Development and application of a Saccharomyces cerevisiae-expressed nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibodies against infectious bronchitis virus

A Saccharomyces cerevisiae-expressed nucleocapsid (N) polypeptide of the M41 strain of infectious bronchitis virus (IBV) was used as antigen in a recombinant yeast-expressed N protein-based enzyme-linked immunosorbent assay (Y-N-ELISA). The Y-N-ELISA was rapid, sensitive, and specific for detecting chicken serum antibodies to IBV, and it compared favorably with a commercial ELISA.     

Recombinant nucleoprotein-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Crimean-Congo hemorrhagic fever virus

The full-length nucleoprotein of Crimean-Congo hemorrhagic fever virus (CCHFV; 482 amino acid residues) was expressed as a His-tagged recombinant protein (His-CCHFV rNP) in the baculovirus system. The His-CCHFV rNP was efficiently expressed in insect cells and purified by Ni(2+) column chromatography. Using this substrate, an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was developed. We evaluated the sensitivity and specificity of the IgG ELISA, using serum samples previously determined to be antibody positive or negative by immunofluorescence tests on CCHFV-infected Vero E6 cells. We found very good correlation between the two tests: 87% for the positive sera (13 of 15) and 99% for the negative sera (107 of 108). These results indicate that the new IgG ELISA using His-CCHFV rNP has high sensitivity and specificity for detecting CCHFV antibodies. The CCHF patients' sera with high titers reacted only with the NP fragment containing amino acid residues between 201 and 306 in Western blotting. It is known that amino acid homologies are high in this region among various isolates. Thus, it is expected that this ELISA can detect antibodies not only for Chinese strains of CCHFV but also for other strains circulating in the world. These results suggest that the IgG ELISA system developed with the recombinant CCHFV NP is a valuable tool for diagnosis and epidemiological investigations of CCHFV infections.     

Development of a recombinant nucleoprotein-based enzyme-linked immunosorbent assay for quantification of antibodies against porcine reproductive and respiratory syndrome virus

A rapid, inexpensive enzyme-linked immunosorbent assay (ELISA) to quantitate antibodies to porcine respiratory and reproductive syndrome virus (PRRSV) in serum was developed using a recombinant PRRSV nucleoprotein (rN). The sensitivity (85.3%) and specificity (81.7%) of the Kansas State University ELISA were good, correlating well (82.4%) with the IDEXX HerdChek ELISA.     

Detection of antibodies to avian infectious bronchitis virus by a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay

The recombinant antigen obtained by cloning and expressing two IBV nucleocapsid protein fragments (143-414 aa, 281-414 aa) in Escherichia coli was used for the detection of avian infectious bronchitis virus (IBV) specific antibodies in chicken sera by the indirect ELISA (rNpIBV-ELISA). As a result of testing 1524 serum samples the diagnostic sensitivity and specificity of rNpIBV-ELISA when comparing those of the routine whole IBV ELISA have been shown to be 93.81% and 87.36%, respectively. The agreement value was 91.5%.     

Comparative studies with an enzyme-linked immunosorbent assay (ELISA) for antibodies to avian infectious bronchitis virus

An indirect enzyme-linked immunosorbent assay (ELISA) was used to detect IgG class-specific antibodies to avian infectious bronchitis virus (IBV) in chickens. The technique detected specific antibody with a high degree of sensitivity and was more sensitive than neutralisation or haemagglutination-inhibition methods of antibody assay. The possible application of the technique for diagnosis and research on IBV is discussed.     

Evaluation of a dipstick enzyme-linked immunosorbent assay for detection of antibodies to dengue virus.

Accurate serological confirmation of dengue (DEN) infection is difficult, because simple reliable assays for the detection of DEN antibodies are not available. To address this problem, a dipstick enzyme-linked immunosorbent assay (ELISA) was evaluated. The dipstick contained dots of serially diluted DEN 2 antigen. To detect immunoglobulin G (IgG), the dipstick was processed through four reaction cuvettes containing test serum, enhancer, enzyme-conjugated anti-human IgG and IgM antibody, and substrate. Total assay time was 45 min. To detect IgM, the serum was passed through a protein G device to remove IgG. The dipstick was then processed as before, except that the incubation times were longer and enzyme-conjugated anti-human IgM was used. The total assay time was 3 h. The dipstick ELISA results were compared with results from microplate ELISA. The IgG dipstick ELISA showed a sensitivity of 95.2% and a specificity of 100% compared to an IgG microplate ELISA with serum samples from 125 individuals living in an area in which DEN is endemic. In tests with 75 serum samples from patients with clinically suspected acute DEN infections, the IgM dipstick ELISA showed a sensitivity of 97.9% and specificity of 100% compared to those of an IgM antibody capture microplate ELISA. These results showed that the dipstick ELISA was a sensitive and specific test for the detection of either DEN IgM or IgG in human serum. The dipstick ELISA was also shown to be useful for detecting seroconversions to DEN IgM or IgG in paired serum samples from 20 patients with virus isolation-confirmed acute DEN infections.     

Evaluation of Vircell enzyme-linked immunosorbent assay and indirect immunofluorescence assay for detection of antibodies against Legionella pneumophila

We evaluated the abilities of the Vircell immunoglobulin G (IgG) and IgM indirect immunofluorescence assay (IFA) for Legionella pneumophila serogroup 1, the IgM and IgG enzyme-linked immunosorbent assay (ELISA) for Legionella pneumophila serogroup 1, and the IgM-plus-IgG ELISA for Legionella pneumophila serogroups 1 to 6 to diagnose Legionnaires' disease (LD) in a well-described sample of patients with and without LD. Also, we determined the agreements, sensitivities, and specificities of the different Vircell assays in comparison to a validated ELISA (Serion classic ELISA). Clinical sensitivity and specificity were 74.6% and 96.6%, respectively, for the IgM IFA, 65.1% and 88.0% for the IgG IFA, 92.3% and 100% for the IgM ELISA, 43.3% and 96.6% for the IgG ELISA, and 90.8% and 100% for the IgM-plus-IgG ELISA. Compared to Serion classic ELISA, agreement, sensitivity, and specificity were 80.0%, 83.1%, and 78.4%, respectively, for the IgM IFA, 75.2%, 66.0%, and 79.5% for the IgG IFA, 89.5%, 82.0%, and 97.6% for the IgM ELISA, 81.9%, 88.9%, and 78.0% for the IgG ELISA, and 93.5%, 90.0%, and 96.6% for the IgM-plus-IgG ELISA. The value of a positive diagnostic result obtained by the Vircell IgM IFA, the Vircell IgG IFA, and the Vircell IgG ELISA might not be acceptable for a diagnostic assay. Both the high specificities and sensitivities of the Vircell IgM ELISA and the IgM-plus-IgG ELISA and the high correlation with the Serion classic ELISA indicate that they are useful in the diagnosis of LD.     

Evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to Junin virus in rodents

Junin virus is the etiological agent of Argentine hemorrhagic fever, a serious rodent-borne disease. An enzyme-linked immunosorbent assay (ELISA) to detect Junin virus IgG antibodies in rodents was evaluated using sera from 27 Calomys musculinus and five Calomys laucha, inoculated experimentally with a live attenuated strain of this arenavirus. The test performance was compared against an indirect immunofluorescence assay (IFA). The ELISA had a sensitivity and specificity of 100% and a reproducibility of 87.9% for samples with titers above the selected cut-off value. IFA had lower sensitivity (53%) with the same specificity. The ELISA results were similar, whether carried out on whole blood or serum samples, thus eliminating the need for serum separation. A high correlation (K=0.86) between ELISA and IFA results was obtained from 1011 wild sigmodontine and murine rodents collected within and outside of the Argentine hemorrhagic fever endemic area. These results indicate that Junin virus IgG ELISA is the most suitable assay for detection of Junin virus antibodies in rodent samples.     

Development of a nucleoprotein-based enzyme-linked immunosorbent assay using a synthetic peptide antigen for detection of avian metapneumovirus antibodies in Turkey sera

Avian metapneumoviruses (aMPV) cause an upper respiratory tract disease with low mortality but high morbidity, primarily in commercial turkeys, that can be exacerbated by secondary infections. There are three types of aMPV, of which type C is found only in the United States. The aMPV nucleoprotein (N) amino acid sequences of serotypes A, B, and C were aligned for comparative analysis. On the basis of the predicted antigenicity of consensus sequences, five aMPV-specific N peptides were synthesized for development of a peptide antigen enzyme-linked immunosorbent assay (aMPV N peptide-based ELISA) to detect aMPV-specific antibodies among turkeys. Sera from naturally and experimentally infected turkeys were used to demonstrate the presence of antibodies reactive to the chemically synthesized aMPV N peptides. Subsequently, aMPV N peptide 1, which had the sequence 10-DLSYKHAILKESQYTIKRDV-29, with variations at only three amino acids among aMPV serotypes, was evaluated as a universal aMPV ELISA antigen. Data obtained with the peptide-based ELISA correlated positively with total aMPV viral antigen-based ELISAs, and the peptide ELISA provided higher optical density readings. The results indicated that aMPV N peptide 1 can be used as a universal ELISA antigen to detect antibodies for all aMPV serotypes.     

An enzyme-linked immunosorbent assay for detection of antibodies against halothane-altered hepatocyte antigens

Patients with massive liver cell necrosis that may follow halothane anaesthesia have a high incidence of circulating antibodies against halothane-induced hepatocyte antigens. In order to provide an objective and quantitative method for the detection of these antibodies, an enzyme-linked immunosorbent assay has been developed. Sera, after absorption with normal rabbit liver microsomal fraction, are tested for binding to microsomal fractions from control and halothane-pretreated rabbits. Those containing antibodies against halothane-induced determinants give significantly enhanced binding to halothane-altered fractions; this specificity was verified by absorption experiments. Using this method, halothane-related antibodies were detected in sera from 16/24 patients with halothane-associated liver failure, at titres ranging from 1:100 to 1:25600. Such antibodies were not detectable in sera from 26 normal blood donors, 5 healthy anaesthetists, 12 patients who had received multiple halothane anaesthetics but had normal liver function tests and 32 patients with a variety of other liver diseases. This rapid and reproducible assay should be of value for the detection of antibodies and for detailed investigation of patient antibody responses, and also for characterization of the route of production and metabolism of the antigen.     

A monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay for identification of infectious bronchitis virus serotypes

An antigen-capture enzyme-linked immunosorbent assay (C-ELISA) was developed for detection and identification of infectious bronchitis virus (IBV) serotypes Arkansas, Connecticut, and Massachusetts using monoclonal antibodies (MAbs) specific to the S1 glycoprotein of the respective serotype. The assay (designed as a double-antibody sandwich assay) gave the best results when the S1-specific MAb, antigen, and chicken serum were of the same serotype. However, when a group-specific (M glycoprotein-specific) MAb was used for antigen capture, a distinctive pattern of cross-reactivity was observed between the antigens and heterologous chicken sera, suggesting a complex distribution of epitopes on the IBV M glycoproteins. Treatment of antigen with NP40 enhanced the ELISA signal only when the M glycoprotein-specific MAb was used for antigen capture. Although C-ELISA was inconsistent in detecting IBV in chicken tissue homogenates, it was highly effective in detecting the virus in allantoic fluid after the homogenates were given one chicken embryo passage.     

A dot enzyme-linked immunosorbent assay for detection of antibodies against Entamoeba histolytica

A visual micro-dot enzyme-linked immunosorbent assay (ELISA) based on detection of antibodies against soluble antigens of axenically grown cultures of Entamoeba histolytica is described. The antigen was spotted on a nitrocellulose sheet, the unsaturated sites blocked with bovine serum albumin (BSA) and incubated with 3-fold dilutions of patient sera followed by incubation with protein A conjugated to peroxidase. Enzymic activity was evidenced using the substrate 4-chloro-1-naphthol. A positive reaction produced a blue spot. The sensitivity of the assay was better and comparable to the indirect haemagglutination assay (IHA) and plate ELISA. The entire assay could be completed within 3 h. Antigen-loaded and pre-blocked nitrocellulose strips could be stored up to 3 months at room temperature and 37 degrees C.     

Validation of a competitive enzyme-linked immunosorbent assay for detection of antibodies against Babesia bovis

A previously developed competitive enzyme-linked immunosorbent assay (cELISA) based on a species-specific, broadly conserved, and tandemly repeated B-cell epitope within the C terminus of rhoptry-associated protein 1 of Babesia bovis was refined and validated for use internationally. Receiver operating characteristic analysis revealed an assay with a specificity and positive predictive value of 100% and a sensitivity of 91.1%, with various negative predictive values depending on the level of disease prevalence. The cELISA was distributed to four different laboratories, along with a reference set of 100 defined bovine sera, including known-positive, known-negative, and field samples. Pairwise concordances among the four laboratories ranged from 94% to 88%. Analysis of variance of the resulting optical densities and a test of homogeneity indicated no significant difference among the laboratories. Overall, the cELISA appears to have the attributes necessary for international application.     

Validation of a blocking enzyme-linked immunosorbent assay for detection of antibodies against porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome (PRRS) continues to be one of the most significant diseases of swine. IDEXX HerdChek PRRS, a commercially available enzyme-linked immunosorbent assay (ELISA), has become the industry standard for the detection of antibodies against PRRS virus (PRRSV). The need to accurately determine the PRRSV serostatus of herds and individual animals has prompted the development of several follow-up assay methods. A highly specific and repeatable blocking ELISA (bELISA) was developed on the basis of the use of an expressed PRRSV nucleocapsid (N) protein as the antigen and a biotinylated monoclonal antibody. Validation of the bELISA used sera from 316 animals experimentally and naturally infected with North American PRRSV and sera from 370 uninfected animals. Receiver operating characteristic analysis of the data calculated a diagnostic sensitivity of 97.8% and a diagnostic specificity of 100%. The between-run coefficient of variation of an internal quality control serum was 4.24%. The bELISA was able to detect seroconversion as well as the IDEXX ELISA and the indirect fluorescent antibody (IFA) assay; kappa values were 0.94 and 0.96, respectively. A collection of 133 serum samples with unexpected positive IDEXX ELISA results was obtained from 4,038 diagnostic samples submitted from farms from which PRRS-negative results were expected. The bELISA identified 97% of the samples as PRRS seronegative, while the IFA identified 100% as seronegative. The anticipated use of the bELISA is as a follow-up test to the IDEXX ELISA for determining the PRRSV serostatus of individual animals with unexpected positive test results from swine herds from which negative results are expected.     

Development of an enzyme-linked immunosorbent assay for immunoglobulin M antibodies against measles virus

Measles is a highly contagious respiratory virus infection, with typical clinical symptoms including maculopapular rash, fever, cough, coryza, and conjunctivitis. Despite implementation of widespread vaccination programs throughout the world, the rates of global morbidity and mortality are still considerable. This study was performed to design a reliable indirect enzyme-linked immunosorbent assay (ELISA) to measure measles-specific immunoglobulin M (IgM). First, human IgM was purified, and then an anti-IgM antibody was produced in rabbits and purified in a multistep process. The rabbit IgG against human IgM was conjugated with peroxidase. Measles virus-infected Vero cells produced viral antigen. One hundred serum samples from infants of 9 to 18 months of age, mostly vaccinated, were evaluated for determining the presence of specific IgM antibodies against measles virus. The samples were also evaluated for neutralizing antibodies against measles virus by a microneutralization test (MNT). By comparing the results of the ELISA with those of MNT, it was demonstrated that ELISA had a sensitivity and specificity of 100 and 92%, respectively. On the other hand, when the results obtained by our ELISA system were compared with those of an imported measles virus IgM ELISA kit (EIAgen; Adaltis Italia SPa, Bologna, Italy), a high level of agreement was shown (k = 0.926).     

Rapid enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies against

A rapid ELISA was developed for the detection of specific IgG against Micropolyspora faeni and Thermoactinomyces vulgaris and compared with counterimmunoelectrophoresis (CIE) in twenty-seven patients with suspected farmer's lung disease (FLD). Seventeen patients had precipitins to M. faeni or T. vulgaris or both, and ten had no precipitins. The optimum conditions for each step in the ELISA were determined: pre-equilibration of reagents at 37⁰C and vigorous agitation during incubation enabled the total test time required for the procedure to he reduced to 4 hr. A serum dilution of 10-fold produced good differentiation between CIE-positive and -negative sera. Little correlation was seen between CIE and ELISA for either M. faeni or T. vulgaris antigens in tests with sera from patients with precipitins: high readings were often recorded in ELISA where no precipitins had been detected with the same antigen. Positive- negative discrimination of unknown sera in ELISA was achieved through the inclusion of CIE-positive and -negative reference sera in each test run. Thirteen CIE-positive sera were classed as positive in ELISA with the M. faeni antigen while eight of thirteen CIE-positive sera were positive in ELISA with the T. vulgaris antigen. For both antigens, four CIE-negative sera were recorded as positive in ELISA.     

Comparison of the enzyme-linked immunosorbent assay (elisa), haemagglutination inhibition test and agar gel precipitation test for detection of antibodies to avian infectious bronchitis virus

The immune response after vaccination with infectious bronchitis virus (IBV) under field conditions was measured by the ELISA, haemagglu-tination-inhibition (HI) and agar-gel precipitin (AGP), tests. Vaccinations were performed in three flocks and one experimental group via the drinking water with the vaccine strains H 120 and H 52. In each flock 40 random serum samples were taken every 2 weeks and tested individually. In the experimental group blood samples were collected every week from each of the 10 chickens. The primary vaccination with H 120 resulted in a rapid increase of antibody titre as detected by ELISA followed by a slow decrease over the next few weeks. By the HI and AGP tests no antibody responses could be seen after this primary vaccination. Revaccination with the H 52 strain provoked a further increase in ELISA titres. In the experimental group, and in flock W, a similar increase occurred by the HI test and precipitating antibodies appeared. The formation of HI antibodies in flock T (nipple waterers) was somewhat retarded and precipitating antibodies were just detectable. In flock F revaccination did not result in the immediate production of HI and AGP antibodies. However, 6 weeks after revaccination a significant rise in ELISA, HI and AGP antibodies was observed, probably as the result of a field infection. It was demonstrated that, based on the higher sensitivity, the ELISA test is more suitable than HI and AGP to monitor antibody responses to vaccination against infectious bronchitis. Strain specificity in the HI test is discussed as a reason for its failure to detect antibodies after primary vaccination with the highly attenuated vaccine strain H 120.     

Evaluation of a commercial enzyme-linked immunosorbent assay for the detection of herpes simplex virus.

A total of 136 specimens were tested for the presence of herpes simplex virus by routine tissue culture and a commercial enzyme-linked immunosorbent assay (Ortho Diagnostic Systems, Inc.). Forty-six (33.8%) of the specimens were positive by tissue culture. The sensitivity and specificity of the commercial system were 69.6 and 93.3%, respectively. The commercial system was rapid and moderately specific but lacked the sensitivity necessary for direct specimen testing.     

Methods for the detection of anti-endothelial antibodies by enzyme-linked immunosorbent assay

In order to obtain a routine simple screening test for the detection of anti-endothelial antibodies (AEA) we developed a highly reproducible and sensitive enzyme-linked immunosorbent assay (ELISA) on fixed endothelial hybridoma cells. Detection of AEA with this type of monolayer appeared to be superior to ELISAs with monolayers of human umbilical vein endothelial cells, unfixed endothelial hybridoma cells or assays with membranes of endothelial cells. Glutaraldehyde treated endothelial hybridoma cells are most appropriate for use in ELISA procedures to detect AEA because the endothelial hybridoma cells are easy to culture, form a constant antigenic source and when plated and fixed to microtitre plates they can be stored without losing their ability to bind AEA.     

Evaluation of two commercial enzyme-linked immunosorbent assay kits for the detection of Mycoplasma gallisepticum antibodies

Sensitivity and specificity of two commercial Mycoplasma gallisepticum (MG) enzyme-linked immunosorbent assay (ELISA) kits, rapid slide agglutination (SA) and haemagglutination inhibition (HI) tests were compared using sera from specific pathogen free chickens, turkeys or ducks which had been inoculated with various avian mycoplasmas, bacteria or with a reovirus. Results show that sensitivity of SA was superior to ELISA and HI tests in the ability to detect antibodies formed in early response to MG infection. However, both ELISA kits and HI tests had a higher degree of specificity.