How to coat plasma sample (Antigen) in ELISA plate for a direct ELISA?

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Alexandr Chernov

Is you have antibody conjugated to a fluorochrome or just an antibody?

If you want to determine the concentration of antigen, then you need to make a sandwich ELISA.

For example, to detect concentration of endoglin.

Prasanna Pal

Alexandr Chernov Sir, I have antibody developed in rabbit and also have anti-rabbit secondary antibody conjugated to HRP. Can I coat the plasma sample (containing Ag) in immuno plate and determine its concentration using these two antibodies?

Michael G. Weller

As Antoni Iborra

already mentioned, the direct coating of plasma on the plate will usually not work if your target is not a major component. For other targets, the use of a sandwich-immunoassay (non-competitive immunoassay) should be used. In most cases, two different antibodies of different species are required. If you have only one antibody, a competitive assay may be applied.

Antoni Iborra

Sir, thank you for your answer.

What should be the percentage of target protein in plasma to be detected in direct ELISA?

Michael G. Weller Sir, can you please share a standard protocol of competitive ELISA stating concentration of primary and secondary antibody to be used, the concentration of antigen for coating etc.

Thank you.

It is not possible to give a protocol without many details of your system. Polyclonal antibodies are not standard reagents. Their dilution factors vary even from batch to batch. Such assays are optimized in a largely empirical way.

Zid Touzani

Michael G. Weller and Antoni Iborra

I have the same question for example for cortisol or any small molecule which competitve ELISA can I use, direct or indirect one and also if you have a good protocol as reagents I have BSA-corticsol that I want to coat on the plaque, Abs against cortisol, cortisol and Abs-HRP against Abs against cortisol.