scholarly journals Serodiagnosis of Human Granulocytic Ehrlichiosis by a Recombinant HGE-44-Based Enzyme-Linked Immunosorbent Assay

1999 ◽  
Vol 37 (11) ◽  
pp. 3540-3544 ◽  
Author(s):  
Jacob W. Ijdo ◽  
Caiyun Wu ◽  
Louis A. Magnarelli ◽  
Erol Fikrig
Keyword(s):  
Immunosorbent Assay ◽  
Fluorescent Antibody ◽  
Immunoblot Analysis ◽  
Normal Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Testing ◽  
Serum Samples ◽  
Granulocytic Ehrlichiosis ◽  
Healthy Humans ◽  
Human Granulocytic Ehrlichiosis

Current antibody testing for human granulocytic ehrlichiosis relies predominantly on indirect fluorescent-antibody assays and immunoblot analysis. Shortcomings of these techniques include high cost and variability of test results associated with the use of different strains of antigens derived from either horses or cultured HL-60 cells. We used recombinant protein HGE-44, expressed and purified as a maltose-binding protein (MBP) fusion peptide, as an antigen in a polyvalent enzyme-linked immunosorbent assay (ELISA). Fifty-five normal serum samples from healthy humans served as a reference to establish cutoff levels. Thirty-three of 38 HGE patient serum samples (87%), previously confirmed by positive whole-cell immunoblotting, reacted positively in the recombinant ELISA. In specificity analyses, serum samples from patients with Lyme disease, syphilis, rheumatoid arthritis, and human monocytic ehrlichiosis (HME) did not react with HGE-44–MBP antigen, except for one sample (specificity, 98%). We conclude that recombinant HGE-44 antigen is a suitable antigen in an ELISA for the laboratory diagnosis and epidemiological study of HGE.

scholarly journals Comparison of Two Recombinant Major Outer Membrane Proteins of the Human Granulocytic Ehrlichiosis Agent for Use in an Enzyme-Linked Immunosorbent Assay

2000 ◽  
Vol 7 (4) ◽  
pp. 652-657 ◽  
Author(s):  
Tomoko Tajima ◽  
Ning Zhi ◽  
Quan Lin ◽  
Yasuko Rikihisa ◽  
Harold W. Horowitz ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Outer Membrane Proteins ◽  
Cross Reactivity ◽  
Immunofluorescence Assay ◽  
Babesia Microti ◽  
Enzyme Linked Immunosorbent Assay ◽  
Granulocytic Ehrlichiosis ◽  
Healthy Humans ◽  
Human Granulocytic Ehrlichiosis ◽  
Negative Controls

ABSTRACT Enzyme-linked immunosorbent assay (ELISA) for human granulocytic ehrlichiosis (HGE) using two different recombinant P44 proteins (rP44 and rP44-2hv) of the HGE agent as antigens was evaluated. Sera from a total of 72 healthy humans both from regions where HGE is nonendemic and regions where HGE is endemic were used as negative controls to determine the cutoff value for ELISA. Sera from a total of 14 patients (nine from whom the HGE agent was isolated and five who were HGE-PCR positive) were used as positive controls. One hundred nine sera from 72 patients in an area where HGE is endemic who were suspected of having HGE were examined by ELISA and indirect immunofluorescence assay (IFA). All IFA-negative sera were negative by both ELISAs. Of 39 sera that were IFA positive, 35 and 27 were positive by ELISA using rP44 and rP44-2hv, respectively, indicating that the use of rP44 is more sensitive. Western blot analysis of the four rP44-ELISA-negative IFA-positive sera using whole HGE agent as antigen suggests that these four sera were false IFA positive. There was no difference in results with or without the preabsorption of sera with Escherichia coli or with or without the cleavage of the fused protein derived from the vector. There was a significant positive correlation between IFA titers and optical densities of ELISAs. Four Ehrlichia chaffeensis-positive and 10 Borrelia burgdorferi-positive sera were negative by ELISA. However, twoBabesia microti-positive sera showed strong cross-reactivity to the fused vector protein, which was eliminated after cleavage of the protein. Thus, ELISA using rP44 nonfusion protein would provide a simple, specific, and objective HGE serologic test which can be easily automated.

scholarly journals Infection with Agents of Human Granulocytic Ehrlichiosis, Lyme Disease, and Babesiosis in Wild White-Footed Mice (Peromyscus leucopus) in Connecticut

1999 ◽  
Vol 37 (9) ◽  
pp. 2887-2892 ◽  
Author(s):  
Kirby C. Stafford ◽  
Robert F. Massung ◽  
Louis A. Magnarelli ◽  
Jacob W. Ijdo ◽  
John F. Anderson
Keyword(s):  
Ixodes Scapularis ◽  
Peromyscus Leucopus ◽  
Fluorescent Antibody ◽  
Babesia Microti ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rrna Gene ◽  
Serum Samples ◽  
Granulocytic Ehrlichiosis ◽  
Human Granulocytic Ehrlichiosis ◽  
Staining Methods

White-footed mice, Peromyscus leucopus, were captured in southern Connecticut during 1997 and 1998 to determine the prevalence of infections caused by granulocytic Ehrlichiasp., Borrelia burgdorferi, and Babesia microti. Of the 50 mice captured and recaptured, 25 of 47 (53.2%) and 23 of 48 (47.9%) contained antibodies to the BDS or NCH-1 Ehrlichiastrains, respectively, as determined by indirect fluorescent antibody (IFA) staining methods. The majority (83.3%) of 48 mice also contained antibodies to B. burgdorferi, as determined by enzyme-linked immunosorbent assay. Moreover, 20 of 26 (76.9%) contained antibodies to B. microti by IFA staining methods. In nested PCR tests using the 16S rRNA gene, the DNA of the human granulocytic ehrlichiosis (HGE) agent was detected in 17 of 47 mice (36.2%), but only 4 (23.5%) of these 17 mice were PCR positive at each capture. Antibody-positive reactions to granulocyticEhrlichia sp. organisms were detected in 17 of 23 (73.9%) of the PCR-positive mice. The sequences from PCR products from nine positive blood samples were identical to the HGE agent.Ehrlichia spp. were cultured from three of five mice captured in April 1998, including one that was PCR positive in April 1997. In addition, 2 of 14 larval Ixodes scapularis pools, which were attached to two PCR-positive mice, contained DNA of the HGE agent. A high percentage of white-footed mice are infected or have been infected naturally by the HGE agent with low-level persistent infection or frequent reinfection in some individual mice. However, the changes noted in the presence of DNA and antibodies in repeated blood and serum samples from individual mice over several months of field collection suggests that infection with granulocytic Ehrlichia is transient in most wild P. leucopus.

scholarly journals Evaluation of an Indirect Enzyme-Linked Immunosorbent Assay for Routine Screening of Theiler's Murine Encephalomyelitis Virus Antibodies in Mice Colonies

2008 ◽  
Vol 20 (6) ◽  
pp. 789-791 ◽  
Author(s):  
Juan M. Laborde ◽  
Cecilia Carbone ◽  
Santiago G. Corva ◽  
Cecilia M. Galosi
Keyword(s):  
Immunosorbent Assay ◽  
Fluorescent Antibody ◽  
Receiver Operating Curve ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Special Equipment ◽  
Theiler's Murine Encephalomyelitis Virus ◽  
Theiler’S Murine Encephalomyelitis Virus ◽  
Encephalomyelitis Virus ◽  
Buffer Control

The current study demonstrates the ability of an indirect enzyme-linked immunosorbent assay (iELISA) to detect antibodies against Theiler's murine encephalomyelitis virus in mice colonies. The antigen was produced from infected baby hamster kidney (BHK)-21 cells and treated with 1% Nonidet P40 in saline buffer. Control antigen was prepared following the same procedure using uninfected BHK-21 cells. The optimal antigen and serum dilutions were established. The reaction was revealed using an anti-mouse-horseradish peroxidase conjugate and 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). Optimized iELISA was validated by detection of antibodies in known positive and negative serum samples before testing the samples of unknown status. Performance of the iELISA was compared with the indirect fluorescent antibody test, and the cutoff value was determined by receiver operating curve. Indirect ELISA showed 100% sensitivity, 99.38% specificity, and 97.78% predictive positive value. The antigen used is easy to produce, and no special equipment is required. The iELISA developed is simple and provides a rapid and less costly tool for diagnosis and research.

scholarly journals Cysticercus antibodies and antigens in serum from blood donors from Pondicherry, India

2005 ◽  
Vol 47 (4) ◽  
pp. 227-230 ◽  
Author(s):  
Subhash Chandra Parija ◽  
N. Balamurungan ◽  
Priyadarshi Soumyaranjan Sahu ◽  
S.P. Subbaiah
Keyword(s):  
Immunosorbent Assay ◽  
Tamil Nadu ◽  
Blood Donors ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Testing ◽  
Specific Detection ◽  
Serum Samples ◽  
Blood Samples ◽  
Tamil Nadu State ◽  
Apparently Healthy

The aim of the present study was to screen the serum of blood donors, which are apparently healthy and residing in Pondicherry or its neighboring districts of Tamil Nadu State, for specific detection of Cysticercus antigens and antibodies. A total of 216 blood samples were collected from blood donors at the Central Blood Bank, JIPMER Hospital, Pondicherry, India during January and February 2004. Enzyme-linked immunosorbent assay (ELISA) was used to demonstrate anti-Cysticercus antibodies and the Co-agglutination (CoA) was used to detect antigen in sera. 14 (6.48 %) males were positive for either anti-Cysticercus antibodies or antigens. Of these eight sera were positive for anti-Cysticercus antibodies and six were positive for antigens. Results of the present study show that serum Cysticercus antigen detection may be a useful adjunct to antibody testing for seroprevalence studies of cysticercosis in the community. The present study is the first kind of study, carried out to determine both cysticercal antibodies as well as antigens in the serum samples collected from the healthy blood donors.

Presence of antibodies against rabies in wild boars

2011 ◽  
Vol 59 (1) ◽  
pp. 149-154 ◽  
Author(s):  
Gorazd Vengušt ◽  
Peter Hostnik ◽  
Mojca Cerovšek ◽  
Polona Cilenšek ◽  
Tadej Malovrh
Keyword(s):  
Wild Boar ◽  
Rabies Virus ◽  
Immunosorbent Assay ◽  
Fluorescent Antibody ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Oral Vaccination ◽  
Hunting Season ◽  
Wild Boars ◽  
Research Reporting

Serum samples of 746 shot wild boars collected throughout Slovenia during the hunting season of 2005/2006 were examined for the presence of antibodies against rabies virus: 541 samples were collected in areas subjected to yearly antirabies vaccination, and 205 samples were collected in areas where preventive antirabies vaccination was not practised. Using a modified enzyme-linked immunosorbent assay (ELISA), in 209 out of 746 sera (28%) the levels of antibodies against rabies virus were higher than 0.5 IU/ml and deemed positive. A total of 173/541 (32%) and 36/205 (18%) samples were positive in the vaccinated and nonvaccinated areas, respectively. Further analysis of 191 out of the 746 samples using the fluorescent antibody virus neutralisation (FAVN) test revealed the presence of antibodies against rabies virus in 122/191 (64%) samples. This is the first extended research reporting that antibodies against rabies virus that originate from preventive oral vaccination targeting the fox population are present in wild boar.

scholarly journals Immunodiagnosis of Human Granulocytic Ehrlichiosis by Using Culture-Derived Human Isolates †

1998 ◽  
Vol 36 (6) ◽  
pp. 1480-1488 ◽  
Author(s):  
M. Dana Ravyn ◽  
Jesse L. Goodman ◽  
Carrie B. Kodner ◽  
Deborah K. Westad ◽  
Lisa A. Coleman ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Immunoblot Analysis ◽  
Cross Reactivity ◽  
Immunofluorescence Assay ◽  
Etiologic Agent ◽  
Immunoglobulin M ◽  
Test Method ◽  
Serum Samples ◽  
Granulocytic Ehrlichiosis ◽  
Human Granulocytic Ehrlichiosis

Human granulocytic ehrlichiosis (HGE) is an emerging infection caused by an Ehrlichia species closely related toEhrlichia equi and Ehrlichia phagocytophila. Recent advances in the isolation and cultivation of this organism have allowed us to develop an immunofluorescence assay (IFA), enzyme immunoassay (EIA), and Western immunoblotting (WB) using HL-60 cell culture-derived human isolates. Antibody was detected in sera from culture-confirmed HGE patients by IFA and EIA, and these samples were reactive when analyzed by immunoblot analysis. HGE patient sera had high antibody titers and did not react with uninfected HL-60 cells. When IFA, EIA, and WB were used to analyze sera from healthy donors or those with a range of other disorders, including infections caused byEhrlichia chaffeensis, Rickettsia rickettsii, and Coxiella burnetti, no significant cross-reactivity could be detected by EIA or immunoblot analysis with the exception of two of four serum samples from R. rickettsii-infected patients that were reactive by IFA only. Sera from HGE patients did not significantly cross-react in serologic tests for Borrelia burgdorferi. Using sera from patients previously enrolled in two clinical trials of treatment for early Lyme disease, we evaluated a two-step approach for estimation of the seroprevalence of antibodies reactive with the etiologic agent of HGE. On the basis of the immunoblot assay results for sera from culture-confirmed HGE patients, WB was used to confirm the specificity of the antibody detected by EIA and IFA. EIA was found to be superior to IFA in the ability to detect WB-confirmed antibodies to the HGE agent. When EIA and WB were used, 56 (19.9%) patients with early Lyme disease (n = 281) had either specific immunoglobulin M (IgM) or IgG antibodies; 38 patients (13.5%) had IgM only, 6 (2.1%) had IgG only, and 12 (4.3%) had both IgM and IgG. Therefore, Lyme disease patients are at high potential risk for exposure to Ehrlichia. Analysis by immunoblotting of serial samples from persons with culture-confirmed HGE or patients with Lyme disease and antibodies to the agent of HGE revealed a reproducible pattern of the immune response to specific antigens. These samples confirmed the importance of the 42- to 45-kDa antigens as early, persistent, and specific markers of HGE infection. Other significant immunogenic proteins appear at 20, 21, 28, 30, and 60 kDa. Use of the two-test method of screening by EIA and confirming the specificity by WB appears to offer a sound approach to the clinical immunodiagnosis of HGE.

scholarly journals Characterization of a Borrelia burgdorferi VlsE Invariable Region Useful in Canine Lyme Disease Serodiagnosis by Enzyme-Linked Immunosorbent Assay

2000 ◽  
Vol 38 (11) ◽  
pp. 4160-4166 ◽  
Author(s):  
Fang Ting Liang ◽  
Richard H. Jacobson ◽  
Reinhard K. Straubinger ◽  
Amy Grooters ◽  
Mario T. Philipp
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Antibody Response ◽  
Immunosorbent Assay ◽  
Protein A ◽  
Immunoblot Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Entire Study ◽  
Variable Surface

Sera collected from dogs experimentally infected withBorrelia burgdorferi by tick inoculation were analyzed for an antibody response to each of the six invariable regions (IRs; i.e., IR1 to IR6) of VlsE, the variable surface antigen of B. burgdorferi. Six synthetic peptides (C1 to C6), which reproduced the six IR sequences were used as peptide-based, enzyme-linked immunosorbent assay (ELISA) antigens. Two IRs, IR2 and IR6, were found to be immunodominant. Studies with serially collected serum samples from experimentally infected dogs revealed that the antibody response to IR6 appears earlier and is stronger than that to IR2. Thus, the IR6 sequence alone appeared to be sufficient for serodiagnosis. When C6 alone was used as antigen, the peptide-based ELISA was positive in 7 of 23 dogs (30%) as early as 3 weeks postinfection. All dogs (n = 33) became strongly positive 1 or 2 weeks later, and this response persisted for the entire study, which lasted for 69 weeks. Of 55 sera submitted by veterinarians from dogs suspected of having Lyme disease, 19 were also positive by the C6 ELISA, compared to 20 positives detected by immunoblot analysis using cultured B. burgdorferi lysates as antigen. The sensitivity of using C2 and C6 together for detecting specific antibody in both experimentally infected and clinically diagnosed dogs was not better than sensitivity with C6 alone, confirming that C6 suffices as a diagnostic probe. Moreover, the C6 ELISA yielded 100% specificity with serum samples collected from 70 healthy dogs, 14 dogs with infections other than B. burgdorferi, and 15 animals vaccinated with either outer surface protein A, whole-spirochete vaccines, or the common puppy-vaccines. Therefore, this C6 ELISA was both sensitive and specific for the serodiagnosis of canine Lyme disease and could be used with vaccinated dogs.

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.

Development and application of an indirect enzyme-linked immunosorbent assay using recombinant S1 for serological testing of porcine epidemic diarrhea virus

2019 ◽  
Vol 65 (5) ◽  
pp. 343-352
Author(s):  
Ying Shan ◽  
Yajie Liu ◽  
Ziqi Liu ◽  
Guowei Li ◽  
Cong Chen ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Porcine Epidemic Diarrhea Virus ◽  
Fluorescent Antibody ◽  
Area Under The Curve ◽  
Antibody Detection ◽  
Economic Losses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV) causes severe infectious diseases in all ages of swine and leads to serious economic losses. Serologic tests are widely accepted and used to detect anti-PEDV antibodies that could indicate PEDV infection or vaccination. In this study, PEDV recombinant S1 protein (rS1) was expressed with the Bac-to-Bac system and purified by nickel-affinity chromatography. An indirect enzyme-linked immunosorbent assay based on rS1 (rS1-ELISA) was then developed and optimized by checkerboard assays with serial dilutions of antigen and serum. Serum samples from 453 domestic pigs and 42 vaccinated pigs were analyzed by the indirect fluorescent antibody (IFA) test and rS1-ELISA. Taking IFA as a gold standard, rS1-ELISA produced a high sensitivity (90.7%) and specificity (94.6%) by a receiver operating characteristic (ROC) curve. In addition, ROC analysis also revealed that rS1-ELISA was consistent with IFA (area under the curve 0.9583 ± 0.0082). This rS1-ELISA was then applied to antibody detection in inactivated PEDV vaccinated pigs. The antibody could be detected 2–4 weeks after the first inoculation. These results indicated that the rS1-ELISA established in this study provides a promising and reliable tool for serologic detection of anti-PEDV IgG antibodies in infected or vaccinated pigs.

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