How do i optimize the Phage-antibody competitive ELISA?

30 October 2023 0 147 Report

Hello,

I am working on scFV phage display library. I got positive monoclonal from Phage display library. Now i did Phage-antibody competitive ELISA. i got following surprising results.

1) Phage affinity calibration curve was optimized with coated antigen on ELISA plate.

2) also calibrate the antibody affinity curve with coated antigen on ELISA plate.

3) use various phage dilution (50ul, 40ul, 30ul, 20ul, 10ul, 0ul) with antibody concentration (500nM) got from step 2.

Problem:

4) When i followed the same protocol from step 3 with serial dilution of Antibody (2uM 5 fold dilution). i got low affinity.

Note: Control is also run along side the experiment.

Whats the possible reason. Need your expert opinion.

Thanks

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