scholarly journals Detection of Antibodies to Anaplasma phagocytophilum and Ehrlichia chaffeensis Antigens in Sera of Korean Patients by Western Immunoblotting and Indirect Immunofluorescence Assays

2003 ◽  
Vol 10 (6) ◽  
pp. 1059-1064 ◽  
Author(s):  
Jin-ho Park ◽  
Eun-jeong Heo ◽  
Kyoung-seong Choi ◽  
J. Stephen Dumler ◽  
Joon-seok Chae
Keyword(s):  
South Korea ◽  
Western Blot ◽  
Anaplasma Phagocytophilum ◽  
Fluorescent Antibody ◽  
Environmental Research ◽  
Ehrlichia Chaffeensis ◽  
Protein Antigens ◽  
Serum Samples ◽  
Western Blot Assay ◽  
Korean Patients

ABSTRACT Two hundred seventy one serum samples from South Korean patients were tested to detect antibodies against Anaplasma phagocytophilum (the human granulocytic ehrlichiosis agent) and Ehrlichia chaffeensis (the human monocytic ehrlichiosis agent) by indirect fluorescent-antibody assay (IFA) and the Western blot assay. These sera were collected from patients with symptoms of high fever. The rate of seropositivity for Orientia tsutsugamushi was 50.9% by IFA at the Public Health & Environmental Research Institute and National Institute of Health in South Korea. By IFA, 30 (11.1%) and 39 (14.4%) of the serum samples reacted with A. phagocytophilum and E. chaffeensis antigens, respectively. By the Western blot assays, 24 (8.9%) and 29 (10.7%) of the serum samples reacted with purified A. phagocytophilum and E. chaffeensis protein antigens, respectively. This report strengthens other evidence regarding the presence of A. phagocytophilum and E. chaffeensis infections in humans in South Korea.

Epidemiological and clinical features of HEV infection: a survey in the district of Foggia (Apulia, Southern Italy)

2013 ◽  
Vol 142 (2) ◽  
pp. 287-294 ◽  
Author(s):  
G. SCOTTO ◽  
D. MARTINELLI ◽  
M. CENTRA ◽  
M. QUERQUES ◽  
F. VITTORIO ◽  
...  
Keyword(s):  
General Population ◽  
Western Blot ◽  
Blood Donors ◽  
Southern Italy ◽  
Hepatitis E ◽  
Hiv Positive ◽  
Italian Population ◽  
Serum Samples ◽  
Western Blot Assay ◽  
Low Prevalence

SUMMARYIn this study we assessed the seroprevalence of hepatitis E virus (HEV) infection in both the Italian population and immigrants from developing countries in Foggia (Apulia, Southern Italy). The seroprevalence of HEV was determined in 1217 subjects [412 (34%) immigrants and 805 Italian subjects (blood donors, general population, HIV-positive, haemodialysis patients)]. Serum samples were tested for anti-HEV and confirmed by Western blot assay; in positive patients HEV RNA and genotype were also determined. There were 8·8% of patients that were positive to anti-HEV, confirmed by Western blot. The prevalence in immigrants was 19·7%, and in Italians 3·9% (blood donors 1·3%, general population 2·7%, HIV-positive patients 2·0%, haemodialysis patients 9·6%). Anti-HEV IgM was found in 38/107 (35·5%) of the anti-HEV-positive serum samples (34 immigrants, four Italians). This study indicates a higher circulation of HEV in immigrants and Italian haemodialysis patients, whereas a low prevalence of HEV antibodies was seen in the remaining Italian population.

scholarly journals Enzyme Immunoassay Detection of Antigen-Specific Immunoglobulin G Antibodies in Longitudinal Serum Samples from Patients with Cryptosporidiosis

2001 ◽  
Vol 8 (2) ◽  
pp. 415-423 ◽  
Author(s):  
Jeffrey W. Priest ◽  
Anna Li ◽  
Mohamad Khan ◽  
Michael J. Arrowood ◽  
Patrick J. Lammie ◽  
...  
Keyword(s):  
Western Blot ◽  
Immunoglobulin G ◽  
Protozoan Parasite ◽  
Epidemiologic Studies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Igg Antibody ◽  
Large Format ◽  
Western Blot Assay ◽  
Wide Range

ABSTRACT Cryptosporidium parvum is a protozoan parasite that causes diarrheal illness in a wide range of mammalian hosts, including humans. Characteristic serum immunoglobulin G (IgG) antibody responses to antigens in the 27- and 17-kDa size ranges have been shown to develop after infection, and several enzyme-linked immunosorbent assay (ELISA) and Western blot assay formats have been used to measure these IgG levels in human serum. Using a collection of serial samples from laboratory-confirmed cryptosporidiosis patients, we compared the results obtained by using two new ELISAs with those obtained with two different Western blot assays. When assayed with the large-format Western blot, 97% of the 67 patients had a demonstrable antibody response on at least one occasion. The Cp23 ELISA correctly identified 93% of the samples that had a 27-kDa response by Western blot and 100% of the negative samples. The Triton antigen ELISA detected 77% of the samples that had a 17-kDa response by Western blot and 88% of the negative samples. The sensitivity of the Triton antigen assay was higher for samples collected between 16 and 92 days after the onset of symptoms (96%). The minigel-format Western blot did not compare favorably with the large-format blot for the detection of antibodies to the 27-kDa antigen (71% sensitivity). A half-life of about 12 weeks was estimated for antibodies to both the 27- and 17-kDa antigens. We believe the Cp23 and Triton antigen ELISAs will be useful in epidemiologic studies of the prevalence ofCryptosporidium infection in the population.

scholarly journals Western blot assay of anti-Echinococcus granulosus antibody positive serum samples by indirect haemagglutination method

2019 ◽  
Vol 76 (2) ◽  
pp. 195-202 ◽  
Author(s):  
Ayşe Semra Güreser ◽  
Gamze Gizem Duman ◽  
Fakhriddin Sarzhanov ◽  
Djursun Karasartova ◽  
Funda Dogruman Al ◽  
...  
Keyword(s):  
Western Blot ◽  
Echinococcus Granulosus ◽  
Serum Samples ◽  
Western Blot Assay ◽  
Indirect Haemagglutination ◽  
Positive Serum

scholarly journals Development of a Western Blot Assay for Detection of Bovine Immunodeficiency-Like Virus Using Capsid and Transmembrane Envelope Proteins Expressed from Recombinant Baculovirus

1999 ◽  
Vol 6 (2) ◽  
pp. 168-172 ◽  
Author(s):  
Y. Abed ◽  
G. St-Laurent ◽  
H. Zhang ◽  
R. M. Jacobs ◽  
D. Archambault
Keyword(s):  
Recombinant Protein ◽  
Fusion Protein ◽  
Western Blot ◽  
Fusion Proteins ◽  
Transmembrane Protein ◽  
Recombinant Baculovirus ◽  
Serum Samples ◽  
Western Blot Assay ◽  
Recombinant Fusion Proteins ◽  
Syncytial Virus

ABSTRACT A 120-amino-acid polypeptide selected from the transmembrane protein region (tTM) and the major capsid protein p26 of bovine immunodeficiency-like virus (BIV) were expressed as fusion proteins from recombinant baculoviruses. The antigenic reactivity of both recombinant fusion proteins was confirmed by Western blot with bovine and rabbit antisera to BIV. BIV-negative bovine sera and animal sera positive for bovine syncytial virus and bovine leukemia virus failed to recognize the recombinant fusion proteins, thereby showing the specificity of the BIV Western blot. One hundred and five bovine serum samples were tested for the presence of anti-BIV antibodies by the recombinant protein-based Western blot and a reference Western blot assay using cell culture-derived virions as test antigens. There was a 100% concordance when the p26 fusion protein was used in the Western blot. However, the Western blot using the tTM fusion protein as its test antigen identified four BIV-positive bovine sera which had tested negative in both the p26 recombinant-protein-based and the reference Western blot assays. This resulted in the lower concordance of 96.2% between the tTM-protein-based and reference Western blot assays. The results of this study showed that the recombinant p26 and tTM proteins can be used as test antigens for the serodetection of BIV-infection in animals.

scholarly journals Identification and Characterization of Avian Retroviruses in Chicken Embryo-Derived Yellow Fever Vaccines: Investigation of Transmission to Vaccine Recipients

2003 ◽  
Vol 77 (2) ◽  
pp. 1105-1111 ◽  
Author(s):  
Althaf I. Hussain ◽  
Jeffrey A. Johnson ◽  
Marcos da Silva Freire ◽  
Walid Heneine
Keyword(s):  
Western Blot ◽  
Yellow Fever ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Serum Samples ◽  
Rna Sequences ◽  
Western Blot Assay ◽  
Specific Pcr ◽  
Show Evidence

ABSTRACT All currently licensed yellow fever (YF) vaccines are propagated in chicken embryos. Recent studies of chick cell-derived measles and mumps vaccines show evidence of two types of retrovirus particles, the endogenous avian retrovirus (EAV) and the endogenous avian leukosis virus (ALV-E), which originate from the chicken embryonic fibroblast substrates. In this study, we investigated substrate-derived avian retrovirus contamination in YF vaccines currently produced by three manufacturers (YF-vax [Connaught Laboratories], Stamaril [Aventis], and YF-FIOCRUZ [FIOCRUZ-Bio-Manguinhos]). Testing for reverse transcriptase (RT) activity was not possible because of assay inhibition. However, Western blot analysis of virus pellets with anti-ALV RT antiserum detected three distinct RT proteins in all vaccines, indicating that more than one source is responsible for the RTs present in the vaccines. PCR analysis of both chicken substrate DNA and particle-associated RNA from the YF vaccines showed no evidence of the long terminal repeat sequences of exogenous ALV subgroups A to D in any of the vaccines. In contrast, both ALV-E and EAV particle-associated RNA were detected at equivalent titers in each vaccine by RT-PCR. Quantitative real-time RT-PCR revealed 61,600, 348,000, and 1,665,000 ALV-E RNA copies per dose of Stamaril, YF-FIOCRUZ, and YF-vax vaccines, respectively. ev locus-specific PCR testing of the vaccine-associated chicken substrate DNA was positive both for the nondefective ev-12 locus in two vaccines and for the defective ev-1 locus in all three vaccines. Both intact and ev-1 pol sequences were also identified in the particle-associated RNA. To investigate the risks of transmission, serum samples from 43 YF vaccine recipients were studied. None of the samples were seropositive by an ALV-E-based Western blot assay or had detectable EAV or ALV-E RNA sequences by RT-PCR. YF vaccines produced by the three manufacturers all have particles containing EAV genomes and various levels of defective or nondefective ALV-E sequences. The absence of evidence of infection with ALV-E or EAV in 43 YF vaccine recipients suggests low risks for transmission of these viruses, further supporting the safety of these vaccines.

scholarly journals Spherical Body Protein 4 Is a New Serological Antigen for Global Detection ofBabesia bovisInfection in Cattle

2010 ◽  
Vol 18 (2) ◽  
pp. 337-342 ◽  
Author(s):  
Mohamad Alaa Terkawi ◽  
Nguyen Xuan Huyen ◽  
Putut Eko Wibowo ◽  
Faasoa Junior Seuseu ◽  
Mahmoud Aboulaila ◽  
...  
Keyword(s):  
Western Blot ◽  
Recombinant Proteins ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Fluorescent Antibody ◽  
Spherical Body ◽  
Babesia Bovis ◽  
Serum Samples ◽  
Body Protein ◽  
Geographical Regions

ABSTRACTFiveBabesia bovisrecombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), C-terminal rhoptry-associated protein 1 (BbRAP-1/CT), truncated thrombospondin-related anonymous protein (BbTRAP-T), spherical body protein 1 (BbSBP-1), and spherical body protein 4 (BbSBP-4), were evaluated as diagnostic antigens to detect the infection in cattle. The recombinant proteins were highly antigenic when tested with experimentallyB. bovis-infected bovine serum in Western blot analysis. Furthermore, five antisera that had been raised against each of the recombinant proteins reacted specifically with the corresponding authentic protein, as determined in Western blot analysis. Next, enzyme-linked immunosorbent assays (ELISAs) using these recombinant proteins were evaluated for diagnostic use, and the sensitivity and specificity of each protein were demonstrated with a series of serum samples from experimentallyB. bovis-infected cattle. Furthermore, a total of 669 field serum samples collected from cattle in regions ofB. bovisendemicity in seven countries were tested with the ELISAs, and the results were compared to those of an indirect fluorescent antibody test (IFAT), as a reference. Among five recombinant antigens, recombinant BbSBP-4 (rBbSBP-4) had the highest concordance rate (85.3%) and kappa value (0.705), indicating its reliability in the detection of specific antibodies toB. bovisin cattle, even in different geographical regions. Overall, we have successfully developed an ELISA based on rBbSBP-4 as a new serological antigen for a practical and sensitive test which will be applicable for epidemiologic survey and control programs in the future.

scholarly journals Detection of Bovine Immunodeficiency Virus Antibodies in Cattle by Western Blot Assay with Recombinant Gag Protein

1997 ◽  
Vol 9 (4) ◽  
pp. 347-351 ◽  
Author(s):  
Shucheng Zhang ◽  
Wenzhi Xue ◽  
Charles Wood ◽  
Qi-Min Chen ◽  
Sanjay Kapil ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Western Blot ◽  
Bovine Immunodeficiency Virus ◽  
Test Results ◽  
Chain Reaction ◽  
Serum Samples ◽  
Western Blot Assay ◽  
Gag Protein ◽  
Immunodeficiency Virus ◽  
Polymerase Chain

A western blot assay using purified recombinant bovine immunodeficiency virus gag protein has been developed for detection of bovine immunodeficiency virus antibodies in bovine serum samples. The test was standardized with known bovine immunodeficiency virus positive and negative bovine serum samples and the monoclonal antibody to gag protein. Both naturally and experimentally infected cattle sera demonstrated positive test results. The result of western blot assay was compared with polymerase chain reaction test results in 134 blood samples collected from Kansas. Twenty-six samples tested positive for bovine immunodeficiency virus DNA with polymerase chain reaction (18.7%) and 25 were positive for the antibody to gag protein by western blot analysis (17.9%). Of 26 cattle testing positive using the polymerase chain reaction assay, 24 were antibody-positive by western blot assay, thus establishing a strong correlation between the two tests. The sensitivity and specificity of western blot relative to polymerase chain reaction are 0.92 and 0.99, respectively. The western blot assay proved to be a specific and sensitive test.

scholarly journals False-Positive Results in a Recombinant Severe Acute Respiratory Syndrome-Associated Coronavirus (SARS-CoV) Nucleocapsid-Based Western Blot Assay Were Rectified by the Use of Two Subunits (S1 and S2) of Spike for Detection of Antibody to SARS-CoV

2006 ◽  
Vol 13 (3) ◽  
pp. 409-414 ◽  
Author(s):  
Mimoun Maache ◽  
Florence Komurian-Pradel ◽  
Alain Rajoharison ◽  
Magali Perret ◽  
Jean-Luc Berland ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Western Blot ◽  
False Positive ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Serum Samples ◽  
Western Blot Assay ◽  
N Protein ◽  
False Positive Diagnosis ◽  
Healthy Donors

ABSTRACT To evaluate the reactivity of the recombinant proteins expressed in Escherichia coli strain BL21(DE3), a Western blot assay was performed by using a panel of 78 serum samples obtained, respectively, from convalescent-phase patients infected with severe acute respiratory syndrome-associated coronavirus (SARS-CoV) (30 samples) and from healthy donors (48 samples). As antigen for detection of SARS-CoV, the nucleocapsid protein (N) showed high sensitivity and strong reactivity with all samples from SARS-CoV patients and cross-reacted with all serum samples from healthy subjects, with either those obtained from China (10 samples) or those obtained from France (38 serum samples), giving then a significant rate of false positives. Specifically, our data indicated that the two subunits, S1 (residues 14 to 760) and S2 (residues 761 to 1190), resulted from the divided spike reacted with all samples from SARS-CoV patients and without any cross-reactivity with any of the healthy serum samples. Consequently, these data revealed the nonspecific nature of N protein in serodiagnosis of SARS-CoV compared with the S1 and S2, where the specificity is of 100%. Moreover, the reported results indicated that the use of one single protein as a detection antigen of SARS-CoV infection may lead to false-positive diagnosis. These may be rectified by using more than one protein for the serodiagnosis of SARS-CoV.

Evidence of Anaplasma phagocytophilum in game animals from Slovenia

2012 ◽  
Vol 60 (4) ◽  
pp. 441-448 ◽  
Author(s):  
Diana Žele ◽  
Jana Avberšek ◽  
Igor Gruntar ◽  
Matjaž Ocepek ◽  
Gorazd Vengušt
Keyword(s):  
Wild Boar ◽  
Cervus Elaphus ◽  
Anaplasma Phagocytophilum ◽  
Ursus Arctos ◽  
Roe Deer ◽  
Fluorescent Antibody ◽  
Brown Bear ◽  
Serum Samples ◽  
Game Species ◽  
Game Animals

Anaplasma phagocytophilumis a tick-borne rickettsial pathogen responsible for granulocytic anaplasmosis in mammalian hosts including humans. Wild animals may play an important role in the epidemiology of this disease. The aim of this study was to estimate the prevalence of infection withA. phagocytophilumamong wildlife in Slovenia. Serum samples (n = 376) from the most important game species [red deer (Cervus elaphus), roe deer (Capreolus capreolus), wild boar (Sus scrofa), chamois (Rupicapra rupicapra) and brown bear (Ursus arctos)] were examined byA. phagocytophilum-specific indirect fluorescent-antibody assay (IFA) and wild boar spleen samples (n = 160) were tested by polymerase chain reaction (PCR).A. phagocytophilum-specific antibodies were found in 72% of sera andA. phagocytophilumDNA was present in 6.2% of spleens. The data indicate thatA. phagocytophilumis present and widespread in Slovenian game animals and that game species are involved in the natural life cycle ofA. phagocytophilum.

scholarly journals Molecular Characterization of Antibody Epitopes of Ehrlichia chaffeensis Ankyrin Protein 200 and Tandem Repeat Protein 47 and Evaluation of Synthetic Immunodeterminants for Serodiagnosis of Human Monocytotropic Ehrlichiosis

2009 ◽  
Vol 17 (1) ◽  
pp. 87-97 ◽  
Author(s):  
Tian Luo ◽  
Xiaofeng Zhang ◽  
William L. Nicholson ◽  
Bing Zhu ◽  
Jere W. McBride
Keyword(s):  
Tandem Repeat ◽  
Fluorescent Antibody ◽  
Protein A ◽  
Enzyme Linked Immunosorbent Assay ◽  
Reference Laboratory ◽  
Ehrlichia Chaffeensis ◽  
Analytical Sensitivity ◽  
Serum Samples ◽  
Ankyrin Protein ◽  
Antibody Epitopes

ABSTRACT Recently, major species-specific antibody epitopes in three immunoreactive tandem repeat proteins (TRPs) of Ehrlichia chaffeensis, TRP32, TRP47, and TRP120, have been identified and molecularly characterized within tandem repeat (TR) regions. In this study, we mapped the major immunodeterminants of the E. chaffeensis 200-kDa ankyrin protein (Ank200) and the minor immunodeterminants in the N- and C-terminal regions of E. chaffeensis TRP47. Major antibody epitopes of Ank200 were localized to four polypeptide regions (18-mer, 20-mer, 20-mer, and 21-mer, respectively) in terminal acidic domains, which reacted with antibodies in sera from human monocytotropic ehrlichiosis (HME) patients and an E. chaffeensis-infected dog. Two minor epitope-containing regions were identified in the N terminus and the C terminus of TRP47. The sensitivities and specificities of synthetic peptides representing these and other well-defined major immunodeterminants of E. chaffeensis were determined by enzyme-linked immunosorbent assay (ELISA). Thirty-one HME patient serum samples that had detectable E. chaffeensis antibodies (titers from 64 to 8,192) by indirect fluorescent-antibody assay (IFA) were tested. All 31 serum samples reacted with at least one E. chaffeensis peptide, 30 (96.8%) with TRP120 peptides, 27 (87.1%) with TRP32 peptides, 24 (77.4%) with TRP47 peptides, 19 (61.3%) with Ank200 peptides, and 28 (90.3%) with recombinant TRP120-TR protein. A mixture of the two most sensitive peptides from TRP120 and TRP32 did not provide enhanced analytical sensitivity compared to that provided by TRP120 alone. Our results demonstrate that the TRP120 peptide can be utilized for development of standardized sensitive point-of-care and reference laboratory immunodiagnostics for HME. This is the first study to compare analysis of molecularly defined major antibody epitopes with IFA for diagnosis of HME.

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