Japanese Encephalitis (JE) ELISA Protocol

1. Take the required number of micro wells from the foil pouch. Number the test strips as 1, 2, 3, 4….)
2. Select the samples to be tested. Dilute serum sample 1:100 or CSF 1:10 in tubes using sample diluents for JE IgM.
3. Prepare wash buffer. Dilute wash buffer concentrate to 1X with distilled water.
4. Wash the strips 3 times with 1x Wash Buffer. Do not allow the wells to dry.
5. Add 50µl of JE Positive control and JE IgM Negative control to respective wells as per the protocol.
6. Transfer 50µl of diluted samples to respective wells.
7. Cover the plate with adhesive plate sealer to prevent evaporation of samples. Keep the plate in a humidified box and incubate the plate at 37ºC for 1 hour.
8. At the end of incubation, wash the plate five times with wash buffer. Tap the plate after last wash on a tissue paper.
9. Add 50µl of JE antigen to each well of the plate.
10. Cover the plate with adhesive plate sealer to prevent evaporation of samples. Keep the plate in a humidified box and incubate the plate at 37ºC for 1 hour.
11. At the end of incubation, wash the plate five times with wash buffer. Tap the plate after last wash on a tissue paper.
12. Add 50µl of Anti JE monoclonal antibody (Biotin labeled) to each well.
13. Cover the plate with adhesive plate sealer and incubate the plate at 37ºC for 1 hour.
14. Wash the wells 5 times with 300µl diluted wash buffer.
15. Add 50µl of Avidin-HRP to each well.
16. Cover the plate with adhesive plate sealer and incubate the plate at 37ºC for 30 minutes.
17. Wash the wells 5 times with 300µl diluted wash buffer.
18. Add 100µl of TMB substrate to each well.
19. Incubate at room temperature in dark for 10 minutes.
20. Add 100µl stop solution to stop the reaction.
21. Read the absorbance within 10 minutes at 450nm with a reference filter of 620nm.

Japanese Encephalitis (JE) ELISA Protocol

Tags: ELISA Protocol, Japanese Encephalitis, Japanese Encephalitis (JE), Japanese Encephalitis (JE) ELISA, JE, JE ELISA