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1. Pipette 1 cm3 of each of the antigen solutions into 5 gamma vials
2. Pipette 1 cm3
of assay sample into 6th
3. Locate the 8 cm3
I (aq) which is in a labelled glass vial
4. Use the 1 mL autopipette to add 1 cm3
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 cm3
of buffer solution to 1st
6 vials and 2 cm3
to the 7th
7. Leave vial A
to one side
8. Add 1 cm3
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 cm3
of supernatant from each vial into a 2nd
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
13. Use 2 cm3
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
1. 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.
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.