Division (b) DASI ELISAF1
United Kingdom
- 1))
Prepare the appropriate dilution of anti- R. solanacearum polyclonal immunoglobulins in coating buffer pH 9.6 (Appendix 4). Add 200 µl to each well. Incubate at 37 °C for four to five hours or at 4 °C for 16 hours.
- 2))
Wash the wells three times with PBS-Tween (Appendix 4).
Add 190 µl of sample extract to at least two wells. Also add positive and negative controls in two wells each per plate. Incubate for 16 hr at 4 °C.
- 3))
Wash the wells three times with PBS-Tween (Appendix 4).
- 4))
Prepare an appropriate dilution of R. solanacearum -specific monoclonal antibodies in PBS (Appendix 4) also containing 0,5 % bovine serum albumin (BSA) and add 190 µl to each well. Incubate at 37 °C for two hours.
- 5))
Wash the wells three times with PBS-Tween (Appendix 4).
- 6))
Prepare an appropriate dilution of anti-mouse immunoglobulins conjugated with alkaline phosphatase in PBS. Add 190 µl to each well. Incubate at 37 °C for two hours.
- 7))
Wash the wells three times with PBS-Tween (Appendix 4).
- 8))
Prepare an alkaline phosphatase substrate solution containing 1 mg p-nitrophenyl phosphate per ml of substrate buffer (Appendix 4). Add 200 µl to each well. Incubate in the dark at ambient temperature and read absorbance at 40 nm at regular intervals within 90 minutes.