Division (a) Indirect ELISA (Robinson Smith et al. , 1995)F1
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- 1))
Use 100 to 200 µl aliquots of sample extract. (Heating at 100 °C for four minutes in a waterbath or heating block may reduce non-specific results in some cases).
- 2))
Add an equal volume of double strength coating buffer (Appendix 4) and vortex.
- 3))
Apply 100 µl aliquots to each of at least two wells of a microtitre plate (e.g. Nunc-Polysorp or equivalent) and incubate for one hour at 37 °C or overnight at 4 °C.
- 4))
Flick out the extracts from the wells. Wash the wells three times with PBS-Tween (Appendix 4), leaving the last washing solution in the wells for at least five minutes.
- 5))
Prepare the appropriate dilution of antibodies against- R. solanacearum in blocking buffer (Appendix 4). For validated commercial antibodies, use the recommended dilutions (usually twice as concentrated as the titre).
- 6))
Add 100 µl to each well and incubate for one hour at 37 °C.
- 7))
Flick out the antibody solution from the wells and wash as before (4).
- 8))
Prepare the appropriate dilution of secondary antibody-alkaline phosphatase conjugate in blocking buffer. Add 100 µl to each well and incubate for one hour at 37 °C.
- 9))
Flick out conjugated antibody from wells and wash as before (4).
- 10))
Add 100 µl alkaline phosphatase substrate solution (Appendix 4) to each well. Incubate in the dark at ambient temperature and read absorbance at 405 nm at regular intervals within 90 minutes.