Introduction

Immunoelectron microscopy (IEM) is the term generally used for techniques that detect the specific binding of antibody to antigen that can be visualized by electron microscopy (1). The use of these techniques results in a 2- to 10,000-fold increase in particle numbers in comparison with methods not using antisera (1) and allows the transmission electron microscope (TEM) to become a sensitive tool, which the plant virus diagnostician or researcher can use to

From: Methods in Molecular Biology, vol. 295: Immunochemical Protocols, Third Edition. Edited by: R. Burns © Humana Press Inc., Totowa, NJ

visualize, specifically detect, and confirm the presence and identity of plant viruses. Only small amounts of even crude antisera and virus extract are necessary with short incubation times for all steps. Carbon-coated electron microscope grids are treated with specific antibody that is used to "trap" virus particles (antigen) that are present in a sap extract, rather like a biological "sticky tape." After trapping, the treated grids are washed thoroughly, negatively stained, and can be visualized in the TEM. Virus particles present can be specifically "labeled" by further incubation with a more concentrated antibody preparation, which has the effect of "decorating" the virus particles with an excess of antibody molecules (2). This can be further enhanced by labeling the decorated virus particles with colloidal gold particles complexed with protein A (3).

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