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United States Department of Agriculture

Agricultural Research Service

Title: Mirror Image in Vivo Electroblotting Technique, a New Technique for Visualizing Virus Particles Electrophoretically Transferred from Infected Leaved to Nitrocellulose Membranes

Authors
item Wagih, Elsayed - UNIV ALEXANDRIA, EGYPT
item Melouk, Hassan
item Sherwood, John - OKLA STATE UNIV

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 8, 1993
Publication Date: N/A

Interpretive Summary: Visualizing virus particles or virus coat protein(s) in vivo, particularly in localized infections, has always been a challenging problem for virologists. Previous methods to accomplish this include electron microscopy and dot enzyme-linked immunosorbent assay. However, these methods are time- and labor-intensive as well as frequently inaccurate. Using a novel approach to an enzyme-linked immunobinding technique, we developed a new method, described in this paper, that results in a mirror image copy of the pattern of virus distribution. This new technique, because of its relative simplicity and sensitivity, will be useful for many fields of biological research where the in vivo distribution of pathogens or cells of specific antigen molecules, such as cancer, needs to be investigated.

Technical Abstract: A new technique was developed for visualizing virus particles electrophoretically transferred from infected leaves to nitrocellulose membranes. This technique was used to study the distribution of peanut mottle virus (PMV) particles in vivo. The immobilized transferable virus proteins from leaf tissue to nitrocellulose membranes were detected by an enzyme-linked immunobinding technique. The free binding groups on the nitrocellulose membrane were blocked with casein. The nitrocellulose membrane was then treated with PMV-specific antiserum. The virus-bound antibodies were located by protein A-peroxidase or protein A-alkaline phosphatase conjugate followed by the peroxidase substrate mixture, 4- chloro-1-naphthol and H2O2, in the first case or the alkaline phosphatase substrate mixture, 5-bromo-4-chloro-3-indolyl phosphate and Nitroblue tetrazolium in the second case. The locations of virus particles were detected by the corresponding reaction product. A mirror image was obtaine on nitrocellulose membrane showing a pattern identical to that of necrotic lesions on leaf pieces but with a significantly larger size of local lesion copy, indicating the presence of virus particles in the apparently healthy tissue outside the necrotic lesion. The new technique is expected, because of its relative simplicity and sensitivity, to be useful for many other fields of biological research where the in vivo distribution of pathogens, antigenic molecules including drugs, or cells of specific antigenic molecules such as cancer or genetically engineered cells needs to be investigated.

Last Modified: 9/20/2014
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