Center for Radiochemistry and Nuclear Materials
Department of Chemistry
To understand the technique of radioimmunoassay by performing a chemical ‘mock-up’ of the procedure.
Explanation and Exercise Guide
Theoretical background for radioimmunoassay (read)
- Pipette 1 cm3 of each of the antigen solutions into 5 gamma vials
- Pipette 1 cm3 of assay sample into 6th vial
- Locate the 8 cm3 of 125I (aq) which is in a labelled glass vial
- Use the 1 mL autopipette to add 1 cm3 of the 125I-antigen to each of the 6 vials
- Add 1 mL of 125I-antigen to 7th vial and label it A – to measure total activity
- Add 1 cm3 of buffer solution to 1st 6 vials and 2 cm3 to the 7th vial (A)
- Leave vial A to one side
- Add 1 cm3 of antibody solution to each of the other 6 vials
- Count sample A on the Triathler (see operating instructions below) for 1 min to determine background 35
- Centrifuge the antigen-antibody complex in the 6 vials at 5000 rpm for 5 min – place 2 red adapters in each of the 6 holes marked with red crosses and place your vials in these wells
- Pipette 2 cm3 of supernatant from each vial into a 2nd set of clean gamma vials and label carefully
- Carefully remove rest of supernatant from each vial and dispose of in the plastic beaker labelled 125I waste
- Use 2 cm3 of buffer solution to wash each precipitate – dispose of the washings in the plastic beaker
- Re-centrifuge the precipitate as above and discard its supernatant into waste beaker
- Count the 6 precipitate samples and all 6 supernatant samples in the Triathler
- For each sample calculate the fraction of the 125I activity found in the antibody-antigen complex, and the fraction found in the solution, i.e. as a free antigen, by dividing your counts by ‘A’. Remember that only half the supernatant liquid was counted in each case.
- Calculate the bound/free antigen ratio for each sample.
- Using the 5 standard antigen concentrations provided plot graphs, using Excel of:
- The bound activity fractions vs. the standard antigen concentration;
- The free activity fraction vs. the standard antigen concentration;
- The bound/free ratio vs. the standard antigen concentration.
- Fit the points with a non-linear trend line. Use the type that fits best. Use the Excel function to obtain the formula for the trendline, and the R2 value.
- Determine the antigen concentration in the sample provided for assay from each calibration graph.
- Calculate a mean value for the assay, and a relative standard deviation from the results from the 3 graphs.
Questions for the Students
- Which was the plot that gave the most sensitive answer?
- What is the major source of error in this experiment?
- What are the other minor sources of error?
Radioactive iodine is potentially very toxic if ingested. Although amounts of activity in this experiment are very low, disposable gloves and eye protection should be worn to avoid direct contact. Under no circumstances should the 125I be used under oxidizing conditions when elemental iodine might be formed with a danger of inhalation.
1 mL autopipette
1 mL of 5 different antigen solutions
1 mL assay solution
14 gamma vials+lids
1 mL tips
10 mL buffer pH 6.9
1 mL antibody times 6
Preparation for the lab Supervisor
The preparation that the lab Supervisor needs to do to ready the lab.
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