I am new in ELISA assays. I am using crude total protein to coat ELISA plates for Indirect ELISA using 100ng conc. of Ag. I already did titration for Ag and Ab's. I am not using any standard antigen, so how can I quantify my protein samples? or make calculation to estimate desired protein concentration?
You need a standard containing a known quantity of the antigen in order to make a standard curve so that you can measure the antigen's concentration in your samples. That is assuming you want an absolute measurement.
If you can get by with a relative measurement, then you can express it as the signal in the ELISA (e.g. absorbance units) per microgram of total protein. But you will have to establish the dynamic range of the ELISA and only use results obtained within that range.
You need a standard containing a known quantity of the antigen in order to make a standard curve so that you can measure the antigen's concentration in your samples. That is assuming you want an absolute measurement.
If you can get by with a relative measurement, then you can express it as the signal in the ELISA (e.g. absorbance units) per microgram of total protein. But you will have to establish the dynamic range of the ELISA and only use results obtained within that range.
You can calculate the total protein in the solution used as antigen by any of methods used for measuring the protein concentration including Lowry et al (1951).
Then draw a standard curve accordingly.
Please follow Shapiro's instruction. It is correct. you may also wish to see some of my relevant publications.
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