[[Preparation before doing procedure for ELISA]] 
       
=====Preparation before starting free T4 ELISA=====
  -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.  

**General procedure for [[ELISA READER]]**

**[[Note on using electronic pipette for ELISA]]**