoglobulin class in sera from 200 healthy blood donors. Elevated Campylobacter antibody titers were shown in 97% of stool culture-positive patients involved in the outbreaks. Rapid changes of IgA and IgM Campylobacter antibodies were typical of the early phase of serologic response in the outbreaks and thus offered the best diagnostic value in the serologic diagnosis of acute campylobacteriosis. In unselected patients with acute enteritis, the assay revealed elevated Campylobacter antibody titers in 37 patients, of whom only 12 had had positive Campylobacter stool cultures. In the sera of patients with other bacterial findings in addition to high titers of Campylobacter antibodies, no cross-reacting antibodies were found, but there was evidence of several mixed infections.     

Prevalence of antibodies against heat-stable antigens from Helicobacter pylori in patients with dyspeptic symptoms and normal persons.

Heat-stable antigens from Helicobacter pylori were investigated for the detection of serum IgG, IgA and IgM antibodies against H. pylori by an ELISA technique. Antibody titers against H. pylori were measured in 167 dyspeptic patients, of whom 96 were H. pylori positive confirmed by culture or microscopy, and in 482 controls (0-98 years). Increased IgG antibody titers were found significantly more often in dyspeptic patients with active chronic gastritis than in patients with normal morphology, as well as in H. pylori-positive patients as compared to H. pylori-negative patients, independent of the endoscopic findings. The heat-stable antigens were compared with acid glycine-extracted antigens and a high degree of concordance was found in the results obtained with the two antigen preparations. The differences in the IgA antibody titers against H. pylori between H. pylori-positive and H. pylori-negative dyspeptic patients were significant and may be useful to confirm a borderline IgG result. No differences were found in IgM antibody titer between H. pylori-positive and -negative patients. The greatest age-dependent increase in IgG and IgA antibody titers was found in children, and if a lower cut-off level is used for children than for adults, as has been proposed, the proportion of people with increased antibody titers against H. pylori would be almost constant from the age of between five and 10 years until the time between 61 and 80 years. Comparison of H. pylori IgG antibodies with IgG antibodies against Campylobacter jejuni and total antibodies against cytomegalovirus (CMV) showed a greater similarity between H. pylori and C. jejuni (R = 0.51) than between H. pylori and CMV (R = 0.22). This may possibly be caused by cross-reactions between H. pylori and C. jejuni. The H. pylori heat-stabile antigen seems not to be very different from other crude H. pylori antigens like acid glycine-extracted antigens, but purification and characterization of the antigens are needed to improve antibody assays.     

No signs of Campylobacter jejuni/coli-related antibodies in patients with active ankylosing spondylitis.

Twenty-two patients with active ankylosing spondylitis were investigated to assess the levels of specific serum IgG, IgA and IgM titres against Campylobacter jejuni/coli before and during treatment with sulfasalazine. An enzyme-linked immunosorbent assay was used, and the results were compared with the antibody levels in 300 healthy blood donors. Three patients had elevated levels of serum anti-Campylobacter-IgA before treatment, and a two-fold decrease in the antibody titre was observed during treatment. Three patients had elevated anti-Campylobacter-IgG titres before treatment. One of these patients also had elevated anti-Campylobacter-IgA and IgM titres. Elevated IgM titres were not seen in any other patient. The results do not support the hypothesis that C. jejuni/coli plays an important role in the pathogenesis of active AS.     

Antibody response to Campylobacter coli in children during intestinal infection and carriage.

In the Central African Republic, etiological studies of diarrhea have shown that Campylobacter coli accounts for almost 40% of Campylobacter enteric isolations. This prompted us to investigate the antibody response to C. coli infection in children. As expected from the literature on Campylobacter jejuni infections, our results show that both infection and carriage elicited antibodies against glycine-extracted membrane antigens, flagella, and cholera toxin. The human antibody response to C. coli resembles the response to C. jejuni, and this similarity will allow comparative studies on larger numbers of infections, both symptomatic and asymptomatic. Anti-cholera toxin antibodies were directed against both the A and B subunits.     

Aberrations in Titre and Avidity of Serum IgM and IgG Antibodies to Microbial and Food Antigens in IgA Deficiency

The antibody levels and relative avidity of serum IgM and IgG antibodies against E. coli O antigens, poliovirus type 1 and beta-lactoglobulin were determined with enzyme-linked immunosorbent techniques in IgA deficient (IgAd) patients with frequent respiratory tract infections and healthy IgAd individuals. Healthy individuals with normal immunoglobulin levels served as controls. The IgM antibody levels against the bacterial, viral and food antigens and the IgG antibody levels against the bacterial antigens were significantly higher in the IgAd group with recurrent infections than in the group of healthy IgAd individuals. The symptomatic IgAd group had significantly higher levels of the IgG antibodies against the bacterial antigen, also when compared with controls. In contrast the healthy IgAd individuals had the highest avidities of IgM antibodies to the viral and food antigens. The high avidities of antibodies could be a compensatory host defence mechanism in IgAd. These aberrations may appear as a consequence of increased mucosal exposure in IgAd to antigens such as E. coli or beta-lactoglobulin, but presumably not to poliovirus which is only exceptionally present in the milieux. They could also be a result of the previously suggested dysregulation of antibody responses in IgAd.     

Kinetics of anti-Campylobacter jejuni monomeric and polymeric immunoglobulin A1 and A2 responses in serum during acute enteritis

The intensity and kinetics of the serum polymeric and monomeric immunoglobulin A1 (IgA1) and IgA2 antibody responses to Campylobacter jejuni were analyzed. A rapid and marked serum IgA antibody response involving both the monomeric and polymeric components of IgA was observed after C. jejuni infections. IgA antibodies reached a peak of activity in serum during week 2 after the first symptoms of enteritis, about 10 days before the peak of IgG activity. Polymeric IgA accounted for most of the anti-C. jejuni activity at the peak of the IgA response (median, 90%; range, 44 to 98%) but rapidly disappeared from serum over a few weeks. In contrast, the serum monomeric IgA antibody response was low and was maintained over a prolonged period of time. Anti-C. jejuni IgA detected in the serum of healthy blood donors was mainly monomeric (median, 83%; range, 17 to 94%). In both the patients and the positive controls, IgA1 was the predominant (greater than 85%) subclass involved, even when the IgA antibody response was mainly polymeric. Our results suggest that polymeric IgA antibody responses are linked to a strong or persisting antigenic stimulation or both. Polymeric IgA antibodies appear to be a potential marker of acute C. jejuni infections, and their determination could provide a useful tool for the serological diagnosis of recent C. jejuni infections.     

Interference by rheumatoid factor activity in the detection of antiavian antibodies in pigeon breeder's disease

The assessment of antiavian antibodies is relevant for the study of pigeon breeder's disease; nevertheless, different factors may hamper their accurate detection. The objective of this study was to determine whether an endogenous interfering effect in pigeon breeder's disease might explain the simultaneous presence of IgM, IgG, and IgA antiavian antibodies in high titers as assessed by ELISA. Fifty-nine patients with pigeon breeder's disease, 80 healthy controls, and 47 asymptomatic breeders were studied. To assess possible interfering effects by endogenous immunoglobulins, serum IgG was separated through protein A-Sepharose CL-4B chromatography. Antiavian antibodies were measured in whole and separated samples by ELISA. Since a decline of IgM antiavian antibodies following IgG removal was consistent with a false-positive effect, the causes were studied. Hig values of IgM, IgG, and IgA antiavian antibodies were found in 17.4% of patients with pigeon breeder's disease. An IgM rheumatoid factor activity against IgG was found through ELISA in sera with false-positive IgM antiavian antibodies. Rheumatoid factor binding was confirmed by Western blot. Experimental addition of purified rheumatoid factor to sera with IgG antiavian antibodies replicated the interfering effect. A control group of rheumatoid arthritis with high rheumatoid factor values did not show positive antiavian antibodies tests. No IgG with anti-IgM or anti-IgA activity was found, and the detection of IgA against IgM and IgG was negative. In conclusion, the study of antiavian antibodies might be affected by different immunoassay conditions. An endogenous rheumatoid factor activity produced false-positive IgM results. Other similar interferences warrant a careful evaluation during the serological assessment of pigeon breeder's disease. Received: 3 December 2001 / Accepted: 22 April 2002     

Antibodies to Campylobacter jejuni in patients with acute enteritis

In 233 patients with acute diarrhoea in paired sera Campylobacter antibodies classes IgG and IgA were assessed by the ELISA method. As antigen the external membrane protein of the strain Campylobacter jejuni was used. Raised IgG levels (greater than 50 u.) and/or IgA (greater than 80 u.) were found in 15% of all examined patients. A quadruple increase of values in one or both classes was recorded in 12.4% of the patients. The incidence of antibodies against Campylobacter jejuni provides evidence that this infectious agent is frequent in this country. Antibodies class IgA suggest by their dynamics acute contact with the Campylobacter antigen. On the other hand, IgG antibodies are not of major importance in newly diagnosed disease.     

Detection of Campylobacter jejuni in healthy monkeys and monkeys with enteric infections by PCR

Campylobacter were detected by PCR in feces of monkeys of different species (clinically healthy, with diarrhea, and dead from acute enteric infections). High prevalence of these bacteria in monkeys was revealed. The incidence of C. jejuni DNA in monkeys with acute enteric infections was higher than in healthy animals (69.6 and 51.3%, respectively). The highest percentage (92.3) of positive results was observed in Macaca mulatta with enteric diseases and in macaque dead of these diseases. The presence of C. jejuni in monkeys with diarrhea and the absence of pathogenic enterobacteria (Shigella, Salmonella, Yersinia) in feces probably attest to etiological relationship of acute enteric infections with Campylobacter.     

Serological responses to papillomavirus group-specific antigens in women with neoplasia of the cervix uteri.

Certain types of human papillomaviruses have been linked to the development of carcinoma of the cervix uteri. We have analyzed 114 serum specimens from women with cervical intraepithelial neoplasia (CIN) or carcinoma of the cervix uteri for the presence of serum antibodies against purified, disrupted bovine papillomavirus (BPV). The titers of immunoglobulin A (IgA) antibodies against BPV were slightly elevated (P less than 0.025) in the sera from CIN or cervical carcinoma patients compared with the titers of 139 serum specimens from sex- and age-matched healthy controls. In contrast, both the IgG and IgM serum antibody titers against BPV were significantly decreased for CIN and cervical carcinoma patients compared with those of healthy controls (P less than 0.001 and P less than 0.005, respectively). These results suggest that the difference between IgA and IgG or IgM antibodies to papillomavirus group-specific antigens may represent interesting serological parameters that could possibly be used in the epidemiologic study of women at risk for CIN.     

Immunoglobulin and complement complexes in blood following infection with human immunodeficiency virus type 1.

Freely soluble and complexed plasma immunoglobulin A (IgA), IgG, IgM, C1q, C3, and factor B in 36 human immunodeficiency virus type I (HIV-1)-seronegative controls, 69 asymptomatic HIV+ subjects, and 117 individuals with symptomatic HIV-associated disease were characterized. Levels of free and complexed IgG and IgA, and to a lesser extent free C1q and complexed IgM, increased with HIV-1 infection. In stark contrast, both HIV+ groups showed three- to sixfold declines in complexed C3, C1q, and factor B levels. The asymptomatic HIV+ population showed declines in levels of C3-bound IgA, IgG2, and IgG4 complexes. The asymptomatic group showed reductions in C3-complexed IgM, IgA, IgG2, and IgG4 levels. HIV infection is associated with complement-deficient immune complexes.     

Expression of Campylobacter jejuni invasiveness in cell cultures coinfected with other bacteria.

Enteroinvasive Salmonella, Shigella, and Escherichia coli strains were found to exert an effect which rendered Campylobacter jejuni capable of intracellular localization in epithelial cells in vitro. When monolayers of HEp-2 or A-549 cells were challenged with pure cultures of C. jejuni or Campylobacter coli, none of the eight strains tested invaded the cells. In contrast, four of these strains were able to localize intracellularly when the cells were challenged with a mixture of campylobacters and enteroinvasive Salmonella typhimurium, Shigella flexneri, Shigella boydii, Shigella sonnei, or E. coli strains. Invasiveness of campylobacters was also induced by one nonenteroinvasive strain of E. coli O124. Coinfection with other nonenteroinvasive E. coli strains did not induce invasiveness in C. jejuni. The degree of internalization induced by S. typhimurium was significantly higher than that induced by Shigella or E. coli strains. The invasive capacity of C. jejuni was found to differ considerably between strains. No evidence of an invasive potential was demonstrable for two C. coli strains or for two enterotoxigenic isolates of C. jejuni examined. C. jejuni was only able to localize intracellularly in cell cultures when the interaction occurred in a microaerobic atmosphere. None of the strains tested evoked keratoconjunctivitis in guinea pig eyes (Sereny test), regardless of the presence of coinfectants. The results indicate that a synergistic interaction that exists between C. jejuni and other enteropathogens facilitates invasion by C. jejuni.     

Humoral immune response to oral microorganisms in periodontitis.

Serum antibody titers from patients with periodontitis were compared with those from periodontally healthy subjects. With the micro-enzyme-linked immunosorbent assay, immunoglobulin G (IgG), IgA, and IgM antibody titers to isolates of Streptococcus sanguis, Actinomyces viscosus, Bacteroides gingivalis, Bacteroides melaninogenicus subsp. intermedius, Bacteroides gingivalis, Bacteroides melaninogenicus subsp. intermedius, Bacteroides ochraceus, and Fusobacterium nucleation were determined. Antibody titers of the IgG and IgA classes to B. melaninogenicus, B. ochraceus, F. nucleatum, and S. sanguis were found to be significantly higher in the controls than in the patients. No correlations were found with serum IgM titers. These findings indicate that periodonitis may be associated with depressed antibacterial serum antibody titers of the IgG and IgA classes.     

Avidity of Aspergillus umbrosus IgG antibodies in farmer's lung disease.

Farmer's lung disease (FL), the commonest form of allergic alveolitis caused by repeated inhalation of mouldy hay, is associated with exposure to the fungus Aspergillus umbrosus among Finnish farmers. The antigen-binding avidity of A. umbrosus-specific IgG antibodies was measured in 12 FL patients in acute phases of initial and recurrent attacks and during 1 year follow up as well as in 12 healthy farmers and five healthy urban controls. The farmers' groups were further divided into two subgroups: subjects with short exposure (< 7 years) and subjects with long exposure (> 25 years). During the first acute phase FL patients with long exposure exhibited a high avidity of A. umbrosus-specific IgG antibodies that remained high during the 1 year follow up, although the A. umbrosus-specific IgG antibody titre decreased. A re-exposure to mouldy hay leading to a recurrence further enhanced the maturation of the antibody avidity, so that an even higher A. umbrosus-specific IgG avidity with a less significant increase of antibody titre occurred than during the first acute attack. Notably higher IgG antibody avidity was observed in FL patients with long exposure than in healthy farmers or in healthy controls.