scholarly journals Laboratory Diagnosis of African Horse Sickness: Comparison of Serological Techniques and Evaluation of Storage Methods of Samples for Virus Isolation

1990 ◽  
Vol 2 (1) ◽  
pp. 44-50 ◽  
Author(s):  
Carol House ◽  
Peter E. Mikiciuk ◽  
Mary Lou Beminger
Keyword(s):  
Virus Isolation ◽  
Fluorescent Antibody ◽  
False Negative ◽  
Laboratory Diagnosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
African Horse Sickness ◽  
Serum Samples ◽  
Negative Results ◽  
Field Samples ◽  
Storage Methods

Five serological methods of diagnosing African horse sickness were evaluated, using a battery of serum samples from experimental horses vaccinated and challenged with each serotype of African horse sickness virus (AHSV1 through AHSV9): agar gel immunodiffusion (AGID), indirect fluorescent antibody (IFA), complement fixation (CF), virus neutralization (VN), and enzyme-linked immunosorbent assay (ELISA). The 5 tests were also compared using a panel of field samples, convalescent equine sera with antibodies to domestic equine viral diseases, and sera from horses awaiting export. The ELISA described in this paper was group specific. It did not require calibration with a standard positive serum but did yield elevated values with negative sera that were repeatedly frozen and thawed or heat inactivated. The IFA test was sensitive but could not be used on some field sera as the control cells exhibited fluorescence, possibly due to the animal being recently vaccinated with cell culture material. Sixty-two experimental sera were compared by VN, CF, AGID, and ELISA. Forty sera, 10 positive and 30 negative, were correctly classified by the 5 serologic assays. The 22 remaining sera gave mixed reactions. The AGID had no false positive results but had false negative results for up to 20% of the samples, depending upon the comparison. The VN, CF, and ELISA were similar in their variability. The length of time that virus could be recovered from a viremic blood sample was compared in an evaluation of storage methods for virus isolation samples. Washed erythrocytes were held at 4 C, washed erythrocytes plus stabilizer were held at −70 C, and blood that was drawn into a preservative (oxalate/phenol/glycerol) was held at 4 C. Virus was isolated for 12 months from a sample stored as washed erythrocytes at 4 C but for only 6 months from the same blood stored by the other 2 methods.

Three highly sensitive "bedside" serum and urine tests for pregnancy compared

1990 ◽  
Vol 36 (9) ◽  
pp. 1686-1688 ◽  
Author(s):  
H Christensen ◽  
H H Thyssen ◽  
O Schebye ◽  
A Berget
Keyword(s):  
Paired Comparison ◽  
False Negative ◽  
Diagnostic Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Negative Results ◽  
False Negative Results ◽  
Significant Difference ◽  
Comparison Of The Results ◽  
Urine And Serum

Abstract We examined three enzyme-linked immunosorbent assay (ELISA) kits for human choriogonadotropin (hCG) (pregnancy tests) for use with urine and serum samples: the Tandem Icon II hCG Urine and Tandem Icon II hCG Serum, the NovoClone Target hCG Test, and the Abbott TestPacks hCG-urine and hCG-serum. Paired comparison of the results from each kit indicated that the NovoClone Target assay showed significantly lower diagnostic sensitivity (P less than 0.05) than did the Tandem Icon II or Abbott TestPack, both for urine and for serum samples. None of the products demonstrated any significant difference (P greater than 0.05) in diagnostic specificity, but the NovoClone Target kit showed several serious false-negative results with both urine and serum. Paired testing of urine kits vs serum kits also showed no significant differences (P greater than 0.05) in diagnostic sensitivity or specificity. We found the Abbott kits to be the most convenient to use and to read.

scholarly journals Comparison between available serologic tests for detecting antibodies againstAnaplasma phagocytophilumandBorrelia burgdorferiin horses in Canada

2015 ◽  
Vol 27 (4) ◽  
pp. 540-546 ◽  
Author(s):  
Gili Schvartz ◽  
Tasha Epp ◽  
Hilary J. Burgess ◽  
Neil B. Chilton ◽  
Katharina L. Lohmann
Keyword(s):  
Point Of Care ◽  
Fluorescent Antibody ◽  
Clinical Status ◽  
False Negative ◽  
Pairwise Comparison ◽  
Cross Reactivity ◽  
Multiplex Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Serologic Tests

To investigate the agreement between available serologic tests for the detection of antibodies against Anaplasma phagocytophilum and Borrelia burgdorferi, 50 serum samples from horses of unknown clinical status and at low risk for infection were tested. In addition to a point-of-care enzyme-linked immunosorbent assay (pocELISA), the evaluated tests included 2 indirect fluorescent antibody tests (IFATs) for antibodies against A. phagocytophilum and an IFAT, an ELISA confirmed with Western blot, and the Lyme multiplex assay for antibodies against B. burgdorferi. For each pair-wise comparison between serologic tests, the difference in the proportion of seropositive results as well as kappa and the prevalence-adjusted, bias-adjusted kappa were calculated. The proportion of seropositive results differed significantly in each pairwise comparison of tests for detection of antibodies against A. phagocytophilum, and between the pocELISA and IFAT as well as between the pocELISA and Lyme multiplex assay for detection of antibodies against B. burgdorferi. Agreement based on kappa varied from poor to fair while agreement was improved when evaluating prevalence-adjusted, bias-adjusted kappa. Lack of agreement may be explained by differences in methodology between the evaluated tests, cross-reactivity or false-positive and false-negative tests. In addition to the limitations of serologic test interpretation in the absence of clinical disease, this data suggest that screening of horses for exposure to tick-borne diseases in nonendemic areas may not be warranted.

scholarly journals Validation of a Blocking Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against Porcine Reproductive and Respiratory Syndrome Virus

2004 ◽  
Vol 11 (3) ◽  
pp. 503-514 ◽  
Author(s):  
Neal H. Ferrin ◽  
Ying Fang ◽  
Craig R. Johnson ◽  
Michael P. Murtaugh ◽  
Dale D. Polson ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Fluorescent Antibody ◽  
Internal Quality ◽  
Respiratory Syndrome Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Characteristic Analysis ◽  
Serum Samples ◽  
Negative Results ◽  
Control Serum

ABSTRACT 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.

scholarly journals Performance of Indirect Immunoglobulin M (IgM) Serology Tests and IgM Capture Assays for Laboratory Diagnosis of Measles

2000 ◽  
Vol 38 (1) ◽  
pp. 99-104
Author(s):  
Samuel Ratnam ◽  
Graham Tipples ◽  
Carol Head ◽  
Micheline Fauvel ◽  
Margaret Fearon ◽  
...  
Keyword(s):  
Measles Virus ◽  
False Negative ◽  
Laboratory Diagnosis ◽  
Immunoglobulin M ◽  
Confirmatory Test ◽  
Serum Samples ◽  
Negative Results ◽  
Capture Assay ◽  
Specimen Collection ◽  
Control And Prevention

ABSTRACT As progress is made toward elimination of measles, the laboratory confirmation of measles becomes increasingly important. However, both false-positive and false-negative results can occur with the routinely used indirect measles immunoglobulin M (IgM) serology tests. The measles IgM capture assay is considered to be more specific, and therefore, its use is indicated for confirmatory testing, but its relative performance has not been fully assessed. Four commercial indirect measles IgM serology test kits (the Behring, Clark, Gull, and PanBio assays) and a commercial IgM capture assay (the Light Diagnostics assay) were evaluated for their abilities to detect measles virus-specific IgM antibody with a total of 308 serum samples from patients involved in a measles outbreak and with confirmed cases of measles and 454 samples from subjects without measles. The Centers for Disease Control and Prevention (CDC) IgM capture assay was also used in a part of the evaluation. Among the indirect assays, the overall sensitivities ranged from 82.8% (Clark assay) to 88.6% (Behring assay) and specificity ranged from 86.6% (PanBio assay) to 99.6% (Gull assay). These rates were 92.2 and 86.6%, respectively, for the Light Diagnostics capture assay and 87.0 and 94.8%, respectively, for the CDC capture assay. While the Light Diagnostics capture assay had the best detection rate (80%) with the acute-phase samples compared with those for the rest of the tests (CDC capture assay, 77%; Behring assay, 70%; Gull assay, 69%; PanBio assay, 58%; and Clark assay, 57%), all tests showed a significantly improved sensitivity in the range of 92% (Clark and PanBio assays) to 97% (Light Diagnostics and CDC capture assays) with the convalescent-phase samples, as expected. The best seropositivity rates (in the range of 92 to 100%) were observed with samples collected 6 to 14 days after the onset of symptoms. The Gull assay showed the highest positive predictive value (99.6%), followed by the Behring assay (97.8%) and the CDC capture assay (96.1%). Overall, the Gull and Behring assays were found to be as good as or better than the capture assays. In conclusion, laboratory diagnosis of measles based on IgM serology varies depending on the timing of specimen collection and the test used, and the case for the use of the IgM capture assay as the confirmatory test appears to be uncertain.

scholarly journals Early Detection of Maedi-Visna (Ovine Progressive Pneumonia) Virus Seroconversion in Field Sheep Samples

2001 ◽  
Vol 13 (4) ◽  
pp. 301-307 ◽  
Author(s):  
R. Varea ◽  
E. Monleón ◽  
C. Pacheco ◽  
L. Luján ◽  
R. Bolea ◽  
...  
Keyword(s):  
Early Detection ◽  
False Negative ◽  
Field Conditions ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Negative Results ◽  
Visna Virus ◽  
False Negative Results ◽  
Ovine Progressive Pneumonia ◽  
Antibody Levels

The aim of this work was to investigate whether an enzyme-linked immunosorbent assay (ELISA) was useful for early detection of maedi-visna virus (MVV) infection in sheep under field conditions. An ELISA based on p25 recombinant protein and a gp46 synthetic peptide was used. Sequentially obtained serum samples ( n = 1,941) were studied for 4 years. ELISA results were compared with those of the agar gel immunodiffusion (AGID) test, and results of both tests were compared with a reference result established using consensus scores for at least 2 of 3 serologic techniques (AGID, ELISA, and western blotting, which was used to resolve result discrepancies between the other 2 techniques). A total of 247 discrepancies were observed between ELISA and AGID. Of these, 131 were due to an earlier detection of 120 sera by the ELISA and 11 sera by AGID. The remaining discrepancies (116) were due to the presence of false reactions in both tests. Fewer false-negative results were found by ELISA than with AGID (6 vs. 69 sera, respectively), whereas the number of false-positive results was virtually the same for ELISA and AGID (21 vs. 20, respectively). In relation to the reference result, ELISA sensitivity and specificity were 97.8% and 98.2%, respectively, whereas values for AGID were 76.3% and 98.3%, respectively. The agreement between ELISA and the reference result was higher than that between AGID and the reference result (κ value: 0.96 and 0.77, respectively). A variation in the ELISA signal (based on optical density) was observed during the study period, suggesting different antibody levels throughout the animal's life. The ELISA was useful for detecting MVV-infected sheep in field conditions and has potential for use in control and eradication programs.

scholarly journals Simultaneous Detection of Antibodies to Mouse Hepatitis Virus Recombinant Structural Proteins by a Microsphere-Based Multiplex Fluorescence Immunoassay

2011 ◽  
Vol 18 (5) ◽  
pp. 758-766 ◽  
Author(s):  
Satoshi Kunita ◽  
Kanako Kato ◽  
Miyuki Ishida ◽  
Kozue Hagiwara ◽  
Shuko Kameda ◽  
...  
Keyword(s):  
False Positive ◽  
Hepatitis Virus ◽  
Fluorescent Antibody ◽  
Laboratory Mouse ◽  
False Negative ◽  
Mouse Hepatitis Virus ◽  
Simultaneous Detection ◽  
False Negative Rate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples

ABSTRACTWe describe a new microsphere-based multiplex fluorescent immunoassay (MFI) using recombinant mouse hepatitis virus (MHV) proteins to detect antibodies to coronaviruses in mouse and rat sera. All the recombinant proteins, including nucleocapsid (N) and 3 subunits of spike protein, S1, S2, and Smid, showed positive reactivity in MFI with mouse antisera to 4 MHV strains (MHV-S, -A59, -JHM, and -Nu67) and rat antiserum to a strain of sialodacryoadenitis virus (SDAV-681). The MFI was evaluated for its diagnostic power, with panels of mouse sera classified as positive or negative for anti-MHV antibodies by enzyme-linked immunosorbent assay (ELISA) using MHV virion antigen and indirect fluorescent antibody assay. The reactivities of 236 naturally infected mouse sera were examined; 227 samples were positive by MFI using S2 antigen (96% sensitivity), and 208 samples were positive using N antigen (88% sensitivity). Based on the assessment by MFI using the S2 and N antigens, only 3 serum samples showed double-negative results, indicating a false-negative rate of 1.3%. In 126 uninfected mouse sera, including 34 ELISA false-positive sera, only 7 samples showed false-positive results by MFI using either the S2 or N antigen (94% specificity). Similarly, the S2 and N antigen-based MFI was 98% sensitive and 100% specific in detecting anticoronavirus antibodies in rat sera. Thus, this MFI-based serologic assay using the S2 and N antigens promises to be a reliable diagnostic method, representing a highly sensitive and specific alternative to traditional ELISA for detection of coronavirus infections in laboratory mouse and rat colonies.

scholarly journals Evaluation of Recombinant Proteins of Burkholderia mallei for Serodiagnosis of Glanders

2012 ◽  
Vol 19 (8) ◽  
pp. 1193-1198 ◽  
Author(s):  
Vijai Pal ◽  
Subodh Kumar ◽  
Praveen Malik ◽  
Ganga Prasad Rai
Keyword(s):  
Sensitivity And Specificity ◽  
Recombinant Proteins ◽  
False Negative ◽  
Enzyme Linked Immunosorbent Assay ◽  
Burkholderia Mallei ◽  
Content Type ◽  
Whole Cells ◽  
Negative Results ◽  
Elisa Format ◽  
Low Sensitivity

ABSTRACTGlanders is a contagious disease caused by the Gram-negative bacillusBurkholderia mallei. The number of equine glanders outbreaks has increased steadily during the last decade. The disease must be reported to the Office International des Epizooties, Paris, France. Glanders serodiagnosis is hampered by the considerable number of false positives and negatives of the internationally prescribed tests. The major problem leading to the low sensitivity and specificity of the complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e., crude preparations of whole cells. False-positive results obtained from other diagnostic tests utilizing crude antigens lead to financial losses to animal owners, and false-negative results can turn a risk into a possible threat. In this study, we report on the identification of diagnostic targets using bioinformatics tools for serodiagnosis of glanders. The identified gene sequences were cloned and expressed as recombinant proteins. The purified recombinant proteins ofB. malleiwere used in an indirect ELISA format for serodiagnosis of glanders. Two recombinant proteins, 0375H and 0375TH, exhibited 100% sensitivity and specificity for glanders diagnosis. The proteins also did not cross-react with sera from patients with the closely related disease melioidosis. The results of this investigation highlight the potential of recombinant 0375H and 0375TH proteins in specific and sensitive diagnosis of glanders.

Fluorescent antibody techniques have allowed for the direct identification and enumeration of individual bacteria in environmental samples without requiring prior growth in culture media (Bahlool and Schmidt 1980, Cloete and Steyn 1988, Macario et al. 1989). The technique involves the use of specific antibodies raised against surface markers of defined pure cultures that are either labelled directly with fluorescent dye molecules or via a fluorescent secondary antibody. This approach has yielded important insights into the spatial distribution of microorganisms, but it suffers from a number of disadvantages. For example, expression of the antigen may be influenced by environmental factors; false-positive and false-negative results may be obtained due to cross-reactivity or lack of reaction; non-specific binding of antibodies may result in high levels of background fluorescence; and production of specific antibodies requires a pure culture of the organism of interest (Cloete and de Bruyn Various recombinant DNA techniques have subsequently been developed that are independent of cultivation methods (Fig. 1). These techniques provide ways of detecting and quantifying specific phylogenetic groups of microbes on 16S rDNA sequences, and relevant structural genes provide ways of monitoring microbial populations of environmental and industrial systems. In addition to these tools, a number of emerging technologies such as the use of biomarker genes are being increasingly used to monitor with great precision and accuracy the behaviour of microbes in the environment.

2003 ◽  
pp. 218-219
Keyword(s):  
Recombinant Dna ◽  
Culture Media ◽  
Specific Binding ◽  
Fluorescent Antibody ◽  
False Negative ◽  
Cross Reactivity ◽  
Phylogenetic Groups ◽  
Specific Antibodies ◽  
Negative Results ◽  
Recombinant Dna Techniques

scholarly journals False negative results in the Roche assay for HDL-cholesterol

2003 ◽  
Vol 40 (5) ◽  
pp. 572-575 ◽  
Author(s):  
Andries J Bakker ◽  
Appie Zijlstra ◽  
Marten P Leemhuis
Keyword(s):  
Cardiovascular Disease ◽  
Risk Evaluation ◽  
False Negative ◽  
Hdl Cholesterol ◽  
Daily Practice ◽  
Vascular Risk ◽  
Serum Samples ◽  
Negative Results ◽  
Risk Estimates ◽  
False Negative Results

Measurement of HDL-cholesterol is important in the risk evaluation of cardiovascular disease. Recently, we encountered a 61-year-old man with a negative result for HDL-cholesterol using the Roche assay. Further analysis revealed that this patient had a monoclonal Ig type IgM κ, with an electrophoresis-based concentration of 55.8 g/L, which apparently caused the negative result for HDL-cholesterol. Analysis of serum samples from other patients with monoclonal or polyclonal hypergammaglobulinaemia revealed that false negative results in the Roche assay for HDL-cholesterol may occur incidentally. Since negative results are detected easily in the laboratory, such patients do not pose a problem in daily practice. However, it is reasonable to suppose that false low results could also be caused by monoclonal as well as polyclonal Igs. Since such false low results remain undetected, vascular risk estimates consequently are altered and unnecessary treatment might be started.

scholarly journals Diagnosis of Visceral Leishmaniasis by Enzyme-Linked Immunosorbent Assay Using Urine Samples

2002 ◽  
Vol 9 (4) ◽  
pp. 789-794 ◽  
Author(s):  
Mohammad Zahidul Islam ◽  
Makoto Itoh ◽  
S. M. Shamsuzzaman ◽  
Rusella Mirza ◽  
Farzana Matin ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Sensitivity And Specificity ◽  
Immunosorbent Assay ◽  
Leishmania Donovani ◽  
Laboratory Diagnosis ◽  
Urine Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Healthy Controls ◽  
Serum Samples ◽  
Crude Antigen

ABSTRACT A diagnostic method has been developed to detect anti-Leishmania donovani immunoglobulin G (IgG) in urine by enzyme-linked immunosorbent assay (ELISA). In measuring anti-L. donovani IgG, IgA, and IgM in urine, the method performed best in the detection of IgG. The sensitivity and specificity of the assay were determined with panels of urine samples from 62 visceral leishmaniasis (VL) patients, 59 healthy controls from areas of endemicity, 53 healthy controls from areas of nonendemicity, 59 malaria patients, 13 tuberculosis patients, 23 cutaneous leishmaniasis patients, and 7 patients with other diseases. Using L. donovani promastigote crude antigen, the test had 93.5% sensitivity (58 positives of 62 VL patient samples) and 89.3% specificity (191 negatives of 214 non-VL patient samples). The ELISA with acetone-treated L. donovani promastigote antigen raised the sensitivity and specificity to 95.0 and 95.3%, respectively. Western blot analysis revealed that most of the samples that cross-reacted with crude antigen in ELISA did not recognize any antigenic component of L. donovani crude antigen. We also checked 40 serum samples from the same group of VL patients for anti-L. donovani IgG and got 90.0% sensitivity with both crude and acetone-treated antigens. As collection of urine is much easier than collection of serum, the detection of anti-L. donovani IgG in urine with acetone-treated antigen will be useful in epidemiological studies. It could be an adjunct of laboratory diagnosis.

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