scholarly journals Development of a Recombinant Nucleoprotein-Based Enzyme-Linked Immunosorbent Assay for Quantification of Antibodies against Porcine Reproductive and Respiratory Syndrome Virus

2000 ◽  
Vol 7 (4) ◽  
pp. 700-702 ◽  
Author(s):  
Steven B. Witte ◽  
Cindy Chard-Bergstrom ◽  
Thomas A. Loughin ◽  
Sanjay Kapil
Keyword(s):  
Immunosorbent Assay ◽  
Respiratory Syndrome Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
State University ◽  
Kansas State University ◽  
Porcine Respiratory

ABSTRACT A rapid, inexpensive enzyme-linked immunosorbent assay (ELISA) to quantitate antibodies to porcine respiratory and reproductive syndrome virus (PRRSV) in serum was developed using a recombinant PRRSV nucleoprotein (rN). The sensitivity (85.3%) and specificity (81.7%) of the Kansas State University ELISA were good, correlating well (82.4%) with the IDEXX HerdChek ELISA.

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 Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay for Detection and Differentiation of Antibodies against European and North American Porcine Reproductive and Respiratory Syndrome Virus

2002 ◽  
Vol 9 (6) ◽  
pp. 1183-1191 ◽  
Author(s):  
Torsten Seuberlich ◽  
Jon-Duri Tratschin ◽  
Barbara Thür ◽  
Martin A. Hofmann
Keyword(s):  
Immunosorbent Assay ◽  
North American ◽  
Nucleocapsid Protein ◽  
Simultaneous Detection ◽  
The United States ◽  
Respiratory Syndrome Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Epidemiological Surveys ◽  
The Us ◽  
The Eu

ABSTRACT Two types of porcine reproductive and respiratory syndrome virus (PRRSV) have been reported, the European type (EU PRRSV) and the North American type (US PRRSV). We developed a dual enzyme-linked immunosorbent assay (ELISA) for the simultaneous detection and differentiation of serum antibodies directed against either of the two PRRSV types. This tandem PRRS ELISA is based on affinity-purified recombinant nucleocapsid protein expressed in Escherichia coli. Sensitivity and specificity were assessed by using the IDEXX HerdChek PRRS ELISA and the indirect immunofluorescence assay as reference tests. A total of 1,571 sera originating from the United States, Europe, and two PRRS-free countries, i.e., Switzerland and New Zealand, were used for validation of the tandem PRRS ELISA. The new test performed at least as well as the reference tests in regard to sensitivity (0.94 for the US PRRS ELISA and 0.93 for the EU PRRS ELISA) and specificity (0.96 for the US PRRS ELISA and 0.99 for the EU PRRS ELISA). Positive sera were correctly differentiated in 582 of 591 cases, indicating a high differentiation capability of this dual ELISA. The robustness and repeatability of the test were assessed and found to be appropriate for diagnostic applications. Taken together, the data indicate that the tandem PRRS ELISA described here is the first differentiation ELISA for PRRSV serology based on recombinant antigen. It is convenient with respect to antigen production, and it is reliable, economical, and highly sensitive and specific. Thus, it is considered to be a powerful tool for routine diagnostics, epidemiological surveys, and outbreak investigations.

Antigen-Capture Blocking Enzyme-Linked Immunosorbent Assay Based on a Baculovirus Recombinant Antigen to Differentiate Transmissible Gastroenteritis Virus from Porcine Respiratory Coronavirus Antibodies

2009 ◽  
Vol 21 (5) ◽  
pp. 598-608 ◽  
Author(s):  
Lissett López ◽  
Angel Venteo ◽  
Marga García ◽  
Ana Camuñas ◽  
Ana Ranz ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Transmissible Gastroenteritis Virus ◽  
Suitable Alternative ◽  
S Protein ◽  
Antigen Capture ◽  
Gastroenteritis Virus ◽  
Transmissible Gastroenteritis ◽  
Respiratory Coronavirus ◽  
Porcine Respiratory

A new commercially available antigen-capture, blocking enzyme-linked immunosorbent assay (antigen-capture b-ELISA), based on baculovirus truncated-S recombinant protein of Transmissible gastroenteritis virus (TGEV) and 3 specific monoclonal antibodies, was developed and evaluated by examining a panel of 453 positive Porcine respiratory coronavirus (PRCoV), 31 positive TGEV, and 126 negative field sera by using another commercially available differential coronavirus b-ELISA as the reference technique to differentiate TGEV- from PRCoV-induced antibodies. The recombinant S protein-based ELISA appeared to be 100% sensitive for TGEV and PRCoV detection and highly specific for TGEV and PRCoV detection (100% and 92.06%, respectively), when qualitative results (positive or negative) were compared with those of the reference technique. In variability experiments, the ELISA gave consistent results when the same serum was evaluated on different wells and different plates. These results indicated that truncated recombinant S protein is a suitable alternative to the complete virus as antigen in ELISA assays. The use of recombinant S protein as antigen offers great advantages because it is an easy-to-produce, easy-to-standardize, noninfectious antigen that does not require further purification or concentration. Those advantages represent an important improvement for antigen preparation, in comparison with other assays in which an inactivated virus from mammalian cell cultures is used.

scholarly journals Cloning of the Bovine Immunodeficiency Virusgag Gene and Development of a Recombinant-Protein-Based Enzyme-Linked Immunosorbent Assay

2000 ◽  
Vol 7 (4) ◽  
pp. 557-562 ◽  
Author(s):  
Ling Zheng ◽  
Michelle Swanson ◽  
Jinghua Liao ◽  
Charles Wood ◽  
Sanjay Kapil ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Immunosorbent Assay ◽  
Protein A ◽  
Bovine Immunodeficiency Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
State University ◽  
Coding Region ◽  
Serum Samples ◽  
Diagnostic Laboratory ◽  
Gag Protein

ABSTRACT An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific bovine immunodeficiency virus (BIV) antibodies in cattle, using recombinant Gag protein as an antigen. Thegag coding region from BIV was cloned into an expression vector, pQE32, which expressed high levels of recombinant protein fromEscherichia coli. The ELISA was standardized by a checkerboard titration against known BIV-positive and -negative sera from cattle and a monoclonal antibody to the Gag protein. A total of 139 cattle serum samples, from the diagnostic laboratory at Kansas State University, Manhattan, and from the Dairy Station, Louisiana State University, Baton Rouge, were compared by ELISA and immunoblot assays for the detection of BIV-specific antibodies. Of 26 cattle sera samples which tested positive using the immunoblot assay, 23 were positive by ELISA, thus establishing a strong correlation between the two tests. The sensitivity and specificity of ELISA relative to immunoblotting were 0.88 and 0.93, respectively. ELISA proved to be as specific as immunoblotting but was much less time-consuming and easier to perform.

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