Dr Piyush Tailor

1. Dispense 200uL samples in wells of dummy plate according to worklist using 200 uL pipette.
2. Take out the microtiter plate. Don't touch the lower surface of the plate.
3. Dispense 50 uL sample from the dummy plate into the microtiter plate using multichannel pipette.Note the time of starting dispensing of sample.
4. Take enzyme conjugate in trough.
5. Dispense 50 uL enzyme conjugate in microtiter plate using multichannel pipette.Note time after last column is dispensed with conjugate.
6. Mix the plate in incubator- shaker for 10 sec.
7. Incubate the plate at 24'C in the incubator for 2 hrs from midpoint of start time of sample dispensing and end of conjugate dispensing..
8. After 2 hours , take the plate from the incubator. And wash the plate in the ELISA washer.
9. After washing, Pat dry the plate on tissue paper.
10. Take TMB(Tetramethyl benzedene) substrate in the trough.
11. Dispense 100 uL TMB substrate in the microtiter plates with the multichannel pipette.
12. Mix in incubator cum shaker for 5 sec.
13. Incubate at 24'C in the incubator for 20 minutes.
14. Take stop solution in the trough.
15. Dispense 150 uL stop solution in the microtiter plate.
16. Mix in incubator cum shaker for 5 sec.
17. Incubate for 5 minutes in the incubator at 24'C.
18. Take the plate from incubator and put it in the ELISA reader.
19. Connect ELISA reader with the computer and set Filter i.e at 450 nm . then read plate for 3 times.
20. Take average of the 3 OD . and sort the data and calculate results from the graph derived from Standard.
21. Whenever OD of any sample is more than OD of lowest value of calibrator,result will calculated as “#NA”,So release it as “<0.2” microU/ml.
22. Whenever OD of any sample is more than OD of highest value of calibrator,Dilute the sample for 1:10 times in next batch and release the report by multiplying result with 10.
23. If the OD of diluted sample is more than OD of highest value of calibrator,release result as “>value of highest calibrator” e.g.If highest calibrator value is 9.5 microU/ml,then release the report as “>95”microU/ml.
24. After complition of use of elisa washer,give prime wash with deionised water.

General procedure for ELISA READER

Note on using electronic pipette for ELISA