Experimental+procedure+for+Introduction+to+Radioimmunoassay


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 * __Experimental__**

1. Pipette 1 cm 3 of each of the antigen solutions into 5 gamma vials

2. Pipette 1 cm 3 of assay sample into 6 th vial

3. Locate the 8 cm 3 of 125 I (aq) which is in a labelled glass vial

4. Use the 1 mL autopipette to add 1 cm 3 of the 125 I-antigen to each of the 6 vials

5. Add 1 mL of 125 I-antigen to 7th vial and label it **A** – to measure total activity

6. Add 1 cm 3 of buffer solution to 1 st 6 vials and 2 cm 3 to the 7 th vial **(A)**

7. Leave vial **A** to one side

8. Add 1 cm 3 of antibody solution to each of the other 6 vials

9. Count sample **A** on the Triathler (see operating instructions below) for 1 min to determine background 35

10. 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

11. Pipette 2 cm 3 of supernatant from each vial into a 2 nd set of clean gamma vials and label carefully

12. Carefully remove rest of supernatant from each vial and dispose of in the plastic beaker labelled 125 I waste

13. Use 2 cm 3 of buffer solution to wash each precipitate – dispose of the washings in the plastic beaker

14. Re-centrifuge the precipitate as above and discard its supernatant into waste beaker

15. Count the 6 precipitate samples and all 6 supernatant samples in the Triathler

__**Write-Up**__

1. For each sample calculate the fraction of the 125 I 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.

2. Calculate the bound/free antigen ratio for each sample.

3. Using the 5 standard antigen concentrations provided plot graphs, using Excel of:

4. The bound activity fractions vs. the standard antigen concentration;

5. The free activity fraction vs. the standard antigen concentration;

6. The bound/free ratio vs. the standard antigen concentration.

7. 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.

8. Determine the antigen concentration in the sample provided for assay from each calibration graph.

9. Calculate a mean value for the assay, and a relative standard deviation from the results from the 3 graphs.