ISEM Combined With Antibody Decoration

Virus particles that have been already adsorbed or "trapped" on the grid by ISEM are further incubated with the same specific anti body but at a lower dilution. This has the effect of "decorating" or coating the virus particles individually, thus specifically identifying them ([2]; see Note 15). Such flexibility is extremely valuable especially in diagnostic situations for the identification of unknown viruses or for differentiating morphologically identical viruses in mixed infections (10).

1. Using a variable volume pipet dilute required specific antiserum (see Note 2) to 1:500 in phosphate buffer pH 6.5 (see Note 4) in a 0.5-mL microfuge tube.

2. In a Parafilm lined Petri dish, place 20-^L drops of the diluted antiserum (see Note 5) and, using the watchmaker's forceps, float carbon-coated grids (see Note 6), carbon-side down, on them. Incubate at room temperature for 15 min (see Note 7).

3. Grind infected material in a muslin-lined bag in 0.06 M phosphate buffer, pH 6.5, with a hand grinder (see Note 10) to a dilution of 1:10.

4. Place 20-^L drops of sap extract on Parafilm in a Petri dish

5. Hold the antiserum coated grids in the forceps and wash with 20 drops of phosphate buffer ph 6.5, in a constant stream using a Pasteur pipet, on the treated side (see Note 11). Drain excess liquid with filter paper and place on sap for 15 min at room temperature (see Note 7)

6. Dilute specific antiserum to 1:100 (see Note 3) in a 0.5-mL microfuge tube and place 20-^L drops on Parafilm in a Petri dish.

7. Wash the grids with 20 drops of buffer and drain as before. Place them on the diluted antibody and incubate for 15 min at room temperature (see Note 7).

8. Wash the grids with 20 drops of distilled water and stain with three to five drops of 2% uranyl acetate (see Note 12), draining as before (see Note 14).

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