Enzyme-linked immunosorbent assay for antibodies against teichoic acid in patients with staphylococcal infections

A highly purified teichoic acid preparation was used in an enzyme-linked immunosorbent assay to measure the specific immunoglobulin G (IgG) and IgM response in staphylococcal disease. Antibody determination in a normal population, showing a difference of up to 20-fold in the mean IgG titers between the youngest children and adults, was used to establish age-correlated upper normal values. IgM antibodies were found to be of little diagnostic value since their response was often low or absent. Increased IgG titers were found in 24 of 27 (89%) patients with endocarditis, in 11 of 14 (79%) with complicated septicemia, and in 10 of 20 (50%) with uncomplicated septicemia with serum samples drawn between days 7 and 30 of disease. With paired samples, the numbers of patients with increased IgG titers were 17 of 17, 3 of 4, and 6 of 7, respectively, in the same patient groups. Increased IgG titers were less often demonstrated in patients with chronic osteomyelitis (7 of 22). The enzyme-linked immunosorbent assay for teichoic acid antibodies was found to be a sensitive and specific method for diagnosing staphylococcal endocarditis and septicemia. For optimal results, both the substantial age-correlated variation in normal titers and the importance of adequately spaced samples should be considered.     

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.     

Enzyme-linked immunosorbent assay for detection of immunoglobulin A and G antibodies toCampylobacter pylori

An enzyme-linked immunosorbent assay (ELISA) employing an acid-glycine extract was used to detect IgG and IgA antibodies toCampylobacter pylori in sera from 179 patients with upper gastrointestinal disease, 174 blood donors and 65 children. The incidence of positive ELISA results clearly increased with the severity of histopathologic findings in the antrum mucosa and was also high in patients with peptic ulcers and gastric cancer. The incidence in blood donors and children was much lower and increased with age. The results achieved with the ELISA were similar to those observed previously using the immunoblot method. Differences between whole cell preparation and acid-glycine extract with respect to their protein profiles and immunoblot reactivities were minor. IgM titres were very low and could not be related to histopathological findings, peptic lesions or culture findings. The ELISA may be particularly useful for monitoring the outcome of therapy aimed at eradication ofCampylobacter pylori.     

Enzyme-linked immunosorbent assay for immunoglobulin G and M antibodies to Chlamydia trachomatis in human sera.

Microimmunofluorescence methods used for detection of immunoglobulin G and M antibodies to Chlamydia trachomatis are not available to many clinical laboratories. We evaluated a simple enzyme-linked immunosorbent assay in which serotype L2 elementary bodies are used as antigen. The enzyme-linked immunosorbent assay proved satisfactory for the detection of serum IgG. A total of 160 human sera were tested, and the results correlated well with those obtained by microimmunofluorescence. Results for IgM antibody detection were not as successful, and correlation with current methods was poor.     

Microenzyme-linked immunosorbent assay for detection of immunoglobulin G and immunoglobulin M antibodies to Legionella pneumophila

The microenzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin M and G (IgM, IgG) antibodies to Legionella pneumophila serogroup 1 antigens was evaluated. IgM antibodies were measured by both double-sandwich and single-sandwich techniques. These assays were compared with the previously standardized indirect immunofluorescence test in four groups of subjects: (i) pneumonia patients with culture-proven Legionnaires disease with serogroup 1 isolates, (ii) pneumonia patients with serogroup 1 organisms detected by direct immunofluorescence testing of respiratory secretions but without culture confirmation, (iii) pneumonia patients with negative culture and direct immunofluorescence tests, and (iv) healthy hospital employees. In addition, the sensitivity and specificity of the IgG ELISA were evaluated with larger groups of controls and Legionnaires disease patients. The ELISA was more sensitive than the indirect immunofluorescence test. However, it detected antibody rises in pneumonia patients without culture or direct immunofluorescence evidence of L. pneumophila serogroup 1 infection, thereby suggesting that the specificity of the ELISA was slightly lower than that of the indirect immunofluorescence test. The double-sandwich ELISA was a sensitive method for detecting IgM antibodies and, as previously reported, appeared to be free from interference by rheumatoid factor. IgM anti-Legionella antibodies detected by the ELISA appeared earlier and were less persistent than IgG antibodies. In addition, the IgM ELISA was useful in detecting antibodies in necropsy serum samples obtained from patients dying acutely of Legionnaires disease. The data presented show that the ELISA is a reliable method for the detection of specific anti-Legionella antibodies.     

Enzyme-linked immunosorbent assay for detection ofMycoplasma pneumoniae specific immunoglobulin M

An ELISA was developed for the detection of IgM antibodies to Mycoplasma pneumoniae in human sera, using microtiter plates coated with rabbit antiserum to human IgM selecting for IgM antibodies in the first reaction step. The specific antibodies were detected using enzyme-labelled, detergent-solubilized antigen. The complement fixation test was used as reference method. In a prospective study of 59 patients with community-acquired pneumonia, 13 of whom had evidence of mycoplasmal etiology, the ELISA was shown to have high specificity (97%). In samples taken seven days after onset of the disease all complement-fixation positive samples (n = 20) but one were positive, demonstrating the diagnostic value of a positive test in samples taken after that period.     

Enzyme-linked immunosorbent assay for immunoglobulin G and immunoglobulin A antibodies to Shigella flexneri antigens.

An enzyme-linked immunosorbent assay has been developed to detect class-specific antibodies to Shigella flexneri lipopolysaccharide antigens. This enzyme-linked immunosorbent assay system has been used to measure antibodies present in serum or intestinal secretions without further purification. It is considerably more sensitive than passive hemagglutination, allowing detection of as little as 1.3 ng of specific immunoglobulin G antibody per ml in immune sera. Optimal conditions for this assay are outlined in this report.     

Enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Escherichia coli Vero cytotoxin 1.

The frequency of Vero cytotoxin 1 (VT1)-neutralizing antibody (NAb) in serum specimens from 790 age-stratified (0 to 70 years) control individuals from Toronto was 61 of 790 (7.7%), with a peak of 19% in the 20- to 30-year-old age group and a second peak of 16.7% in the 60- to 70-year-old age group. A total of 568 serum specimens, including 538 from the 790 Toronto control subjects, 21 from patients from three outbreaks of VT-producing Escherichia coli (VTEC) infection, and 9 known VT1-NAb-positive serum specimens from patients with hemolytic-uremic syndrome (HUS), were then tested for the presence of anti-VT1 immunoglobulin G (IgG) by an enzyme-linked immunosorbent assay (ELISA). The mean ELISA values of 522 VT1-NAb-negative serum specimens and 46 VT1-NAb-positive serum specimens were 0.09 +/- 0.06 (range, 0 to 0.56) and 0.78 +/- 0.66 (range, 0.16 to 2.91), respectively (P < 0.001; Student's t test). With a breakpoint of 0.21 (mean ELISA value of the VT1-NAb-negative sera + 2 standard deviations), the sensitivity, specificity, positive predictive value, and negative predictive value of the VT1 IgG ELISA compared with those of the VT1-NAb assay were, respectively, 95.7, 98.7, 86.3, and 99.6%. There were nine discrepant serum specimens, of which seven were anti-VT1 IgG positive and VT1-NAb negative and two were anti-VT1 IgG negative and VT1-NAb positive. The ELISA was also used for testing 238 control serum specimens from The Netherlands, Japan, and India and acute- and convalescent-phase serum specimens from 42 Toronto patients with HUS. The frequencies of anti-VT1 IgG (with VT1-NAb frequencies in parantheses) in control sera from the Netherlands, Japan, and India were 6% (3%), 1.1% (0%), and 12% (10%), respectively, with no age clustering. The frequencies of anti-VT1 IgG seropositivity in HUS patients were 5 of 14 (35.7%) in patients with unknown toxin exposure, 2 of 22 (9.1%) in individuals with known exposure to VT1 plus VT2 or VT1 alone, and 0 of 6 (0%) in patients exposed to only VT2. Development of serum anti-VT1 IgG response appears to be the exception rather than the rule in sporadic HUS patients infected with VTEC expressing VT1. However, in two family outbreaks associated with VTEC strains expressing VT1 alone and VT1 plus VT2, respectively, the presence of anti-VT1 IgG in virtually all exposed individuals who remained symptom free suggests that the presence of antibody was associated with protection.     

Enzyme-linked immunosorbent assay for immunoglobulin G antibody to encephalomyocarditis virus

An enzyme-linked immunosorbent assay for immunoglobulin G antibody to encephalomyocarditis virus was developed. This assay was comparable to antibody assay by neutralization. Its adaptability should be useful for laboratory and epidemiological studies of infections due to encephalomyocarditis virus.     

Enzyme-linked immunosorbent assay for detection of immunoglobulin M antibody to hepatitis B core antigen.

An enzyme-linked immunosorbent assay for detection of specific immunoglobulin M (IgM) antibodies against the core antigen of the hepatitis B virus (anti-HBc IgM) is described. The interference of IgM rheumatoid factor was evaluated quantitatively. In the anti-HBc IgM test, the rheumatoid factor gave false-positive results when the concentration exceeded 20 IU/ml. The rheumatoid-positive sera were disclosed by a control and retested for anti-HBc IgM after absorption of rheumatoid factor with latex particles aggregated with human IgG. In five of seven selected patients with acute hepatitis B followed to biochemical and clinical recovery, anti-HBc IgM was present transiently until antibodies against hepatitis B surface antigen (anti-HBs) appeared. Two patients had persistent anti-HBc IgM during the follow-up period. Four patients with hepatitis B surface antigenemia and progression to chronic liver disease did not clear their anti-HBc IgM in the period of observation (11 to 24 months). Anti-HBc IgM could not be demonstrated in 223 of 225 Danish blood donors. The two donors found positive for anti-HBc IgM also had anti-HBs. Twenty patients with acute A or non-A non-B hepatitis were negative for anti-HBc IgM. The enzyme-linked immunosorbent assay for anti-HBc IgM described here has a high specificity and sensitivity. The diagnostic relevance needs further evaluation, including quantitation of anti-HBc IgM, but the results presented indicate that anti-HBc IgM may be helpful in differentiating between prior and recent or ongoing hepatitis B infection.     

Enzyme-linked immunosorbent assay for streptococcal M protein antibodies.

The enzyme-linked immunosorbent assay (ELISA) described by Engvall and Perlmann, which uses antigen-coated tubes and enzyme-labeled anti-immunoglobulin, has been used for the detection of antibodies against streptococcal M protein. The antigen used in the assay was obtained by guanidine extraction of type M-12 streptococcal cell walls followed by hydroxyapatite chromatography. This antigen has the capacity to elicit bactericidal antibodies in rabbits. The results show that the ELISA is specific and highly sensitive for the detection of antibodies in rabbit and human antisera. Preliminary results suggest that, when M-12 antigen is used, the antibodies detected by ELISA are the same antibodies detected in the bactericidal test. The assay has been performed with human and rabbit sera. There was a 96% agreement between bactericidal and ELISA results with rabbit sera and 97.5% agreement with human sera. All bactericidal antibody-positive sera tested thus far yielded positive ELISA results.     

Enzyme-linked immunosorbent assay for immunoglobulin G antibody to Pasteurella multocida in rabbits.

Three antigen preparations of Pasteurella multocida, lipopolysaccharide antigen, boiled-cell extract antigen, and boiled whole-bacterium antigen, were used in an enzyme-linked immunosorbent assay (ELISA) to detect rabbit immunoglobulin G antibody to P. multocida. The sensitivity of each antigen preparation was compared by using sera from P. multocida-infected and uninfected rabbits and sera from two rabbits immunized with different serotypes of P. multocida. In the ELISA, all three antigen preparations detected high titers of antibodies in infected rabbits and markedly lower levels in uninfected rabbits. When whole-bacterium or boiled-cell extract antigens were used, the ELISA detected antibodies in sera from both immunized rabbits, but with lipopolysaccharide antigen, only antibody to the homologous serotype was detected. Sera absorbed with P. multocida and Bordetella bronchiseptica, another respiratory pathogen of rabbits, revealed that antibodies detected in the ELISA did not cross-react. Since the lipopolysaccharide antigen was more difficult to prepare and may be type specific, and since the whole-bacterium antigen was the least sensitive, the boiled-cell extract was chosen as the best antigen preparation to use in the ELISA.     

Enzyme-linked immunosorbent assay for detection of human immunoglobulin G to lipopolysaccharide of spotted fever group rickettsiae.

An enzyme-linked immunosorbent assay for detecting human immunoglobulin G to spotted fever group rickettsiae was developed and tested. The assay uses proteinase K-resistant material, characteristic of the rickettsial lipopolysaccharides shown to be group specific by immunoblots, as the antigen. The results indicate that the assay provides a sensitive, yet specific, alternative method for diagnosing rickettsial diseases.     

Detection of antichlamydial immunoglobulin G and M antibodies by enzyme-linked immunosorbent assay

Chlamydia trachomatis causes a wide range of infections in adults and conjunctivitis and pneumonia in neonates. The complement fixation test for chlamydial antibody is broadly reactive, but possesses low sensitivity, whereas the microimmunofluorescence test is highly sensitive, but technically difficult to perform. A simple, rapid enzyme-linked immunosorbent assay (ELISA) has been developed for the measurement of immunoglobulin G (IgG) and IgM antibodies to C. trachomatis. Wells of microtiter plates were coated with Renografin-purified elementary bodies (serotype L2) grown in cycloheximide-treated McCoy cells, and serum antibody was detected with peroxidase-labeled goat antihuman IgG and IgM antibody. Of 41 sera tested from patients with lymphogranuloma venereum, pelvic inflammatory disease, cervicitis, or urethritis there was a 90 and 63% correlation of positive results for IgG and IgM, respectively, by microimmunofluorescence and ELISA. Of the positive correlates, ELISA titers were up to 128 times higher than microimmunofluorescence titers for IgG and IgM. The ELISA detected no false-positive results, but missed two positive results for IgG. Both of these sera were reactive against serotypes C and J, suggesting that the ELISA with LGV L2 antigen may not measure antibodies to serotypes within the C serogroup. The IgM ELISA detected 7 negative and 4 positive results not detected by the microimmunofluorescence test. Of four paired sera examined by ELISA, three showed a fourfold rise in IgG antibody titer, and one showed a twofold rise. Further evaluation of this ELISA will be required to determine how useful it will be in seroepidemiological studies and as a diagnostic tool.     

Enzyme-linked immunosorbent assay for detection of streptolysin O antibodies.

An enzyme-linked immumosorbent assay (ELISA), based upon the detection of streptolysin O antibodies in human sera, was developed. Disposable polystyrene tubes, sensitized with streptolysin O antigen, were used as the test vehicles. Corresponding antibodies, present in test sera, were detected by binding of the antibodies to goat anti-human immunoglobulin G conjugated to horseradish peroxidase. Demonstration of bound conjugate was accomplished by monitoring peroxidase activity spectrophotometrically at 450 nm, using 5-aminosalicylic acid as the indicator. A total of 97 human sera, previously analyzed by means of the anti-streptolysin O titration technique, were evaluated with the ELISA procedure. A direct quantitative relationship, found to be statistically significant, was demonstrated between Todd units and absorbance values obtained with ELISA.     

Enzyme-linked immunosorbent assay for the detection of influenza type-specific antibodies

An enzyme-linked immunosorbent assay (ELISA) using a purified ribonucleoprotein antigen is described for detection of type-specific anti-influenza virus antibodies. ELISA was found to be more sensitive than indirect immunofluorescent and complement fixation tests. A significant increase in antibody titer could be demonstrated by ELISA between serum specimens collected prior to and during the influenza season. ELISA appears to be useful for rapid and sensitive diagnosis of influenza infections.     

Enzyme-linked immunosorbent assay for detection of immunoglobulin M antibody against the Reiter treponeme flagellum in syphilis

An enzyme-linked immunosorbent assay (ELISA) for detection of immunoglobulin M (IgM) antibodies against the periplasmatic flagellum of the Reiter treponeme is described. IgM in the test samples was bound to anti-IgM-coated microtest plates, and flagellum-specific IgM antibody was subsequently detected by incubation with a purified flagellum preparation and monospecific anti-flagellum conjugate. Rheumatoid factor, antinuclear antibodies, or flagellum-specific IgG did not interfere. The specificity of the ELISA for IgM antibodies was 99.5% for sera from 200 blood donors and 98.6% for 147 patient sera that gave false-positive reactions in other syphilis serological tests. The sensitivity was 88.5% for sera from 87 patients with first-time primary syphilis, 93.5% for sera from 62 patients with first-time secondary syphilis, 21.4% for sera from 42 patients who were reinfected, and 0% for sera from 13 patients with late syphilis. Of the sera from 153 patients with treated syphilis, 7.2% had IgM antibodies, and sera from patients with primary or secondary syphilis generally had no IgM antibodies 6 months after treatment. The finding of IgM antibodies indicates that patients should receive antisyphilis treatment if they have not been treated recently, but a negative result does not exclude the possibility of active syphilis. The method may prove useful for the diagnosis of congenital syphilis in newborns.