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.     

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.     

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.     

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.     

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.     

Indirect ELISA for the detection of HSV-2 specific IgG and IgM antibodies with glycoprotein G (gG-2).

The glycoprotein G (gG-2) purified from HSV-2 infected cells has been reported to be useful for determination of HSV-2 type-specific antibodies using conventional ELISA formats. This study further confirmed the specificity of gG-2 and demonstrated the feasibility of a specific IgM assay. The gG-2 ELISA was developed to detect HSV-2 specific IgG and IgM antibodies in human sera with high levels of sensitivity and specificity. Of 45 patients with culture-proven recurrent HSV-2 genital infection 44 were reactive for gG-2 IgG. Of 30 sera from patients with culture-proven recent initial HSV-2 genital infection 29 were positive for gG-2 IgM. Three patients with primary HSV-2 genital infection showed gG-2 IgM in the convalescent but not in the acute sera. The IgG- and IgM-gG-2 ELISA showed high specificity. None of 40 sera from children were reactive by either assay. Only one of 94 sera from patients with antibody to herpesviruses other than HSV reacted in the IgG assay but none reacted in the IgM assay. There was no cross-reaction with sera from patients with proven HSV-1 infection with the gG-2 antigen. The results suggest that the IgG assay can be used for demonstration of past HSV-2 infection and the IgM assay for the diagnosis of HSV-2 in neonatal herpes and primary genital herpes, when cultures or rapid diagnostic techniques are unavailable.     

Enzyme-linked immunosorbent assay for mumps and parainfluenza type 1 immunoglobulin G and immunoglobulin M antibodies

A solid-phase enzyme-linked immunosorbent assay (ELISA) for detection of mumps and parainfluenza type 1 antibodies (immunoglobulin G [IgG] and IgM classes) is described and compared with the conventional complement fixation (CF) test. A highly positive correlation was found between mumps IgG ELISA and the mumps CF test, whereas parainfluenza type 1 IgG ELISA had only a moderate positive correlation with the respective CF test. Mumps IgM antibodies could be demonstrated in all patients with serologically verified and clinically typical (parotitis, meningitis, or orchitis) mumps virus infection, but not in patients with rises in parainfluenza CF titers. Mumps IgM was already present in the acute-phase sera if they were not taken during the first 2 days after onset of disease. Mumps IgM was also found in some paired sera that were taken too late to demonstrate any significant increase in the antibody titers by CF. Therefore, mumps IgM ELISA provides an improvement over the conventional laboratory diagnosis of mumps infection, since the measurement of specific IgM antibodies in a single serum by ELISA is diagnostic, rather than the identification of a fourfold or greater rise in CF antibody titer. An unexpected finding was that parainfluenza type 1 IgM antibodies could not be demonstrated by ELISA in paired sera with rises in parainfluenza CF titers, suggesting a different antibody response from that occurring in mumps infection.     

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.     

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.     

Enzyme-linked immunosorbent assay for immunological diagnosis of human tularemia.

The enzyme-linked immunosorbent assay (ELISA) was applied for immunological diagnosis of human tularemia, using lipopolysaccharide from Francisella tularensis as antigen. Sera collected from patients, healthy individuals, and vaccinated volunteers were investigated for antibodies against F. tularensis by ELISA and tube agglutination. In ELISA all sera were titrated with a polyspecific anti-immunoglobulin enzyme conjugate. A limited number of consecutive sera from individual patients were also investigated for immunoglobulin G (IgG) and IgM antibodies by means of immunoglobulin class-specific conjugates. On an average ELISA was more than 10-fold as sensitive as tube agglutination. Two weeks after onset of disease, sera from patients had significantly higher titers in ELISA than sera from healthy controls. High titers persisted after more than 2 years. Significant amounts of both IgG and IgM antibodies were present within 1 to 2 weeks after infection. The antibody activity increased during the first month, without any significant change of the relation between IgG and IgM titers. After 2.5 years the IgG/IgM titer ratio of sera from patients was significantly increased. Within 6 weeks after vaccination sera from about half of the vaccinees had significantly elevated titers in ELISA. Titers observed after vaccination were generally lower than those found after infection. An elevated ELISA titer can be of diagnostic importance by the end of the first week of illness. A significant increase of titer in consecutive serum samples indicates a diagnosis of tularemia. Determination of IgG and IgM antibodies may be of value in determing whether a positive titer of a single serum sample is of longstanding or recent origin.     

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     

Performance of three microimmunofluorescence assays for detection of Chlamydia pneumoniae immunoglobulin M,G, and A antibodies

The microimmunofluorescence (MIF) test is considered the "gold standard" for laboratory diagnosis of acute and chronic Chlamydia pneumoniae infection. The performance of a MIF test based on C. pneumoniae antigen from Washington Research Foundation (WRF) was compared with those of assays from Labsystems (LAB) and MRL Diagnostics (MRL) by investigation of sera from three groups of patients: group I, 83 sera from 28 patients with atypical pneumonia; group II, 37 sera from 16 patients with acute C. pneumoniae or Chlamydia psittaci respiratory tract infection confirmed by PCR or culture; group III, 100 sera from 100 persons enrolled in the Copenhagen City Heart Study. The accordance among the results of the WRF assay and the two commercial assays was excellent for the immunoglobulin M (IgM) antibody detection rate (98%). The accordance in detection rates for IgG and IgA antibodies in sera from patients with acute infections was acceptable (87 and 88%), and in sera from group III, it was excellent (95 and 97%). The determinations of endpoint titers were reproducible with <1 dilution step difference for all three methods, except that the mean IgM antibody titer found by the LAB assay was almost 2 dilution steps higher than that found by the other two methods. Although the three assays use different C. pneumoniae strains as antigens, the detection rates and IgG and IgA endpoint titers were similar. The difference in endpoint titers of IgM antibodies is of no major concern, as the diagnosis of acute C. pneumoniae infection rests on the presence of IgM antibodies, not on their level.     

Immunoglobulin class-specific antibody response in respiratory syncytial virus infection measured by enzyme immunoassay

Immunoglobulin class-specific enzyme immunoassay (EIA) was used for determination of antibody responses in sera collected from 26 children with acute primary respiratory syncytial virus (RSV) infections. All 26 patients had IgG antibody responses with a significant titer increase in 24 (92%); an IgM antibody response was detected in 19 of the 26 (73%). From patients aged 6 months or less only 5 of 8 produced detectable IgM antibodies, whereas all patients aged 1–2 years did so. IgM antibodies appeared within 1 week after onset of illness and persisted from 20 days to 2–3 months. An IgA antibody response was observed in 20 of 26 (77%) patients with a significant titer increase detected in 17 of 26 (65%) patients. In some patients the persistence of IgA antibodies followed that of the IgM antibodies, but in others the IgA antibody titers remained stable up to the end of the follow-up. The most sensitive assay system for serological diagnosis of acute RSV infection in children was the determination of titer increases by IgG antibody.     

Determination of IgA antibodies to human cytomegalovirus by enzyme-linked immunosorbent assay (ELISA)

A sensitive enzyme-linked immunosorbent assay (ELISA) is described for detection of IgA antibodies cytomegalovirus (CMV). The antigen consisted of a sonicated extract of CMV infected human embryo cells. The tested sera were absorbed with staphylococcus aureus (strain Cowan 1) before analysis. Rabbit antihuman IgA peroxidase conjugate was used to detect human IgA bound to viral antigen. In parallel, Igm and IgG antibodies to CMV were studied by ELISA and by the immunoperoxidase antibody to membrane antigen (IPAMA) technique, respectively. CMV IgA was detected in high titers by ELISA in eight of nine CMV patients. The maximal IgA titers were generally lower than the IgM titers detected by ELISA. No CMV IgA antibodies (titer less than 20) were detected by ELISA in 57 control sera (healthy adults, hospitalized patients with various other diseases), paired sera of five patients with acute herpes simplex, infection, two patients with Epstein-Barr infections, one patients with varicella, and one with zoster infections. The potential application of the ELISA CMV IgA technique in serodiagnosis of CMV infections is discussed.     

Demonstration of antibodies against Brucella melitensis 16M lipopolysaccharide and native hapten in human sera by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of human immunoglobulin G (IgG), IgA, and IgM antibodies against Brucella melitensis 16M by using lipopolysaccharide (LPS) and native hapten (NH) as antigens is described. The results obtained with the LPS ELISA were compared with the results of the NH ELISA. A good statistically significant correlation was established between the antibody titers of the IgG class against both antigens. A total of 104 (99%) of the 105 serum samples of patients with brucellosis exhibited specific anti-NH antibodies by the ELISA technique. In 52 (50%) of these positive samples, antibodies against NH were detected by radial immunodiffusion (RID). In 100% of these RID-positive sera, the antibody titers of the IgG class with ELISA-determined anti-NH specificity were equal to or greater than 160. These results point to a higher sensitivity of the ELISA technique as compared with RID. Inhibition experiments revealed that the assay was specific for LPS and NH from B. melitensis 16M.     

Serum antibody response to filamentous hemagglutinin in patients with clinical pertussis measured by an enzyme-linked immunosorbent assay

Titers of antibodies to filamentous hemagglutinin (FHA) were determined by enzymelinked immunosorbent assay in acute and convalescent phase serum samples from 158 patients with clinical symptoms typical of whooping-cough. In 96 of the patients the diagnosis was verified by culture. Significant changes in serum levels of IgG, IgM and/or IgA antibodies against FHA were demonstrated in 126 patients (80%). Thus, demonstration of significant changes in FHA antibody titers in serum can be used for serological diagnosis of pertussis. The results also show that high levels of IgG, IgM and/or IgA antibodies in a single serum sample suggest current pertussis infection, but if the diagnosis is based on determinations of FHA antibody titers in a single serum sample the sensitivity is low. The levels of antibody to FHA were compared with previously determined levels of antibodies to pertussis toxin. A significant antibody response against both FHA and pertussis toxin was seen in 111 patients (70 %) while 147 patients (93 %) developed a significant increase in antibodies against one or both antigens.     

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.     

Anti-transglutaminase antibodies and the serological diagnosis of coeliac disease

Tissue transglutaminase (tTG) has recently been identified as the antigenic target recognised by anti-endomysial antibodies in patients with coeliac disease. In this study, an enzyme-linked immunosorbent assay (ELISA) is used to measure IgA, IgG and IgM antibodies to tTG in patients with coeliac disease and a variety of other inflammatory disorders; and is compared to the standard immunofluorescence test used to detect endomysial antibodies (EMA). In the samples tested, 3% control sera (n=146), 83% EMA-positive sera (n=29), 9% patients with Graves' disease (n=94), 12% antimitochondrial antibody-positive sera (n=53), 11% rheumatoid arthritis patients (n=53) and 22% systemic lupus erythematosus (SLE) patients (n=46) were positive for anti-tTG antibodies. In contrast, none of the controls, 1% of patients with Graves' disease, 2% antimitochondrial antibody-positive sera, 2% rheumatoid arthritis patients and none of the SLE patients were positive for EMA. Measurement of IgG or IgM antibodies to tTG was much less reliable than IgA anti-tTG antibody for the serological diagnosis of coeliac disease. The addition of calcium to the coating buffer improved the assay characteristics of the anti-tTG ELISA. However, the IgA anti-tTG ELISA, with and without calcium, performed less well than the standard EMA test used for the serological diagnosis of coeliac disease. In particular, the anti-tTG ELISA gave a higher rate of non-specific positive reactions.     

Characterization of the humoral immune response in Sudanese leishmaniasis: specific antibody detected by class- and subclass-specific reagents

Sera from Sudanese patients with either visceral or mucosal manifestations of infection with Leishmania were investigated to determine the class of antibodies against the causative parasite in an enzyme-linked immunosorbent assay (ELISA) with intact promastigotes as antigen. All patient sera had significantly more IgG and more antiparasite IgG than did control Sudanese or control Dutch sera; little or no IgM or IgA antiparasite antibody activity was detected. When sera specific for the subclasses of IgG were used to detect the anti-promastigote antibodies, these were found in IgG1- and IgG3-specific ELISA but not in those for IgG2 or IgG4.     

Antilipopolysaccharide antibodies and differential diagnosis of chronic Pseudomonas aeruginosa lung infection in cystic fibrosis

Chronic lung infection in cystic fibrosis is characteristically associated with polyagglutinable, serum-sensitive, mucoid strains of Pseudomonas aeruginosa. Enzyme-linked immunosorbent assay (ELISA) methods for standard-free quantitation of immunoglobulin G (IgG) and IgM antibodies to P. aeruginosa lipopolysaccharides (LPSs) have been developed. We now report the development of assays for quantitation of monomer and dimer total IgA and IgA anti-LPS antibodies. Use of these methods in diagnosis of early chronic P. aeruginosa lung infection was assessed. IgG and IgA anti-LPS levels increased significantly at the onset of chronic infection and continued to increase to very high levels in the later stages of infection. IgM anti-LPS levels also rose at the onset of chronic infection but did not increase further. The function of true- and false-positive rates was illustrated by using various concentrations of IgG, IgA, and IgM anti-LPS for discrimination of patients. Values that gave optimum separations were used for statistical evaluation of the diagnostic sensitivities and specificities of anti-LPS antibody concentrations. The results obtained in these assays were compared with a diagnosis, based on the number of precipitins in crossed immunoelectrophoresis, of serum samples from cystic fibrosis patients. In 64 paired serum samples taken before and immediately after the onset of chronic infection, as defined by crossed immunoelectrophoresis precipitins, the predictive values of a positive ELISA were 86% for IgG and 89% for IgA. The predictive values for a negative ELISA were 98% for IgG and 97% for IgA. Results of the IgM anti-LPS ELISA had a lower predictive value. Immunoblotting and absorption studies showed that IgG anti-LPS antibodies were directed specifically against LPS of P. aeuruginosa. ELISAs were developed to determine the specific IgG sublclasses involved. The increase in IgG anti-LPS involved all four subclasses. Highest anti-LPS titers were seen with IgG1 and IgG4, but the largest relative increases were seen with IgG2 and IgG3.