scholarly journals Characterization of Toxoplasma gondii SAG2 Expressed in Insect Cells by Recombinant Baculovirus and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

2002 ◽  
Vol 9 (6) ◽  
pp. 1343-1347 ◽  
Author(s):  
Xiaohong Huang ◽  
Xuenan Xuan ◽  
Hiroshi Suzuki ◽  
Chihiro Sugimoto ◽  
Hideyuki Nagasawa ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Immunosorbent Assay ◽  
Insect Cells ◽  
Fluorescent Antibody ◽  
Recombinant Baculovirus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Conformational Structure ◽  
Serum Samples ◽  
Weak Reaction ◽  
Diagnostic Potential

ABSTRACT A baculovirus carrying the SAG2 gene of Toxoplasma gondii was constructed, and recombinant SAG2 protein (S-rSAG2) was expressed in insect cells. S-rSAG2 was recognized by sera from cats and pigs infected with T. gondii. Mice immunized with S-rSAG2 produced high titers of specific immunoglobulin G2a (IgG2a) and IgG1 antibodies. In an indirect fluorescent antibody test, all mouse antisera against S-rSAG2 reacted strongly to the natural parasites, but those against rSAG2 expressed in Escherichia coli (E-rSAG2) only showed very weak reaction, although no markedly difference was found in the reaction to denatured antigen, T. gondii lysate, in Western blot analysis. The results suggest that S-rSAG2 is better than E-rSAG2 in both antigenicity and immunogenicity. Enzyme-linked immunosorbent assay (ELISA) with S-rSAG2 could differentiate clearly between sera from 30 specific-pathogen-free cats and 4 experimentally infected cats. Serum samples from domestic cats in Japan were tested by the ELISA and compared with a latex agglutination test (LAT) and ELISA with E-rSAG2. Of 187 samples, all 35 LAT-positive sera had strong reactions to S-rSAG2 and E-rSAG2. Of the 152 LAT-negative sera, 18 were positive in the ELISA with S-rSAG2, whereas only 2 were positive in the ELISA with E-rSAG2. Although there were significant correlations among the three methods, the ELISA with S-rSAG2 was more sensitive than the others, which could be attributed to the fact that S-rSAG2 shares some common conformational structure with the native antigen. The results suggest that S-rSAG2 would be a useful reagent for the detection of T. gondii infection in cats.

scholarly journals  Toxoplasma gondii and Neospora caninum antibodies in goats in the Czech Republic

2012 ◽  
Vol 57 (No. 3) ◽  
pp. 111-114 ◽  
Author(s):  
E. Bartova ◽  
K. Sedlak
Keyword(s):  
Czech Republic ◽  
Toxoplasma Gondii ◽  
Immunosorbent Assay ◽  
Competitive Inhibition ◽  
Neospora Caninum ◽  
Fluorescent Antibody ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Antibodies ◽  
Serum Samples ◽  
The Czech Republic

Toxoplasma gondii is zoonotic protozoan parasite that causes infections in many vertebrate species. The present study determined the seroprevalence of T. gondii and N. caninum in goats from the Czech Republic. Serum samples were collected from 251 healthy adult goats in the Czech Republic during the years 2006 to 2009. Sera samples were tested for serum antibodies to Toxoplasma gondii by an enzyme-linked immunosorbent assay with cut off equal to or higher than 50% S/P. The same samples were tested for serum antibodies to Neospora caninum by a competitive-inhibition enzyme-linked immunosorbent assay with cut off equal to or higher than 30% inhibition; positive sera were confirmed by an indirect fluorescent antibody test with cut-off titre equal to or higher than 40. Sera positive in both tests were marked as positive. In total, 166 (66%) and 15 (6%) goat sera reacted positively for T. gondii and N. caninum antibodies, respectively. All sera positive for N. caninum antibodies were simultaneously positive for T. gondii antibodies. This is the first detection of N. caninum antibodies in goats in the Czech Republic. Our findings indicate that goats in the Czech Republic are frequently exposed to T. gondii, but less frequently to N. caninum.  

scholarly journals Development of an Enzyme-Linked Immunosorbent Assay for the Detection of Antibody to Epizootic Hemorrhagic Disease of Deer Virus

2000 ◽  
Vol 12 (2) ◽  
pp. 142-145 ◽  
Author(s):  
James O. Mecham ◽  
Michael M. Jochim
Keyword(s):  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hemorrhagic Disease ◽  
Structural Protein ◽  
Serum Samples ◽  
Epizootic Hemorrhagic Disease ◽  
Diagnostic Potential ◽  
The Us ◽  
Serotype 1 ◽  
Infected Cells

An enzyme-linked immunosorbent assay has been developed to detect antibodies to epizootic hemorrhagic disease of deer virus (EHDV). The assay incorporates a monoclonal antibody to EHDV serotype 2 (EHDV-2) that demonstrates specificity for the viral structural protein, VP7. The assay was evaluated with sequential sera collected from cattle experimentally infected with EHDV serotype 1 (EHDV-1) and EHDV-2, as well as the four serotypes of bluetongue virus (BTV), BTV-10, BTV-11, BTV-13, and BTV-17, that currently circulate in the US. A competitive and a blocking format as well as the use of antigen produced from both EHDV-1-and EHDV-2-infected cells were evaluated. The assay was able to detect specific antibody as early as 7 days after infection and could differentiate animals experimentally infected with EHDV from those experimentally infected with BTV. The diagnostic potential of this assay was demonstrated with field-collected serum samples from cattle, deer, and buffalo.

scholarly journals Comparison of Enzyme-Linked Immunosorbent Assay, Indirect Fluorescent Antibody Test, and Direct Agglutination Test for Detecting Toxoplasma Gondii Antibodies in Naturally Aborted Ovine Fetuses

1989 ◽  
Vol 1 (2) ◽  
pp. 124-127 ◽  
Author(s):  
Sheryl L. Seefeldt ◽  
Clyde A. Kirkbride ◽  
Jitender P. Dubey
Keyword(s):  
Toxoplasma Gondii ◽  
Immunosorbent Assay ◽  
Indirect Fluorescent Antibody Test ◽  
Fluorescent Antibody ◽  
Antibody Test ◽  
Agglutination Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Direct Agglutination Test ◽  
Indirect Fluorescent Antibody ◽  
Special Equipment

Results obtained in an enzyme-linked immunosorbent assay (ELISA), an indirect fluorescent antibody test (IFA), and a modified direct agglutination test (MAT) for Toxoplasma gondii antibodies from examination of fetal fluids from 377 aborted ovine fetuses were compared. Sixty-seven samples were positive by MAT (titers 1:16 to > 1:65,536), 58 were positive by ELISA, and 62 were positive by immunoglobulin G-IFA. The MAT was preferred because it required less time, labor, and special equipment. It was simple to run, could be done on serum from any species without modification, and it was more effective than the IFA for detecting toxoplasma antibodies in severely autolyzed fetuses. No advantage was found in determining immunoglobulin M antibodies in ovine fetal sera.

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 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.

Expression of sheep pathogen Babesia sp. Xinjiang rhoptry-associated protein 1 and evaluation of its diagnostic potential by enzyme-linked immunosorbent assay

Parasitology ◽  
2016 ◽  
Vol 143 (14) ◽  
pp. 1990-1999 ◽  
Author(s):  
QINGLI NIU ◽  
ZHIJIE LIU ◽  
JIFEI YANG ◽  
PEIFA YU ◽  
YUPING PAN ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Post Infection ◽  
Small Ruminants ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Diagnostic Potential ◽  
Positive Rate ◽  
Ct Antigen ◽  
High Level ◽  
Potential Use

SUMMARYOvine babesiosis is one of the most important tick-borne haemoparasitic diseases of small ruminants. The ovine parasite Babesia sp. Xinjiang is widespread in China. In this study, recombinant full-length XJrRAP-1aα2 (rhoptry-associated protein 1aα2) and C-terminal XJrRAP-1aα2 CT of Babesia sp. Xinjiang were expressed and used to evaluate their diagnostic potential for Babesia sp. Xinjiang infections by indirect enzyme-linked immunosorbent assay (ELISA). Purified XJrRAP-1aα2 was tested for reactivity with sera from animals experimentally infected with Babesia sp. Xinjiang and other haemoparasites using Western blotting and ELISA. The results showed no cross-reactivities between XJrRAP-1aα2 CT and sera from animals infected by other pathogens. High level of antibodies against RAP-1a usually lasted 10 weeks post-infection (wpi). A total of 3690 serum samples from small ruminants in 23 provinces located in 59 different regions of China were tested by ELISA. The results indicated that the average positive rate was 30·43%, and the infections were found in all of the investigated provinces. This is the first report on the expression and potential use of a recombinant XJrRAP-1aα2 CT antigen for the development of serological assays for the diagnosis of ovine babesiosis, caused by Babesia sp. Xinjiang.

scholarly journals High-Level Expression of Truncated Surface Antigen P50 of Babesia gibsoni in Insect Cells by Baculovirus and Evaluation of Its Immunogenicity and Antigenicity

2003 ◽  
Vol 10 (4) ◽  
pp. 596-601 ◽  
Author(s):  
Shinya Fukumoto ◽  
Xuenan Xuan ◽  
Kimie Kadota ◽  
Ikuo Igarashi ◽  
Chihiro Sugimoto ◽  
...  
Keyword(s):  
Insect Cells ◽  
Recombinant Baculovirus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Babesia Gibsoni ◽  
Gene Encoding ◽  
Hydrophobic Region ◽  
Immunodominant Antigen ◽  
Diagnostic Potential ◽  
Highly Hydrophobic ◽  
High Level

ABSTRACT Previously, we identified an immunodominant antigen, P50 of Babesia gibsoni. In the present study, the gene encoding the truncated P50 (rP50t) without a C-terminal hydrophobic region (29 amino acids [aa]) was expressed in insect cells by a recombinant baculovirus. The highly hydrophobic C-terminal 20-aa regions seems to be a transmembrane region, which was evidenced by the fact that rP50t was effectively secreted into the supernatant of insect cells infected with the recombinant baculovirus. N-terminal amino acid sequence analysis of rP50t indicated that N-terminal 19 aa function as a signal peptide. The expression level of rP50t reached up to 2 mg per 108 cells infected with the recombinant baculovirus. The immunogenic property of rP50t was evaluated by an immunization test in mice. Mice immunized with rP50t induced a high-level antibody titer against the B. gibsoni merozoite. Monoclonal antibodies (MAbs) to rP50t were produced in mice to determine the immunogenic regions of P50. The epitope(s) recognized by all five MAbs were located between aa 190 and 273, suggesting that the central part of P50 is a highly immunogenic region. The diagnostic potential of rP50t was evaluated using an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate clearly (P < 0.0001) between B. gibsoni-infected dog serum and B. canis-infected dog serum or noninfected dog serum. Our results indicated that the rP50t may provide a useful potential immunogenic reagent for use in diagnosis and as a subunit vaccine to control B. gibsoni infection in dogs.

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 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.

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