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?