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8) Add 100 µl of TMB substrate to each well, and incubate for 10 min in the dark. 9) Stop the reaction with the addition of 50 µl of 5% sulphuric acid. 10) Read the plate visually and spectrophotometrically at 450 nm on a microplate reader to obtain an OD reading. Interpretation of results Determine the ratio of the OD readings obtained for the two subgroup Mabs. 0 } positive Manual of Rotavirus detection and characterization methods 4. 1. 2, using a screw-capped microcentrifuge tube. 5. 2) Add an equal volume of Vertrel XF, vortex for 1 minute, and centrifuge for 10 min, 8,000 g at RT.

Electron microscopy of viruses. In: Cann AJ, ed. Cell culture: a practical approach. Oxford University Press, 1999. 4) Observe in the electron microscope, and identify rotavirus particles through their characteristic morphology (Figure 5). 5 22 This method was provided by the West African Regional Rotavirus Laboratory Manual of Rotavirus detection and characterization methods Figure 5. 0 Add 1 g of SDS to 10 ml of distilled water. Dissolve in a 65°C water bath. 2 g of sodium acetate in 60 ml of distilled water.

Detection procedure 1) Make up TMB substrate buffer, and add 100 µl of buffer to all wells. 2) Incubate plates for 10 min at room temperature; positive wells turn blue. 3) Stop the reaction by adding 50 µl/well of 2M H2SO4; positive wells turn yellow. , Titertek Multiscan MCC/340 MKII) using a 450-nm filter. 2 and is at least twice the absorbance of the background control. A list of possible results for the G serotyping EIA is provided in Table 3. Notes: 1) Wash buffer can be added with a squirt bottle or a microtiter plate washer.

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