Dispense 100uL samples in wells of dummy plate according to worklist using 100 uL pipette.
Take out the microtiter plate. Don't touch the lower surface of the plate.
Dispense 25 uL sample from the dummy plate into the microtiter plate using multichannel pipette.Note the time of starting dispensing of sample.
Take enzyme conjugate in trough.
Dispense 100 uL enzyme conjugate in microtiter plate using multichannel pipette.Note time after last column is dispensed with conjugate.
Mix the plate in incubator- shaker for 10 sec
Incubate the plate at 24'C in the incubator for 1 hr from midpoint of start time of sample dispensing and end of conjugate dispensing.
Wash buffer:dilute 1 volume of wash buffer(25X)with 24 volumes of DI water.
To make 500 ml,Take 20 ml of wash buffer and make upto 500 ml with DI water.
After 1 hour , take the plate from the incubator. And wash the plate in the
ELISA washer.
After washing, Pat dry the plate on tissue paper.
Take TMB(Tetramethyl benzedene) substrate in the trough.
Dispense 100 uL TMB substrate in the microtiter plates with the multichannel pipette.
Mix in incubator cum shaker for 10sec at room temperature.
Incubate at 24'C in the incubator for 20 minutes.
Take stop solution in the trough.
Dispense 150 uL stop solution in the microtiter plate.
Mix in incubator cum shaker for 10 sec.
Incubate for 5 minutes in the incubator at 24'C.
Take the plate from incubator and put it in the ELISA reader.
Connect ELISA reader with the computer and set Filter i.e at 450 nm . then read plate for 3 times.
Take average of the 3 OD . and sort the data and calculate results from the graph derived from Standard.
Whenever result of any sample comes negetive,it's OD will be more than OD of “0” standard OD.So,result will come negetive.So,release the result as “<0”pmol/L.
Whenever OD of any sample comes less than OD of highest Free T4 calibrator,release the result as “>value of highest calibrator”.e.g.If highest calibrator value is 95pmol/L,than release the report as “>95”pmol/L.