PMID- 10856758
OWN - NLM
STAT- MEDLINE
DCOM- 20000808
LR  - 20190831
IS  - 0166-0934 (Print)
IS  - 0166-0934 (Linking)
VI  - 87
IP  - 1-2
DP  - 2000 Jun
TI  - Competitive ELISA for detection of antibodies to porcine reproductive and 
      respiratory syndrome virus using recombinant E. coli-expressed nucleocapsid 
      protein as antigen.
PG  - 109-22
AB  - The 15 kDa nucleocapsid (N) protein is the most abundant protein of the porcine 
      reproductive and respiratory syndrome virus (PRRSV), and is highly antigenic, 
      which therefore makes it a suitable candidate for the detection of virus-specific 
      antibodies and diagnosis of the disease. In this study, complementary DNA 
      corresponding to the entire N gene of the IAF-Klop strain of PRRSV was cloned 
      into the pGEX-4T-1 vector, and the N protein was expressed in Escherichia coli 
      fused to the glutathione S-transferase (GST) protein. The resulting GST-N 
      recombinant fusion protein was purified by affinity chromatography and used as 
      antigen for serological testing by indirect enzyme-linked immunosorbent assay 
      (ELISA). Two anti-N specific monoclonal antibodies (MAbs) (IAF-K8 and IAF-2B4), 
      obtained following fusion experiments with spleen cells of BAlb/c mice that were 
      immunized with the purified virus, were used in a competitive assay to increase 
      the specificity of the ELISA. Both MAbs were found to be directed against highly 
      conserved conformational epitopes of North American isolates of PRRSV. Optimal 
      concentration of GST-N protein was determined by checkerboard titration, using 
      hyperimmune pig antiserum to the homologous PRRSV strain, and corresponded to a 
      range of 0.1-0.5 microg protein per well. When tested on 95 sera from pigs that 
      were experimentally infected with the IAF-Klop strain, the competitive ELISA 
      (K8-ELISA) was capable of detecting anti-PRRSV antibodies in 86.7% (65/75) and 
      92.6% (63/68) of pig sera known to be seropositive by indirect immunofluorescence 
      (antibody titers >16) and a currently used commercial ELISA (HerdCheck(R); 
      Idexx), with specificity values of 100 and 96.2%, respectively. When tested on 
      clinical samples (542 sera) from 28 positive and 28 negative pig herds, the 
      K8-ELISA performed in a similar way to HerdCheck(R) and immunofluorescence (IF) 
      tests as shown by kappa values of 0.762 and 0.803. The sensitivity and 
      specificity of K8-ELISA were 100% on a herd basis, whereas sensitivity values of 
      80 and 82% with a specificity of 98.7% were determined on an individual basis in 
      comparison with HerdCheck(R) and IF tests.
FAU - Dea, S
AU  - Dea S
AD  - Centre de Microbiologie et Biotechnologie, INRS-Institut Armand-Frappier, 
      Universite du Quebec, Laval, Canada. serge.dea@inrs-iaf.uquebec.ca
FAU - Wilson, L
AU  - Wilson L
FAU - Therrien, D
AU  - Therrien D
FAU - Cornaglia, E
AU  - Cornaglia E
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Netherlands
TA  - J Virol Methods
JT  - Journal of virological methods
JID - 8005839
RN  - 0 (Antibodies, Monoclonal)
RN  - 0 (Antibodies, Viral)
RN  - 0 (Antigens, Viral)
RN  - 0 (Epitopes)
RN  - 0 (Nucleocapsid Proteins)
RN  - 0 (Recombinant Fusion Proteins)
RN  - EC 2.5.1.18 (Glutathione Transferase)
SB  - IM
MH  - Animals
MH  - Antibodies, Monoclonal
MH  - Antibodies, Viral/*blood
MH  - Antigens, Viral/genetics
MH  - Enzyme-Linked Immunosorbent Assay/*veterinary
MH  - Epitopes/immunology
MH  - Escherichia coli/genetics
MH  - Europe
MH  - Fluorescent Antibody Technique, Indirect/veterinary
MH  - Genetic Vectors
MH  - Glutathione Transferase/metabolism
MH  - Mice
MH  - Mice, Inbred BALB C
MH  - North America
MH  - Nucleocapsid Proteins/genetics
MH  - Porcine Reproductive and Respiratory Syndrome/blood/*virology
MH  - Porcine respiratory and reproductive syndrome virus/genetics/*immunology
MH  - Recombinant Fusion Proteins/genetics
MH  - Swine
EDAT- 2000/06/17 09:00
MHDA- 2000/08/12 11:00
CRDT- 2000/06/17 09:00
PHST- 2000/06/17 09:00 [pubmed]
PHST- 2000/08/12 11:00 [medline]
PHST- 2000/06/17 09:00 [entrez]
AID - S0166093400001580 [pii]
AID - 10.1016/s0166-0934(00)00158-0 [doi]
PST - ppublish
SO  - J Virol Methods. 2000 Jun;87(1-2):109-22. doi: 10.1016/s0166-0934(00)00158-0.