Development of an enzyme-linked immunosorbent assay for serological diagnosis of tick-borne encephalitis using subviral particles

The similarity of symptoms produced by tick-borne encephalitis (TBE) and Japanese encephalitis (JE) and the high degree of cross-reactivity between TBE and JE viruses by serological tests make the development of a differential diagnostic test a priority. In this study, recombinant prM/E proteins of TBE virus strain Oshima 5-10 expressed in mammalian cells resulted in the release of subviral particles (SPs) into the culture medium. Using the SPs as antigens, enzyme-linked immunosorbent assay (ELISA) systems were developed to detect TBE virus-specific IgM and IgG antibodies, designated SP-IgG and SP-IgM ELISAs, respectively. Of 83 serum samples from encephalitis patients in Khabarovsk, Russia, which were positive with the neutralization test (NT), 82 were positive by the SP-IgG ELISA, for a sensitivity of 98.8%, which was higher than that of a commercial ELISA kit. All 12 NT-negative samples were also negative by the SP-IgG ELISA (specificity, 100%). Of 17 patient samples that were NT-positive, 16 (94.1%) were positive by the SP-IgM ELISA. Of 15 paired serum samples that yielded equivocal results by NT, 11 had positive results with the SP-IgM ELISA, indicating a diagnosis of TBE infection. The SP-IgG and SP-IgM ELISAs showed no cross-reactivity with antibodies to the JE virus. The results indicate that these ELISAs will be useful for the detection of TBE-specific antibodies.     

The accuracy of an alternative confirmatory strategy for detection of antibodies to HIV-1: experience from a regional laboratory in Kagera, Tanzania.

BACKGROUND: Constant improvement of HIV tests often results in withdrawal of poorer quality tests by the manufacturing companies. It is thus often necessary to evaluate new HIV testing kits and modify the existing testing strategies. OBJECTIVES: To evaluate an alternative HIV antibody testing strategy which involves consecutive testing of sera by two enzyme-linked immunosorbent assays (ELISA), which both are recombinant antigen-based but utilise different test principles, followed by re-testing of sera giving discordant results. STUDY DESIGN: Sera (n = 1558) from a cross-sectional study of the HIV-1 seroprevalence in the Kagera region of Tanzania were tested using two ELISAs in parallel: Enzygnost anti-HIV-1/2 plus and Wellcozyme HIV-1 recombinant. Western blot analysis was done on all concordantly reactive and repeatedly discordant reactive samples as well as on 10% of concordantly ELISA negative sera. RESULTS: Two hundred and four sera (13.1%) were confirmed HIV-1-antibody positive. Both ELISAs had a sensitivity of 100%. The specificities of the ELISAs at initial and repeated testing were 99.8 and 99.9%, respectively, for Enzygnost and 97.7 and 99.5%, respectively, for Wellcozyme. None of the sera was concordantly false positive in both ELISAs. The mean ratio of the optical density of a sample to the cut off value of the test run (OD/CO ratio) was lower for samples giving false positive reactions than for confirmed HIV-1-antibody-positive samples. It is therefore important to interpret with caution HIV antibody ELISA test results on samples giving low OD/CO ratios. None of 10% of randomly selected concordantly ELISA negative sera gave a positive Western blot reaction. CONCLUSIONS: This field evaluation of an HIV antibody testing strategy involving the use of a recombinant antigen-based sandwich ELISA (Enzygnost) followed by a recombinant antigen-based competitive ELISA (Wellcozyme) showed that it had a sensitivity and specificity of 100%.     

Evaluation of two enzyme-linked immunosorbent assays for detecting Salmonella enterica subsp. enterica Serovar Dublin antibodies in bulk milk.

Two enzyme-linked immunosorbent assays (ELISAs) for the detecting Salmonella enterica subsp. enterica serovar Dublin antibodies in bulk milk were developed and evaluated for potential use in control programs. The ELISAs were based on either lipopolysacharide (LPS ELISA) or flagellar antigen (GP ELISA). Sensitivity was determined with 79 case herds with a wide range of clinical signs. Specificity was determined with 125 Dutch and 200 Swedish control herds. The relation between antibodies in bulk milk, antibodies in serum, and the level of milk production of individual cows was studied with 61 case herds. The optimal optical density (OD) values of the LPS ELISA and the GP ELISA were determined to be 0.2 and 0.5, respectively. The sensitivities of the LPS ELISA and the GP ELISA were 54 and 63%, respectively, with a specificity of 98% for both ELISAs with samples from the Dutch control herds. The specificities for samples from the Swedish herds were 100% for the LPS ELISA and 95% for the GP ELISA. The sensitivity of the combination of tests was 65% when samples were run in parallel, and the specificity was 100% when samples were run in series, irrespective of whether the samples came from Dutch or Swedish control herds. The variance (R(2)) in the OD value for bulk milk samples could be explained by the percentage of seropositive lactating cows in a herd with the LPS ELISA for 51% of the samples and with the GP ELISA for 72%. The variance in the OD value was best explained by the combination of the percentage of seropositive lactating cows in the herd and the mean log(10) serum antibody titer for that herd (R(2) = 62% for the LPS ELISA and R(2) = 75% for the GP ELISA). Case herds more often tested negative by the ELISA with bulk milk when the percentage of seropositive lactating cows was less than 5%. It is concluded that both ELISAs with bulk milk can be used in control programs to distinguish between infected and noninfected herds. Specificity can be increased by using the two tests in combination. Sensitivity was relatively low for both single tests and both tests combined.     

Enzyme-linked immunosorbent assay detection of immunoglobulins G and M to Venezuelan equine encephalomyelitis virus in vaccinated and naturally infected humans.

Enzyme-linked immunosorbent assays (ELISAs) were developed for detection of immunoglobulin G (IgG) and IgM antibodies to Venezuelan equine encephalomyelitis (VEE) virus in vaccinated and naturally infected humans. A total of 441 sera found negative for VEE antibodies by plaque reduction neutralization were examined by IgG ELISA and gave a 1.0% false-positive rate; no false-positives were found in the IgM ELISA. Sera with neutralizing antibody to western or eastern equine encephalomyelitis virus did not react with VEE antigen in the IgG ELISA. Sensitivity of the IgG ELISA was determined by testing 100 coded pre- and postvaccination human sera. Sixty-two were positive by ELISA; 58 of these 62 were also positive by neutralization tests, and 38 were negative by both tests. No neutralization-positive, ELISA-negative sera were found. Comparison of titers obtained by ELISA and neutralization tests indicated that 88% varied randomly by a fourfold dilution factor or less, while 61% were identical or varied only twofold. In sera obtained sequentially from 10 vaccinees and 5 naturally infected patients, both IgG and IgM antibodies appeared between 2 and 3 weeks after vaccination or onset of symptoms. The IgG and IgM antibody ELISAs described are rapid, specific, and sensitive indicators of VEE antibody status in vaccinated and naturally infected individuals.     

Characterization of rubella virus-specific antibody responses by using a new synthetic peptide-based enzyme-linked immunosorbent assay.

Rubella virus (RV)-specific immunoglobulin G antibodies were studied by enzyme-linked immunosorbent assay (ELISA) techniques in sera from RV (RA 27/3)-vaccinated individuals, patients experiencing natural RV infection, congenital rubella syndrome patients, and individuals failing to respond to repeated RV immunization. Results obtained by using whole-RV ELISAs (detergent-solubilized M33 strain or intact Gilchrist strain) and hemagglutination inhibition (HAI) and neutralization (NT) assays were compared with results obtained with the same sera by using ELISAs employing a synthetic peptide, BCH-178, representing a putative neutralization domain on the RV E1 protein. Murine RV E1-specific monoclonal antibodies with HAI and NT activities exhibited strong reactivity in ELISAs with BCH-178 peptide. In sera from RA 27/3-vaccinated individuals collected at 0 (prevaccine), 1, 2, 3, 4, 5, 6, 12, and 24 to 52 weeks postvaccine, the development of E1-peptide-reactive antibodies closely paralleled increases in RV-specific antibodies measured by whole-RV ELISAs and HAI and NT assays. Similarly, sequential serum samples obtained from patients during acute and convalescent phases of natural RV infection showed a coordinate increase in RV-specific antibodies as measured by whole-RV and peptide ELISAs. Conversely, congenital rubella syndrome patient sera, although exhibiting high levels of antibody in whole-RV ELISAs, had little or no antibody directed to the neutralization domain peptide. Sera from patients failing to respond to repeated RV immunization contained very low levels of RV-specific antibody in all ELISAs. Our results that the sequence represented by BCH-178 peptide may be a previously unidentified neutralization epitope for human antibodies on the RV E1 protein and may prove useful in determining effective RV immunity.     

Serologic diagnosis of Lyme borreliosis by using enzyme-linked immunosorbent assays with recombinant antigens

Class-specific enzyme-linked immunosorbent assays (ELISAs) with purified recombinant antigens of Borrelia burgdorferi sensu stricto and Western blot analyses with whole cells of this spirochete were used to test human sera to determine which antigens were diagnostically important. In analyses for immunoglobulin M (IgM) antibodies, 14 (82%) of 17 serum samples from persons who had erythema migrans reacted positively by an ELISA with one or more recombinant antigens. There was frequent antibody reactivity to protein 41-G (p41-G), outer surface protein C (OspC), and OspF antigens. In an ELISA for IgG antibodies, 13 (87%) of 15 serum samples had antibodies to recombinant antigens; reactivity to p22, p39, p41-G, OspC, and OspF antigens was frequent. By both ELISAs, serum specimens positive for OspB, OspE, and p37 were uncommon. Analyses of sera obtained from persons who were suspected of having human granulocytic ehrlichiosis (HGE) but who lacked antibodies to ehrlichiae revealed IgM antibodies to all recombinant antigens of B. burgdorferi except OspB and IgG antibodies to all antigens except OspE. Immunoblotting of sera from the study group of individuals suspected of having HGE reaffirmed antibody reactivity to multiple antigens of B. burgdorferi. There was minor cross-reactivity when sera from healthy subjects or persons who had syphilis, oral infections, or rheumatoid arthritis were tested by ELISAs with p37, p41-G, OspB, OspC, OspE, and OspF antigens. Although the results of class-specific ELISAs with recombinant antigens were comparable to those recorded for assays with whole-cell antigen and for individuals with confirmed clinical diagnoses of Lyme borreliosis, immunoblotting is still advised as an adjunct procedure, particularly when there are low antibody titers by an ELISA.     

Development and evaluation of ELISA procedures to detect antibodies against the major envelope protein (G(L)) of equine arteritis virus

Enzyme-linked immunosorbant assays (ELISAs) were developed for the detection of antibodies against the major envelope glycoprotein (G_L) of equine arteritis virus (EAV). A 6-Histidine tagged recombinant protein expressing the complete G_L ectodomain (G_L-6His), a glutathione-S-transferase recombinant protein expressing amino acids 55–98 of G_L (G_L-GST) and an ovalbumin-conjugated synthetic peptide representing amino acids 81–106 of G_L (G_L-OVA) were used as diagnostic antigens. An ELISA procedure was developed and optimised for each antigen. The G_L-OVA and G_L-6His assays showed the greatest specificity while the G_L-GST assay was slightly more sensitive that the G_L-OVA and G_L-6His assays; results based on the analysis of 50 virus neutralisation positive and 50 virus neutralisation negative sera. The G_L-OVA ELISA was selected for further evaluation since it was simpler to use than ELISAs based on recombinant antigens and did not suffer from background reactivity. The final sensitivity and specificity of the G_L-OVA ELISA were 96.75 and 95.6%, respectively, results based on the analysis of 400 virus neutralisation positive and 400 virus neutralisation negative sera. It also detected EAV antibody (100% efficiency) in seropositive shedding stallions and, in ponies infected experimentally with the UK93 isolate of EAV, the appearance of virus neutralising antibodies and G_L-OVA ELISA-specific immunoglobulins coincided.     

Disagreement between the results from three commercial tests for the detection of <Emphasis Type="Italic">Borrelia</Emphasis>-specific serum antibodies in the Netherlands associated with antibiotic treatment for Lyme borreliosis: a retrospective study

The diagnosis of Lyme borreliosis is challenging because of the often non-specific symptoms and persisting antibodies after infection. We investigated the diagnostic characteristics of two enzyme-linked immunosorbent assays (ELISAs) and an immunoblot for the detection of Borrelia-specific serum antibodies using different test strategies in individuals with and without antibiotic treatment for Lyme borreliosis. This retrospective study included healthy individuals, patients with active Lyme neuroborreliosis and patients treated for Lyme neuroborreliosis. Two ELISAs were compared: the C6 ELISA and the SERION ELISA. Equivocal and positive results were confirmed by immunoblot. We included 174 healthy individuals, of whom 27 (15.5%) were treated for Lyme borreliosis in the past, 36 patients were treated for Lyme neuroborreliosis and 27 patients had active Lyme neuroborreliosis. All the active Lyme neuroborreliosis patients were reactive in both ELISAs (100% sensitivity); less reactivity was seen in the other three groups (range 17.7% to 69.4%). The concordance between the ELISA results was high in active Lyme neuroborreliosis patients (26/27; 96.3%) and healthy individuals (131/147; 89.1%), but lower in treated healthy individuals (18/27; 66.7%) and treated Lyme neuroborreliosis patients (18/36; 50.0%) (p ≤ 0.005). This study showed that antibiotic treatment against Lyme borreliosis was strongly associated with discordant ELISA and test strategy results (odds ratio: 10.52; p < 0.001 and 9.98; p = 0.014, respectively) suggesting antibiotic treatment influences the pace at which the various antibodies directed to the different antigens used in both ELISAs wane. Among treated neuroborreliosis patients, the SERION ELISA stayed positive for a longer period after infection compared to the C6 ELISA. This should be taken into consideration when requesting and/or interpreting Lyme serology.     

Reliable confirmation of antibodies to human immunodeficiency virus type 1 (HIV-1) with an enzyme-linked immunoassay using recombinant antigens derived from the HIV-1 gag, pol, and env genes.

An enzyme-linked immunoassay (ELISA) using six recombinant proteins corresponding to large segments of the human immunodeficiency virus type 1 (HIV-1) gag, pol, and env gene products (HIVAGEN; SmithKline Bio-Science Laboratories, Van Nuys, Calif.) was developed to confirm the presence of antibodies to HIV-1 in sera reactive in the whole-cell-derived virion screening ELISAs. Serum samples for testing were obtained from healthy seronegative blood donors and from the different categories of HIV-infected individuals (asymptomatic, acquired immunodeficiency syndrome [AIDS]-related complex, and AIDS). A positive reaction was defined as reactivity against an env and at least one other (either gag or pol) HIV-1 gene product; negative was defined as no reaction with any antigen; and indeterminate was defined as reactivity with gag or pol (or both) or with env alone. None of the 1,180 serum samples from healthy seronegative blood donors gave a positive result, and only 49 of these samples (4%) gave indeterminate results. The recombinant HIV-1 antigen ELISA panel identified seropositive individuals with a high degree of accuracy, as a positive reaction was seen with 99.3% of asymptomatic healthy seropositive individuals, 98.1% of patients with AIDS-related complex, and 90.4% of patients with AIDS. None of the 725 HIV-1-seropositive subjects had a negative test result. Reactivity with the Kp41 antigen, corresponding to an amino-terminal portion of the gp41 envelope glycoprotein, by itself demonstrated 100% sensitivity and specificity in distinguishing seronegative from seropositive sera. A subset of seronegative and seropositive samples were tested both with the recombinant HIV-1 antigen ELISA panel and by Western blot (Du Pont Co.). The recombinant HIV-1 antigen ELISA panel accurately identified more seropositive and seronegative samples and had fewer indeterminate results than did Western blot (interpreted by Du Pont criteria).     

Development and evaluation of enzyme-linked immunosorbent assays for detection of shiga-like toxin I and shiga-like toxin II.

Shiga-like toxin (SLT)-producing Escherichia coli has been associated with a spectrum of human illnesses, including hemorrhagic colitis and hemolytic uremic syndrome. It produces at least two antigenically distinct toxins designated SLT-I and SLT-II, which have been implicated in disease. Currently available toxin assays, however, are not suitable for most clinical or public health laboratories. In this study, we have developed two sandwich enzyme-linked immunosorbent assays (ELISAs) based on toxin-specific murine monoclonal capture antibodies and rabbit polyclonal second antibodies which are specific for SLT-I and SLT-II. The SLT-I ELISA detected 200 pg of purified SLT-I, and the SLT-II ELISA detected 75 pg of purified SLT-II. The types of SLT produced by 166 human and 54 animal isolates of E. coli that produced moderate to high levels of toxin were determined by the ELISA, and results were confirmed by cytotoxin neutralization assays. With the exception of results from three strains, the tests agreed on the types of toxin present. DNA probe assays of 86 of 87 isolates also agreed with the ELISA and neutralization results. Although the SLT-II ELISA was specific for the SLT-II variant produced by porcine edema strains, most of the isolates examined produced levels of toxin (less than 50 50% cytotoxic doses [CD50] per ml) below the detection limit of the test. The ELISAs were not sufficiently sensitive to consistently detect low levels of toxin (less than 50 CD50 per ml) found in fecal extracts. On the basis of these findings, both ELISAs appeared to detect significant levels of SLT-I ( > 100 CD50 per ml) and SLT-II ( > 50 CD50 per ml) in E. coli culture extracts and should be useful diagnostic tools in many microbiology laboratories.     

Immunoglobulin G, A, and M responses to BK virus in renal transplantation

Immunoglobulin G (IgG), IgA, and IgM antibodies were measured in serum samples from 71 organ donors, 81 kidney transplant recipients at transplantation, and 67 patients during the posttransplant period by using a virus-like particle-based enzyme-linked immunosorbent assay (ELISA). BK virus (BKV) and JC virus DNA were detected in urine and plasma by real-time PCR. IgG antibodies to BKV were demonstrated in the majority (80.3 to 100%) of patients irrespective of clinical category, but titers were highest in patients with active viral replication. IgA antibodies were present with greater frequency (72.7 to 81.3% versus 0 to 23.6%; P < 0.001) and higher titer (mean optical density, 0.11 to 0.15 versus 0.05 to 0.08; P < 0.001) in patients who were BKV DNA positive than those who were BKV DNA negative. IgM antibodies showed a similar pattern of reactivity but lower frequency in the setting of active viral replication (9.1 to 43.7% versus 0 to 1.4%; P < 0.001). A rise in IgG level of >0.577 optical density (OD) units or a rise in IgA or IgM level of >0.041 OD units was strongly associated with active viral replication. Urine viral load showed a positive correlation with IgM titer (r = 0.22) but a negative correlation with IgG titer (r = -0.28) and IgA titer (r = -0.1). Chronic dialysis patients typically did not have serologic or virologic evidence of active BKV infection. Anti-BKV titers did not rise in patients with JC viruria. In conclusion, measurement of anti-BKV antibody titer and class response can be used to detect the onset of viral replication. ELISAs can be quite specific despite considerable sequence homology between BK virus and JC virus.     

Evaluation of Two Enzyme-Linked Immunosorbent Assays for Detecting Salmonella enterica subsp. enterica Serovar Dublin Antibodies in Bulk Milk

Two enzyme-linked immunosorbent assays (ELISAs) for the detecting Salmonella enterica subsp. enterica serovar Dublin antibodies in bulk milk were developed and evaluated for potential use in control programs. The ELISAs were based on either lipopolysacharide (LPS ELISA) or flagellar antigen (GP ELISA). Sensitivity was determined with 79 case herds with a wide range of clinical signs. Specificity was determined with 125 Dutch and 200 Swedish control herds. The relation between antibodies in bulk milk, antibodies in serum, and the level of milk production of individual cows was studied with 61 case herds. The optimal optical density (OD) values of the LPS ELISA and the GP ELISA were determined to be 0.2 and 0.5, respectively. The sensitivities of the LPS ELISA and the GP ELISA were 54 and 63%, respectively, with a specificity of 98% for both ELISAs with samples from the Dutch control herds. The specificities for samples from the Swedish herds were 100% for the LPS ELISA and 95% for the GP ELISA. The sensitivity of the combination of tests was 65% when samples were run in parallel, and the specificity was 100% when samples were run in series, irrespective of whether the samples came from Dutch or Swedish control herds. The variance (R²) in the OD value for bulk milk samples could be explained by the percentage of seropositive lactating cows in a herd with the LPS ELISA for 51% of the samples and with the GP ELISA for 72%. The variance in the OD value was best explained by the combination of the percentage of seropositive lactating cows in the herd and the mean log₁₀ serum antibody titer for that herd (R² = 62% for the LPS ELISA and R² = 75% for the GP ELISA). Case herds more often tested negative by the ELISA with bulk milk when the percentage of seropositive lactating cows was less than 5%. It is concluded that both ELISAs with bulk milk can be used in control programs to distinguish between infected and noninfected herds. Specificity can be increased by using the two tests in combination. Sensitivity was relatively low for both single tests and both tests combined.     

Evaluation of two enzyme immunoassays for detection of immunoglobulin G antibodies to mumps virus

To determine suitability for national serosurveys, we compared two commercial enzyme-linked immunosorbent assays (ELISAs) for mumps antibody, Enzygnost Anti-Parotitis-Virus/IgG (which uses a whole-virus antigen) and Microimmune Mumps IgG Screen ELISA (which uses a recombinant nucleoprotein antigen), by testing 1,915 opportunistically collected sera submitted to diagnostic laboratories across Australia in 1997 to 1998. The proportion of positive results increased with age in both ELISAs but was significantly higher with the Microimmune than with the Enzygnost ELISA overall (88% versus 63%; P < 0.01) and in all age groups. However, the proportion of equivocal results was significantly higher with the Enzygnost than with the Microimmune ELISA (9% versus 4%; P < 0.01). Of the 572 sera with discrepant or equivocal results, 508 had sufficient sample remaining to perform the neutralization test (NT). A proportion with concordant results in both ELISAs were also tested by the NT. For sera with discrepant results, there was significantly better agreement between the NT and Microimmune than between the NT and Enzygnost (310/444 [70%] versus 135/348 [39%]; P < 0.01). Of 64 sera with equivocal Microimmune results, 45 (70%) were positive in the NT compared with 140 of 160 (88%) equivocal Enzygnost results (P < 0.01). Compared with the NT, the Microimmune ELISA is more sensitive (96% versus 80%) but apparently less specific (36% versus 85%) than the Enzygnost ELISA. However, this is likely to be due to the generally lower sensitivity of the NT, since the Microimmune results reflect expected seroprevalence, based on vaccine uptake in the age groups studied. We conclude that the Microimmune ELISA is a more appropriate assay than the Enzygnost ELISA for estimation of mumps seroprevalence.     

Evaluation of five antibody detection tests for diagnosis of bovine paratuberculosis

Five diagnostic tests based on enzyme-linked immunosorbent assay (ELISA) technology for bovine paratuberculosis were evaluated by using individual serum or milk samples from 359 dairy cattle in seven paratuberculosis-free herds and 2,094 dairy cattle in seven Mycobacterium paratuberculosis-infected dairy herds. Three independent laboratories using three different culture procedures completed fecal cultures for M. paratuberculosis on these cattle and found 417 cows to be shedding M. paratuberculosis in their feces. An animal that was fecal culture positive for M. paratuberculosis by any of the three laboratories was considered a confirmed case of infection. The specificity of three ELISAs (two on serum and one on milk) was > or =99.8%. The specificity of the remaining two ELISAs, both done on serum, was 94.9 and 84.7%. Four of the five ELISAs evaluated produced similar sensitivity in detecting fecal culture-positive cattle (27.8 to 28.9%). Serum ELISA "D" had the lowest specificity (84.7%) and the highest sensitivity (44.5%), but if the cutoff value defining a positive test was changed from 125 to 250% (of the positive control) the sensitivity and specificity, 31.8 and 97.5%, respectively, were comparable to those of the other four assays. If the case definition for M. paratuberculosis infection was based on the culture results of a single laboratory instead of the combined results of three laboratories, ELISA sensitivity estimates were 45.7 to 50.0%. With the exception of ELISA D, assay agreement was high (kappa 0.66 to 0.85) for categorical assay interpretations (positive or negative), but linear regression of quantitative results showed low correlation coefficients (r(2) = 0.40 to 0.68) due to the fact that ELISA results for some cows were high in one assay but low in another assay. Likelihood ratio analysis showed a direct relationship between the magnitude of ELISA result and the odds of a cow shedding M. paratuberculosis in its feces. If used judiciously and interpreted quantitatively, these ELISAs are useful tools in support of paratuberculosis control programs in dairy herds.     

Optimized enzyme-linked immunosorbent assay for detecting cytomegalovirus infections during clinical trials of recombinant vaccines

BackgroundIn clinical trials of cytomegalovirus (CMV) glycoprotein B (gB) vaccines, CMV infection is detected by first depleting serum of anti-gB antibodies and then measuring anti-CMV antibodies with a commercially available enzyme-linked immunosorbent assay (ELISA) kit, with confirmation of positive findings by immunoblot.ObjectivesIdentification of CMV immunoantigens for the development of an ELISA that detects specifically CMV infection in clinical samples from individuals immunized with gB vaccines.Study designSensitivity and specificity of ELISAs using antigenic regions of CMV proteins UL83/pp65, UL99/pp28, UL44/pp52, UL80a/pp38, UL57, and UL32/pp150 were measured.ResultsAn IgG ELISA using a UL32/pp150 [862–1048] capture peptide was the most specific (93.7%) and sensitive (96.4%) for detecting CMV-specific antibodies in sera. The ELISA successfully detected CMV-specific antibodies in 22 of 22 sera of subjects who had been vaccinated with a gB vaccine but who had later been infected with CMV. The ELISA was linear over a wide range of CMV concentrations (57–16,814 ELISA units/mL) and was reproducible as indicated by a 5% intra-day and 7% inter-day coefficients of variation. The signal was specifically competed by UL32/pp150 [862–1048] peptide but not by CMV-gB or herpes simplex virus 2 glycoprotein D. Lipid and hemoglobin matrix did not interfere with the assay.ConclusionThe UL32/pp150 [862–1048] IgG ELISA can be used for the sensitive and specific detection of CMV infection in gB-vaccinated individuals.     

Development and evaluation of serological assays for detection of Hantaanvirus-specific antibodies in human sera using yeast-expressed nucleocapsid protein

Indirect and capture enzyme-linked immunosorbent assays (ELISAs) for detection of Hantaan virus (HTNV)-specific immunoglobulins G (IgG) and M (IgM) in human serum samples were developed on the basis of recombinant yeast-expressed nucleocapsid (N) protein of HTNV. The sensitivities and specificities of the indirect and capture ELISAs were evaluated by comparing the reactivity of sera from patients with hemorrhagic fever with renal syndrome (HFRS) from China with that of a commercial IgG/IgM kit. The sensitivity of the indirect IgG and IgM ELISA tests was both 100% and the specificity of the indirect IgM and IgG ELISA test was 98% and 99%, respectively. The sensitivity and specificity of the capture IgM ELISA was 100% and 97%, respectively. The novel assays were found to detect HTNV-specific antibodies in acute phase sera from suspected HFRS patients in China. The results indicate that these novel ELISAs are suitable for the diagnosis of HTNV and for sero-epidemiological studies.     

Validation of five commercially available ELISAs for the detection of antibodies against infectious bursal disease virus (serotype 1)

In this study, the results are reported from a validation study of five commercially available enzymelinked immunosorbent assays (ELISAs) for the detection of antibodies against infectious bursal disease virus (IBDV), serotype 1. The specificity of the ELISAs varied from 63.8 to 100%. All ELISAs reached a sensitivity of 100% on sera between 14 and 21 days post-vaccination (d.p.v.) with two classical vaccines and a Delaware variant-E virus. Overall, most birds became positive between 8 and 11 d.p.v. As expected, the ELISA with the lowest specificity showed the highest sensitivity at 5 d.p.v. When the decrease in maternally derived antibodies against IBDV was measured, a highly significant correlation (P < 0.001) was found for all ELISAs and the virus neutralization test (VNT). R ² varied from 0.44 to 0.76, whereas the slope varied from 0.33 to 0.57. This indicates that there is a certain correlation between VNT and ELISA titres, but that the correlation differs from ELISA to ELISA. For all ELISAs, no significant (P < 0.05) differences in level of antibodies were detected between antibody titres in breeder serum, egg yolk and progeny at 1 and 4 days of age. This validation of five commercially available ELISAs for the detection of antibodies against IBDV (serotype 1) shows that there are significant differences in their performance. Therefore, choosing an ELISA or interpreting ELISA results without knowing the technical performance should be avoided.     

Common and different antigenic properties of the rabies virus glycoprotein of strains SAD-Vnukovo and Pitman-Moore.

Two fixed rabies virus strains, SAD-Vnukovo and Pitman-Moore (PM) were used as combined immunogens for the generation of hybridomas secreting specific monoclonal antibodies (MoAbs). The obtained hybridomas were primarily screened by an ELISA for production of MoAbs to antigen of SAD-Vnukovo strain. Six positive clones were established. A panel of MoAbs has been characterized according to reactivity in immunofluorescence, immunoblot, ELISA and neutralization tests. All MoAbs were positive in immunofluorescence when cells infected with the SAD-Vnukovo strain were used. By immunoblot, four MoAbs showed specificity for the viral glycoprotein of both SAD-Vnukovo and PM rabies strains. This pattern of reactivity indicated the existence of shared conformation-independent epitopes located on the related antigens. However, in ELISA, the tested MoAbs did not recognize viral glycoproteins of the PM strain. This indicates, that the different strain-specific conformations of the native glycoprotein determine the accessibility of the common linear determinants for respective antibodies. Only one antibody, with conformation-dependent glycoprotein specificity, was capable to neutralize the CVS strain of rabies virus.     

Evaluation of Five Antibody Detection Tests for Diagnosis of Bovine Paratuberculosis

Five diagnostic tests based on enzyme-linked immunosorbent assay (ELISA) technology for bovine paratuberculosis were evaluated by using individual serum or milk samples from 359 dairy cattle in seven paratuberculosis-free herds and 2,094 dairy cattle in seven Mycobacterium paratuberculosis-infected dairy herds. Three independent laboratories using three different culture procedures completed fecal cultures for M. paratuberculosis on these cattle and found 417 cows to be shedding M. paratuberculosis in their feces. An animal that was fecal culture positive for M. paratuberculosis by any of the three laboratories was considered a confirmed case of infection. The specificity of three ELISAs (two on serum and one on milk) was ≥99.8%. The specificity of the remaining two ELISAs, both done on serum, was 94.9 and 84.7%. Four of the five ELISAs evaluated produced similar sensitivity in detecting fecal culture-positive cattle (27.8 to 28.9%). Serum ELISA “D” had the lowest specificity (84.7%) and the highest sensitivity (44.5%), but if the cutoff value defining a positive test was changed from 125 to 250% (of the positive control) the sensitivity and specificity, 31.8 and 97.5%, respectively, were comparable to those of the other four assays. If the case definition for M. paratuberculosis infection was based on the culture results of a single laboratory instead of the combined results of three laboratories, ELISA sensitivity estimates were 45.7 to 50.0%. With the exception of ELISA D, assay agreement was high (kappa 0.66 to 0.85) for categorical assay interpretations (positive or negative), but linear regression of quantitative results showed low correlation coefficients (r² = 0.40 to 0.68) due to the fact that ELISA results for some cows were high in one assay but low in another assay. Likelihood ratio analysis showed a direct relationship between the magnitude of ELISA result and the odds of a cow shedding M. paratuberculosis in its feces. If used judiciously and interpreted quantitatively, these ELISAs are useful tools in support of paratuberculosis control programs in dairy herds.