Development and evaluation of a novel antigen capture assay for the detection of classical swine fever virus antigens

Clavijo, Alfonso; Zhou, En‐Min; Vydelingum, Soopayah; Heckert, Robert A.

doi:10.1016/s0378-1135(98)00160-6

Cited by 22 publications

(17 citation statements)

References 21 publications

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“…SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by the method described by Laemmli (16), with 4% stacking gels and 12% resolving gels and a Bio-Rad minigel apparatus. The separated proteins were either stained with Coomassie blue or analyzed by use of Western blots (19) probed with MAb WH303 or IgG antibodies (hyperimmune serum) from an experimentally CSFV-infected pig (4). Bound antibodies were detected by using horseradish peroxidase (HRP)-conjugated goat antimouse (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.) or rabbit anti-swine IgG (ICN Pharmaceuticals Inc., Aurora, Ohio) with a 4-chloro-1-naphthol-H 2 O 2 substrate kit (Bio-Rad, Mississauga, Ontario, Canada) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Deletions of Structural Glycoprotein E2 of Classical Swine Fever Virus Strain Alfort/187 Resolve a Linear Epitope of Monoclonal Antibody WH303 and the Minimal N-Terminal Domain Essential for Binding Immunoglobulin G Antibodies of a Pig Hyperimmune Serum

Lin¹,

Fan²,

Mallory³

et al. 2000

J Virol

110

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The major structural glycoprotein E2 of classical swine fever virus (CSFV) is responsible for eliciting neutralizing antibodies and conferring protective immunity. The current structural model of this protein predicts its surface-exposed region at the N terminus with a short stretch of the C-terminal residues spanning the membrane envelope. In this study, the N-terminal region of 221 amino acids (aa) covering aa 690 to 910 of the CSFV strain Alfort/187 E2, expressed as a fusion product in Escherichia coli, was shown to contain the epitope recognized by a monoclonal antibody (WH303) with affinity for various CSFV strains but not for the other members of the Pestivirus genus, bovine viral diarrhea virus (BVDV) and border disease virus (BDV). This region also contains the sites recognized by polyclonal immunoglobulin G (IgG) antibodies of a pig hyperimmune serum. Serial deletions of this region precisely defined the epitope recognized by WH303 to be TAVSPTTLR (aa 829 to 837) of E2. Comparison of the sequences around the WH303-binding site among the E2 proteins of pestiviruses indicated that the sequence TAVSPTTLR is strongly conserved in CSFV strains but highly divergent among BVDV and BDV strains. These results provided a structural basis for the reactivity patterns of WH303 and also useful information for the design of a peptide containing this epitope for potential use in the detection and identification of CSFV. By deletion analysis, an antigenic domain capable of reacting with pig polyclonal IgG was found 17 aa from the WH303 epitope within the N-terminal 123 residues (aa 690 to 812). Small N-or C-terminal deletions introduced into the domain disrupt its reactivity with pig polyclonal IgG, suggesting that this is the minimal antigenic domain required for binding to pig antibodies. This domain could have eliminated or reduced the cross-reactivity with other pestiviruses and may thus have an application for the serological detection of CSFV infection; evaluation of this is now possible, since the domain has been expressed in E. coli in large amounts and purified to homogeneity by chromatographic methods.

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Section: Methodsmentioning

confidence: 99%

Deletions of Structural Glycoprotein E2 of Classical Swine Fever Virus Strain Alfort/187 Resolve a Linear Epitope of Monoclonal Antibody WH303 and the Minimal N-Terminal Domain Essential for Binding Immunoglobulin G Antibodies of a Pig Hyperimmune Serum

Lin¹,

Fan²,

Mallory³

et al. 2000

J Virol

110

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“…Rapid, accurate, and pre-clinical laboratory diagnosis of CSFV is therefore a matter of urgency in order to prevent and control the epidemics. Current laboratory diagnoses of CSFV rely on virus isolation, serological methods, detection of antigen and nucleic acid amplification [9][10][11][12]. The PCRbase procedures are generally considered to be the most sensitive in vitro method for detecting CSFV infection.…”

Section: Introductionmentioning

confidence: 99%

Detection of nucleic acid of classical swine fever virus by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Wongsawat¹,

Dharaku²,

Narat³

et al. 2011

Health

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Classical swine fever virus (CSFV) is the causative agent of Classical swine fever which is a highly contagious disease affecting swine and resulting in severe economic losses. In this study, we developed reverse transcription loopmediated isothermal amplification (RT-LAMP) assay targeting the 5'UTR gene for the detection of CSFV. This amplification method can be obtained in 1 h under isothermal conditions (65°C) employing a set of six specific primers mixtures. Amplification product was visualized by using hydroxynaphthol blue (HNB) dye and agarose gel electrophoresis. The sensitivity was 100 copy numbers. No cross-reactivity related to Japanese encephalitis virus (JEV) and porcine reproductive and respiratory syndrome virus (PR-RSV) was demonstrated. The results demonstrated that the RT-LAMP assay is a useful tool for the rapid and sensitive for CSFV detection in swine.

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“…Current diagnostic methods, including detection of viral antigens in tonsils by using fluorescent antibody (18), antigen capture enzyme-linked immunosorbent assay (4,21), and detection of genomic RNA by reverse transcription-PCR (3,8,11,12,24), are relatively rapid diagnostic tests; however, these techniques require centralized laboratory facilities and clinical specimen submissions, which delay disease diagnosis, thus affecting the efficiency of emergency disease management measures.…”

mentioning

confidence: 99%

Diagnostic Evaluation of a Real-Time Reverse Transcriptase PCR Assay for Detection of Classical Swine Fever Virus

Risatti

Holinka

et al. 2005

J Clin Microbiol

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A fluorogenic-probe hydrolysis (TaqMan)-reverse transcriptase (RT) PCR for classical swine fever virus (CSFV) was evaluated for diagnostic sensitivity and specificity by using clinical samples obtained from the Dominican Republic, where the disease is enzootic. The sensitivity of this test, using nasal swab samples taken from both symptomatic and asymptomatic animals, exceeded the diagnostic sensitivity of virus isolation (100% versus 72.4%, respectively) with little loss of specificity (98.9% versus 100%, respectively). At the herd level, three of four infected farms were identified by virus isolation, while the CSFV real-time RT-PCR assay identified all four infected premises. This simple and accurate test permits rapid detection of CSFV in affected herds.

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Development and evaluation of a novel antigen capture assay for the detection of classical swine fever virus antigens

Cited by 22 publications

References 21 publications

Deletions of Structural Glycoprotein E2 of Classical Swine Fever Virus Strain Alfort/187 Resolve a Linear Epitope of Monoclonal Antibody WH303 and the Minimal N-Terminal Domain Essential for Binding Immunoglobulin G Antibodies of a Pig Hyperimmune Serum

Deletions of Structural Glycoprotein E2 of Classical Swine Fever Virus Strain Alfort/187 Resolve a Linear Epitope of Monoclonal Antibody WH303 and the Minimal N-Terminal Domain Essential for Binding Immunoglobulin G Antibodies of a Pig Hyperimmune Serum

Detection of nucleic acid of classical swine fever virus by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Diagnostic Evaluation of a Real-Time Reverse Transcriptase PCR Assay for Detection of Classical Swine Fever Virus

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