Question 1:

Problem: Polyclonal antibodies may bind to overlapping or closely situated epitopes, leading to competition and interference.

Using polyclonal antibodies from the same species can lead to non-specific binding and increased background signal

Solutions

Cross-reactivity: Ensure that the capture and detection antibodies recognize different, non-competing epitopes on the antigen

Background Signal: Adding steps like pre-absorption of the polyclonal serum with unrelated proteins can also help.

Recommendations: Optimize antibody concentrations, incubation times, and washing steps to minimize non-specific binding and background noise. Use effective blocking agents like BSA, casein, or commercial blocking buffers to reduce non-specific binding.

Question 2: Solutions provided for probable issues.

Question 3:

Solution:

The detection antibody (e.g., HRP) could be labeled, but it may cause non-specific binding if not optimized properly.

Using a secondary antibody (e.g., anti-rabbit HRP) as indirect labeling can amplify the signal and reduce background noise.

You can follow this article as reference

https://www.mdpi.com/2615486

Best Wishes

@ Swagata Sarkar- Thankful.