Determination of Enzyme Activity by ELISA

Dilute anti-(mouse-lgG)-lgG (in the case of monoclonal antibodies for testing conjugates prepared from antibodies of other origin, use the respective anti-species specific antibody) to 5 pg/ml in Soln. A. Coat the wells of a microtest plate with 100 pl/well of this dilution and incubate at 4 °C overnight. Remove antibody solution and wash once with TBS. Add 150 pi Soln. C per well and incubate at 

RT for 30 min. Remove blocking solution, rinse once with Soln. B and add 100 pl/well of a dilution series (e.g., 1 500,1 1500,1 4500, 1 13 500,1 40 500,1 121 500,1 364 500 in TBS) of antibody-enzyme conjugate to the wells. Shake at RT for 30 min, remove conjugate solution, knock out the plate on paper tissue, and rinse three times with Soln. B. 

Perform the enzymatic reaction with Soln. D for 10-30 min (each well has to react for the same time and at the same temperature) as described above, stop with Soln. E and read O.D. at 450 nm. Plot O.D. against conjugate dilution. The working dilution of the conjugate should give an O.D. of 1.5-2.5. 

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