Hello

RIA is a type of competitive assay. In this assay keeping the concentration of labelled antigen and unlabeled antibody fixed, the amount of unlabeled antigen (either standard or antigen in the sample) can vary. The unlabeled antigen (either standard or sample) when added to the mixture of labelled antigen and unlabeled antibody, the unlabeled antigen will compete with the labelled antigen for the antibody binding site in the reaction tube and displace the labelled antigen in solution.

"So higher the concentration of the unlabeled antigen in the sample more will be the displacement of the labelled antigen from the labelled antigen-antibody complex and lesser will be the radioactivity measured for the unlabeled antigen-antibody complex." There is an inverse relationship between concentration of unlabeled antigen in the sample and the radioactive measurement of the unlabeled antigen-antibody complex.

Calculations:

Plotting the standard curve.

Determine the normalized percent bound (%B/B0) for each standard and sample as follows:

%B/B0 = net cpm of std or sample /net cpm of 'zero' std

The above ratio multiplied by 100.

Plot %B/B0 for each std v/s the corresponding amounts of unlabeled calibrator. Then determine the amount of analyte in each sample from the standard curve.

I hope I have answered all the points mentioned in your question.

Best Wishes.

Malcolm is spot on. Once you have generated the standard curve, you can then curve fit to find the best fit and excel will generate the equation of the curve. You will then plug your OD values of your unknowns into the equation to determine the concentration of your analyte

Thank you for your answers. Does this mean there is at the same time labelled antigen and unlabelled antigen and how to tell the difference between them?