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Indirect rec ELISA optimisation

indirect ELISA cut-off OD interpretation

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#1 LadyA2A

LadyA2A

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Posted 02 February 2013 - 02:38 PM

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. Posted Image





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