Antigen Coating

Ag diluted in the ELISA coat buffer is added into the polystyrene wells. The dilution of the Ag should be in the such a pattern that the amount of Ag absorbed to the polysterine plate must be max at such dilution. The concentration taken should be 10^g/ml. Incubation for the 2 hours overnight at 370c. Store the Ag solution until the immunoassay is performed. Before performing the immunoassay the wells should be washed in the following way PBS-TWEEN wash. Add 300 ц1 of the PBS-TWEEN buffer mix it well and leave it for 2 min after that aspirate the buffer from the wells.


Add 300 ц1 of the BSA-ELISA coat buffer to the wells and incubate at RT for about two hours. Repeat the PBS-TWEEN wash and also repeat the BSA_ELISA wash

Test Sample Addition

Add 300 ц1 of the undiluted monoclonal supernatant. Incubate it for 4 hours at RT or for overnight at 40C. PBS-TWEEN washes for 3 times

Conjugate Addition
Add 300 ц1 of the 1:5000 v/v diluted (in PBS-TWEEN Buffer) conjugated. Incubate at RT for 2 hours. PBS-TWEEN Washes for 3 times. 0.15M NaCl wash for 3 times. Then Add 300 ц1 of the 0.15M NaCl. Mix the solution 2 and leave it for one to two hours. Aspirate the solution and follow the successive washes.

Substrate Addition

Add 300 ц1 of the substrate to each well. Watch for the development of the colours. Here the substrate is present in the sodium-corbonate buffer ((pH 9.8) in the concentration of 1mg/ml where the buffer is also containing MgCl2 at the molarity value of 1/1000 M

ELISA readings

The sodium colour development is measured by the help of ELISA reader at the wave length of 400nm.