08 August 2016 12 3K Report

I have been running ELISAs for a week but I can't get it to work.

I coated the 96well plate with 100ul of 20ug/ml of campylobacter jejuni in 1M bicarbonate buffer overnight at 4C. Washed 3 times with 0.5% PBS-Tween20. Blocked with 200ul milk solution containing 0.1g/ml non fat dry milk in PBS-Tween20. Added 100ul of chicken bile diluted in PBS-Tween20 in 1:100. Incubated at 37C for 2 hours. Washed 3 times with 0.5% PBS-Tween20. Added 100ul secondary antibodies which is goat anti-chicken IgA diluted in 1:1000 in PBS-Tween20. Incubated at 37C for 2 hours. Washed 3 times with 0.5% PBS-Tween20. Added 100ul of TMB in 0.1M sodium acetate and Hydrogen peroxide. Incubated in room temperature fro 2 hours. Added 100ul of H2SO4 to stop reaction.

Prior to the stop solution. None of the wells turned blue at all. Might try positive control using chicken serum and IgY as secondary antibodies tomorrow.

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