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[LadyA2A]

Hey every body,  .
I've been working on the developpment of an indirect ELISA test based on 2 recombinant protein to cover the 2 main subtypes of Mycoplasma pneumoniae.
I achieved the cloning part and I have the 2 proteins purified and with good quantity but my problem was how to start the ELISA test.
I did a serial dilution of the 2 proteins and i choosed the concentration that gives me an OD=3*negatif control. For this step, I used a plyclonal rabbit anti-Mycoplasma pneumonie antibodies.
After that I run the ELISA test with this concentration using human sera.
I did 3 dilutions for each serum 1/100, 1/200 and 1/400.
My problem now is :

• How to fix the right dilution for human sera?
  
• is the determinantion of cut-off necessary? how to fix its value?

Any help  that you can provide would be very much appreciated because the whole ELISA story is new for me. Thank you.