A quantitative enzyme-linked immunosorbent assay for honeybee venom-specific immunoglobulin G

A quantitative enzyme-linked immunosorbent assay (ELISA) for honeybee venom-specific IgG is reported. This ELISA surmounts the problem of poor reproducibility due to nonparallelism of dilution curves in previously reported ELISAs. The assay is performed in polyvinyl "U" microtiter plates in which HBV is physically adsorbed to the wells. The antigen is sequentially overlaid with human serum albumin, unknown serum diluted in 10% normal goat serum (NGS), and peroxidase-labeled anti-human IgG in NGS. o-Phenylenediamine is used a substrate. The addition of stabilizing protein (NGS) is shown to be crucial in producing parallel dilution curves in this heterogeneous antigen-antibody system. Sensitivity is at or below 1 ng/ml. Coefficient of variation (using a midrange serum) is 12.1% for triplicate wells, 15.5% for sequential dilution, and 15.0% for six entirely separate assays. The assay correlates well with other methods, notably with the Staphylococcus aureus protein A solid-phase radioimmunoassay (r = 0.976).     

Severely impaired production performance in broiler flocks with high incidence of Clostridium perfringens-associated hepatitis

Historical meat inspection data and production performance data collected from a large poultry processing plant in Norway were used to study the association between Clostridium perfringens infection and production performance in commercial broiler flocks. The study period was limited to the first 2.5 years after withdrawal of the in-feed antibiotic avoparcin, when necrotic enteritis was frequently seen. High levels of C. perfringens-associated hepatitis at slaughter were used as an indicator of C.perfringens infection. The contribution margins were 25 to 43% lower from flocks with high levels of C. perfringens-associated hepatitis than those with low levels. Impaired feed conversion ratio and reduced bodyweight at slaughter were major causes of the losses, while condemnations per se contributed to one-fifth of the losses in profit. Incidence of liver lesions showed remarkable variation throughout the study period, and there was considerable difference in frequency of liver lesions between farms.     

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.     

Serological detection of varicella-zoster virus-specific immunoglobulin G by an enzyme-linked immunosorbent assay using glycoprotein antigen

Since the introduction of varicella vaccination in several countries, there has been an urgent need for commercially available test procedures that allow highly sensitive and specific quantitative determination of the varicella-zoster virus (VZV)-specific immune status, including immunity postimmunization. This study compared the performance of two enzyme-linked immunosorbent assays (ELISAs) for the sensitive and specific determination of VZV-specific immunoglobulin G (IgG) in seronegative and latently infected persons, as well as in vaccinees. One ELISA is based on the detection of antibody to VZV-specific envelope glycoproteins (gp), and the other comprises the whole antigen extract prepared from VZV-infected cells. A modified standard fluorescent-antibody-to-membrane-antigen (FAMA) assay was used as a reference. An excellent sensitivity (100%) in relation to FAMA was demonstrated for the gpELISA (Virion\Serion), while the non-gpELISA (Dade Behring) had a lower sensitivity (83%) when sera from latently infected persons were tested. After postvaccinal immunity was measured, a sensitivity of 87% was achieved with gpELISA, whereas the ELISA incorporating antigen extract of VZV-infected cells had a sensitivity of 78%. Excellent specificity (100%) was calculated for both the gpELISA and the non-gpELISA. In conclusion, SERION ELISA classic VZV IgG is useful for the sensitive and specific quantitative determination of VZV immune status after natural infection. The test can also be recommended for measuring antibody response after varicella vaccination, particularly after the cutoff value was optimized.     

Clostridium perfringens antigens recognized by broiler chickens immune to necrotic enteritis

Little is known about immunity to necrotic enteritis (NE) in chickens. A recent study of broiler chickens showed that protection against NE was associated with infection-immunization with virulent but not with avirulent Clostridium perfringens.In the current study, six secreted antigenic proteins unique to virulent C. perfringens that reacted to serum antibodies from immune birds were identified by mass spectrophotometry; three of these proteins are part of the VirR-VirS regulon.     

Immunization of broiler chickens against Clostridium perfringens-induced necrotic enteritis

Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens. Currently, no vaccine against NE is available and immunity to NE is not well characterized. Our previous studies showed that immunity to NE followed oral infection by virulent rather than avirulent C. perfringens strains and identified immunogenic secreted proteins apparently uniquely produced by virulent C. perfringens isolates. These proteins were alpha-toxin, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase (PFOR), fructose 1,6-biphosphate aldolase, and a hypothetical protein (HP). The current study investigated the role of each of these proteins in conferring protection to broiler chickens against oral infection challenges of different severities with virulent C. perfringens. The genes encoding these proteins were cloned and purified as histidine-tagged recombinant proteins from Escherichia coli and were used to immunize broiler chickens intramuscularly. Serum and intestinal antibody responses were assessed by enzyme-linked immunosorbent assay. All proteins significantly protected broiler chickens against a relatively mild challenge. In addition, immunization with alpha-toxin, HP, and PFOR also offered significant protection against a more severe challenge. When the birds were primed with alpha-toxoid and boosted with active toxin, birds immunized with alpha-toxin were provided with the greatest protection against a severe challenge. The serum and intestinal washings from protected birds had high antigen-specific antibody titers. Thus, we conclude that there are certain secreted proteins, in addition to alpha-toxin, that are involved in immunity to NE in broiler chickens.     

Normalized enzyme-linked immunosorbent assay for determining immunoglobulin G antibodies to cytomegalovirus

A normalized enzyme-linked immunosorbent assay for the determination of immunoglobulin G antibodies to cytomegalovirus is described. The rapid assay involves three 30-min incubations and permits the quantitation of antibody levels with a single-specimen dilution in conjunction with a reference antibody preparation. The results obtained with the normalized enzyme-linked immunosorbent assay correlated closely with the results of complement fixation titrations and another commercially available enzyme-linked immunosorbent assay. The specificity of the procedure was further demonstrated by viral absorption, using cytomegalovirus from two different sources and other viral antigen preparations, including rubella and influenza. The reproducibility of the normalized test results is good and allows for greater uniformity of reporting on a day-to-day basis, as well as between laboratories.     

Demonstration of shared antigens in the genus Clostridium by an enzyme-linked immunosorbent assay

Antigens prepared from several strains of each of 10 Clostridium species were used in an indirect enzyme-linked immunosorbent assay with antisera raised against whole cells of a representative strain from each of the 10 species killed by ultra-violet irradiation. With the exception of C. cadaveris, the antisera gave similar results with antigens prepared from all strains of the homologous species. Antigens prepared from 13 other clostridial species were then investigated in an ELISA system with the 10 representative antisera. The results showed many cross-reactions, particularly in the C. perfringens group, the C. difficile/sordelli group and the C. botulinum/novyi/sporogenes group.     

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.     

Detection of anti-arboviral immunoglobulin G by using a monoclonal antibody-based capture enzyme-linked immunosorbent assay

Monoclonal antibody (MAb)-based capture enzyme-linked immunosorbent assays (ELISAs) for the detection of anti-arboviral immunoglobulin G (IgG ELISAs) were developed for a comprehensive array of medically important arboviruses from the Alphavirus, Flavivirus, and Bunyavirus genera. Tests were optimized and standardized so that maximum homology could be maintained among working parameters for the different viral agents, enabling a wide range of viruses to be easily tested for at one time. MAbs were screened for suitability as capture vehicles for antigens from the three genera. The final test configuration utilized group-reactive MAbs eastern equine encephalitis virus 1A4B-6, dengue 2 virus 4G2, and La Crosse encephalitis virus 10G5.4 to capture the specific inactivated viral antigens. Serum IgG was detected by using alkaline phosphatase-conjugated anti-human IgG (Fc portion). A dilution of 1:400 was chosen as the universal screening serum dilution, with endpoint titrations of serum samples testing positive eliminating occasional false-positive results. IgG ELISA results correlated with those of the standard plaque-reduction neutralization assays. As expected, some test cross-reactivity was encountered within the individual genera, and tests were interpreted within the context of these reactions. The tests were standardized for laboratory diagnosis of arboviral infections, with the intent that they be used in tandem with the corresponding IgM antibody-capture ELISAs.     

Detection of immunoglobulin G to Pasteurella haemolytica capsular polysaccharide by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of immunoglobulin G (IgG) to the capsular polysaccharide (CP) of Pasteurella haemolytica serotype 1. Purified CP was first covalently coupled to poly-L-lysine and then optimally adsorbed at a concentration of 5 micrograms/ml to microtiter plates in the presence of carbonate-bicarbonate buffer (pH 9.8). The ELISA was used to evaluate and compare the CP-specific IgG response of calves vaccinated with different P. haemolytica-derived experimental vaccines. Elevated levels of ELISA IgG titers were detected in postvaccination sera and lung lavage from calves vaccinated intradermally with live logarithmic-phase organisms or the culture supernatants. The ELISA was found to be a rapid, reproducible, and sensitive technique for the detection of CP-specific antibodies and may be useful to delineate the protective role of these antibodies in bovine pneumonic pasteurellosis.     

Recombinant polypeptide antigen-based immunoglobulin G enzyme-linked immunosorbent assay for serodiagnosis of dengue

We have developed an indirect enzyme-linked immunosorbent assay for detection of anti-dengue virus (DENV) immunoglobulin G antibodies using four recombinant DENV envelope polypeptides as antigens, which demonstrated a sensitivity of 89.4% and a specificity of 93.3%. These easily produced antigens are a feasible, cost-effective alternative for generating reagents for dengue serological tests.     

Elimination of nonspecific cytomegalovirus immunoglobulin M activities in the enzyme-linked immunosorbent assay by using anti-human immunoglobulin G.

Direct enzyme-linked immunosorbent assay methods offer several advantages in assessing past or recent exposure to cytomegalovirus (CMV) infection, but there persist many pitfalls in the use of these methods for determining specific immunoglobulin M (IgM). The efficiency of absorption of sera by IgG-coated latex beads, aggregated human IgG, or Staphylococcus aureus, i.e., for removing nonspecific CMV IgM activities, was evaluated in comparison with the effect of an anti-human IgG hyperimmune serum. Large routine series comprising serum samples from patients of various clinical groups and healthy individuals were examined. The CMV IgM-positive samples were at first treated with latex or aggregated IgG, but these absorptions left too many CMV IgM-positive individuals. S. aureus increased the nonspecific activity of some sera and, in other cases, removed or impaired specific IgM activities. The anti-IgG treatment caused the disappearance of nonspecific CMV IgM activities that had resisted the other treatments, whereas specific activities remained intact. Utilizing this method, only 1.03% of the routine series patients remained CMV IgM positive by the enzyme-linked immunosorbent assay, a figure in good agreement with a mean probability of CMV antibody acquisition of 0.33% for the population living in Belgium. On the other hand, in a series of patients who were investigated for serological response to several viruses, eight individuals displayed multiple IgM activities after anti-IgG treatment. In these cases, most IgM activities were found in patients who had IgG toward the related antigen for a long time before transient IgM was detected. This result implies that to assess a diagnosis of primary infection, it is necessary to examine serial specimens for IgG acquisition accompanying specific IgM.     

Mumps-specific immunoglobulin M and G antibodies in natural mumps infection as measured by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay for the demonstration of mumps immunoglobulin G (IgG ELISA) and immunoglobulin M antibodies (IgM ELISA) in serum was compared with complement fixation (CF), hemagglutination inhibition (HI), and hemolysis-in-gel (HIG) tests. The antibody levels measured by IgG ELISA had a high positive correlation with the CF and HIG tests, whereas only a moderate correlation was found between IgG ELISA and HI. Similar patterns of antibody response were observed with IgG ELISA, CF, and HIG: the antibody titres increased rapidly after the onset of symptoms and reached the maximal values in about three weeks. The HI antibodies developed more slowly during the first week of disease, after which the titres increased rapidly up to the fourth week. IgM antibodies measured by ELISA developed soon after onset of symptoms; most patients had IgM antibodies from the second day, and the highest titres were reached within the first week. The antibody response in mumps parotitis did not differ from that in mumps meningitis/encephalitis, while relatively higher antibody titres were found in patients with orchitis/epididymitis. The diagnostic efficiencies of the methods were compared with serum specimens from 33 patients who had a serologically verified mumps infection by at least one of the five methods used (rising antibody titres in paired sera or detectable IgM): IgM ELISA detected all 33 cases, IgG ELISA 29, HIG 28, HI 23, and CF 13. In 27 cases, IgM antibodies were already present in the acute phase serum specimens. It was concluded that mumps IgM ELISA is a more rapid and sensitive means for the serological diagnosis of mumps infection than the conventional tests.     

Quantitation of rabbit immunoglobulin G antibodies to Salmonella minnesota Re by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was developed which allowed the measurement of rabbit immunoglobulin G antibodies directed to Salmonella minnesota Re glycolipid. Efficient adsorption of the antigen to polystyrene could only be effected provided it had been previously dialyzed against 0.2 M EDTA (pH 7.0) and subsequently treated with 0.2% sodium deoxycholate (15 min at 56 degrees C) in 0.05 M diethanolamine buffer (pH 9.6). The method is by far more sensitive than quantitative precipitation in the determination of IgG antibodies. Inhibition by glycolipid of binding S. minnesota Re antibodies to immobilized glycolipid attests to the specificity of the assay and permits the detection of as little as 100 ng of glycolipid.     

Detection by enzyme-linked immunosorbent assay of specific immunoglobulin G isotypes in primary and established cytomegalovirus infections

An enzyme-linked immunosorbent assay using monoclonal antibodies was developed to study the subclass distribution of immunoglobulin G (IgG) to cytomegalovirus (CMV) in individuals from a number of clinical groups. Most CMV-seropositive individuals had IgG1 and IgG3. IgG2 and IgG4 were detected less frequently at very low levels of activity, mostly among mothers at delivery and renal patients. Most seroconversions were accompanied by an important increase of the IgG1 activity, whereas IgG3 appeared at lower levels; neither IgG2 nor IgG4 occurred. This suggests that these isotypes play a secondary role in the response to the CMV infection and that they may be considered markers of past infections. Anti-CMV IgG1 is the most efficiently transmitted through the placenta. Whether infected or not, newborns had the same subclass distribution and activity levels as their mothers. Isotype determination did not offer a decisive explanation of a number of discrepancies observed between CMV IgG enzyme-linked immunosorbent assay and complement fixation test results.     

Detection of anti-West Nile virus immunoglobulin M in chicken serum by an enzyme-linked immunosorbent assay

The emergence of West Nile (WN) virus in New York and the surrounding area in 1999 prompted an increase in surveillance measures throughout the United States, including the screening of sentinel chicken flocks for antibodies. An enzyme-linked immunosorbent assay (ELISA) for the detection of chicken immunoglobulin M (IgM) to WN virus was developed, standardized, and characterized as a rapid and sensitive means to detect WN viral antibodies in sentinel flocks. Serum specimens from experimentally infected chickens were analyzed by using this assay, and IgM was detected as early as 3 to 7 days postinfection. Persistence of IgM varied from at least 19 to more than 61 days postinfection, which indicates the need to bleed sentinel flocks at least every 2 weeks for optimal results if this method is to be used as a screening tool. The ELISA was compared to hemagglutination-inhibition and plaque reduction neutralization tests and was found to be the method of choice when early detection of WN antibody is required. House sparrows and rock doves are potential free-ranging sentinel species for WN virus, and the chicken WN IgM-capture ELISA was capable of detecting anti-WN IgM in house sparrow serum samples from laboratory-infected birds but not from rock dove serum samples. The chicken WN IgM-capture ELISA detected anti-WN antibodies in serum samples from naturally infected chickens. It also detected IgM in serum samples from two species of geese and from experimentally infected ring-necked pheasants, American crows, common grackles, and redwinged blackbirds. However, the test was determined to be less appropriate than an IgG (IgY)-based assay for use with free-ranging birds. The positive-to-negative ratios in the ELISA were similar regardless of the strain of WN viral antigen used, and only minimal cross-reactivity was observed between the WN and St. Louis encephalitis (SLE) IgM-capture ELISAs. A blind-coded serum panel was tested, and the chicken WN IgM-capture ELISA produced consistent results, with the exception of one borderline result. A preliminary test was done to assess the feasibility of a combined SLE and WN IgM-capture ELISA, and results were promising.     

Evaluation of specific immunoglobulin E by enzyme-linked immunosorbent assay in hydatid disease

The diagnostic significance of elevated hydatid-specific immunoglobulin E (IgE) as measured by enzyme-linked immunosorbent assay (ELISA) was evaluated against hydatid-specific IgG ELISA and Casoni's intradermal test in surgically proven cases of hydatidosis. A specific IgE ELISA did not correlate with IgG ELISA or Casoni's intradermal test, although its recorded sensitivity was 87%. The specific IgE ELISA was at the same time false-positive in 57.14% of the cases because of its cross-reactions with cases of ascariasis and taeniasis, unlike Casoni's intradermal test and specific IgG ELISA. It is suggested that, in a helminth-infested population, the determination of specific IgE levels is not better than Casoni's intradermal or specific IgG ELISA tests.     

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.     

Demonstration of circulating pneumococcal immunoglobulin G immune complexes in patients with community-acquired pneumonia by means of an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was developed for quantitation of circulating immune complexes (CICs) containing specific antipneumococcal immunoglobulin G (IgG). These CICs were detected in 17 (85%) of 20 patients with bacteremic pneumococcal pneumonia, 4 (36.4%) of 11 patients with probable pneumococcal pneumonia, 3 (16.7%) of 18 patients with pneumonia of other (nonpneumococcal) etiology, and 13 (41.9%) of 31 patients with pneumonia of unknown etiology. There was no correlation between CICs and serum IgG antibody levels. Pneumococcal capsular antigen was demonstrated in dissociated CICs by latex agglutination.