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J Virol Methods. 2014 Feb;196:7-14. doi: 10.1016/j.jviromet.2013.09.016. Epub 2013 Oct 24.

Production of cocktail of polyclonal antibodies using bacterial expressed recombinant protein for multiple virus detection.

Author information

1
Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012, India.
2
Amity Institute of Biotechnology, Amity University, Noida 201303, India.
3
Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012, India. Electronic address: rakeshjain56@yahoo.co.in.

Abstract

Cocktail of polyclonal antibodies (PAb) were produced that will help in multiple virus detection and overcome the limitation of individual virus purification, protein expression and purification as well as immunization in multiple rabbits. A dual fusion construct was developed using conserved coat protein (CP) sequences of Cucumber mosaic virus (CMV) and Papaya ringspot virus (PRSV) in an expression vector, pET-28a(+). The fusion protein (∼40kDa) was expressed in Escherichia coli and purified. Likewise, a triple fusion construct was developed by fusing conserved CP sequences of CMV and PRSV with conserved nucleocapsid protein (N) sequence of Groundnut bud necrosis virus (GBNV) and expressed as a fusion protein (∼50kDa) in pET-28a(+). PAb made separately to each of these three viruses recognized the double and triple fusion proteins in Western blot indicating retention of desired epitopes for binding with target antibodies. The fusion proteins (∼40kDa and ∼50kDa) were used to produce cocktail of PAb by immunizing rabbits, which simultaneously detected natural infection of CMV and PRSV or CMV, PRSV and GBNV in Cucurbitaceous, Solanaceous and other hosts in DAC-ELISA. This is the first report on production of a cocktail of PAb to recombinant fusion protein of two or three distinct viruses.

KEYWORDS:

Cocktail antibodies; DAC-ELISA; Recombinant fusion protein; Western blot

PMID:
24161814
DOI:
10.1016/j.jviromet.2013.09.016
[Indexed for MEDLINE]
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