IMMUNO ELECTRON MICROSCOPY (IEM)

LABORATORY PROCEDURES FOR PLANT CELL VIRUSES

Appendix

PVC/1998/2.03 Appendix 2

IMMUNO ELECTRON MICROSCOPY (IEM)

INTRODUCTION

In immuno electron microscopy an adsorption preparation is incubated with a virus specific antiserum, subsequently contrasted with an electron dense solution and subjected to electron microscopical examination. The immunological reaction given by the precipitation of the virus specific antibodies with the coat protein of the virus and visible in electron microscopy proves the identity of the virus present and provides further evidence for the composition of the viruses in the sample, e.g. indicates for undecorated virus-like particles.

PROCEDURE

A leaf dip sample is prepared according to PVC/1998/2.02 Appendix 5 and the homogenate is incubated on the grids for 5 min. After incubation with virus suspension, the grids are washed gently with approximately 40 drops of 0.1 M sodium/potassium phosphate buffer, pH 7.0, and are subsequently incubated with virus specific antisera diluted 1:50 (v/v) in the same buffer for 15 min. After thorough washing with a gentle stream of 40 drops of deionised water, the virus grids are contrasted for EM as in PVC/1998/2.02 Appendix 5.

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