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Peptides for Detection of Antibody to Porcine Reproductive Respiratory Syndrome Virus

a technology antibody, applied in the field of detection and quantification of porcine reproductive respiratory syndrome virus, can solve the problems of increased secondary bacterial infections, respiratory distress, and failure to thrive, and achieve the effect of reducing the incidence of false positives

Inactive Publication Date: 2009-12-10
IDEXX LABORATORIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012]Another embodiment of the invention provides a method of decreasing the incidence of false positives in a diagnostic assay that detects PRRSV antibodies specific for PRRSV ORF 7. The method comprises using a PRRSV ORF 7 polypeptide comprising about 19 to about 28 N-terminal amino acid deletions as an antibody capture antigen in the diagnostic assay. The PRRSV ORF7 polypeptide can be SEQ ID NO:1 or SEQ ID NO:18. The polypeptide can further comprise one or more amino acids at either terminus that are not contiguously associated with a PRRSV ORF7 in nature.

Problems solved by technology

The virus can cause major reproductive problems in adult pigs resulting in abortion.
In neonatal pigs the virus can cause respiratory distress, a failure to thrive, and increased secondary bacterial infections.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0071]PEXUSorf7 was expressed and purified as described previously (EMBO J. 1984, 3: 1429-1434). Recombinant full length U.S.ORF7, N-terminal deletion derivative U.S.ORF7 and Carboxy-terminal deletion derivative U.S.ORF7 proteins were expressed using a Studier pET expression system using methods described by the manufacturer (EMD Biosciences, Ind., Madison, Wis. 53719). The nucleic acids encoding the proteins described below were cloned into the pET200 expression system using methods described by the manufacturer. The recombinant proteins were expressed with a histidine tag at the amino terminus that is encoded by the vector allowing for rapid affinity purification. The proteins were expressed and purified from the E. coli strain BL21(star) using methods described by the manufacturer (EMD Biosciences). Crude lysates of E. coli were separated using SDS-PAGE gels, the separated proteins were blotted to nitrocellulose, and the nitrocellulose blot blocked using standard techniques known...

example 2

Reactivity in Western Blot

[0077]This data demonstrates the C-Terminal deletion derivative of U.S.ORF7 (SEQ ID NO:7) does not react with positive swine sera in a Western blot, where as the full length U.S.ORF7 and N-Terminal (a.k.a. amino-terminal) deletion derivative of U.S. ORF 7 (SEQ ID NO:6) both react with positive swine sera.

TABLE 3Positive SwineNegative SwineSeraSeraPEXUSorf7PositiveNegative(SEQ ID NO: 4)Full length U.S. ORF7PositiveNegative(SEQ ID NO: 5)N-Terminal derivativePositiveNegativeU.S. ORF7 (SEQ ID NO: 6)C-Terminal derivativeNegativeNegativeU.S. ORF7 (SEQ ID NO: 7)

example 3

Reactivity in ELISA

[0078]Recombinant protein was expressed using a Studier pET expression system using methods described by the manufacturer (EMD Biosciences, Ind., Madison, Wis. 53719). The genes encoding the proteins described above were cloned into the pET200 expression system using methods described by the manufacturer. The recombinant proteins are expressed with a histidine tag at the amino terminus that is encoded by the vector that allows rapid affinity purification. The protein was expressed and purified from the E. coli strain BL21(star) using methods described by the manufacturer (EMD Biosciences).

[0079]Immulon 1 plates were coated overnight at 4° C. with purified recombinant protein at 1 ug / ml in carbonate buffer, pH9.5. Plates were emptied by “flicking” and patting dry. Plates were blocked overnight using 2.5% BSA in PBS. Plates were “flicked” and patted dry and over-coated with 2.5% sucrose in 10 mM Tris buffer (pH7.5). After flicking and patting dry, plates were vacuum...

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PUM

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Abstract

The invention provides compositions and methods for the detection and quantification of PRRSV antibodies and antibody fragments using polypeptides.

Description

PRIORITY[0001]This application is a divisional application of U.S. Ser. No. 11 / 362,599, filed Feb. 24, 2006 (now allowed), which claims the benefit of U.S. Appl. No. 60 / 656,348, filed Feb. 25, 2005, both of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION[0002]The invention relates to methods and compositions for the detection and quantification of porcine reproductive respiratory syndrome virus (PRRSV) antibodies and antibody fragments using PRRSV polypeptides.BACKGROUND OF THE INVENTION[0003]Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus (RNA enveloped virus) that causes porcine reproductive and respiratory syndrome (PRRS). The virus can cause major reproductive problems in adult pigs resulting in abortion. In growing pigs the symptoms include increased mortality, decreased appetite, fever, respiratory problems, pneumonia, increased secondary bacterial infections, and atrophic rhinitis. In neonatal pigs the virus ca...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/70
CPCC07K14/005C07K2319/21G01N2469/20G01N33/56983G01N2469/10C12N2770/10022C07K14/08C07K16/10C12N15/11G01N33/569
Inventor KRAH, III, EUGENE REGIS
Owner IDEXX LABORATORIES
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