I am performing cell binding experiment of Tc99m-labeled precursor for calculating Kd and Bmax value. I have hard time in order to calculate the specific activity of Tc99m.
Here are experimental procedure and my observation:
- Before conducting cell binding experiment, the precursor is labeled with 25 mCi of Tc99m and purified by HPLC to remove unlabeled precursor for accurate calculation of kd value. After purification 30uCi of Tc99m-labeled precursor was added to 15ml of cell media and prepared different concentration in a serial dilution.
- I know the non carrier specific activity of Tc99m (5.2×106 Ci/g or 5.15×105 Ci/mmol) calculated using A= Nl, however the Tc99m don’t exist in carrier free form due to presence of long lived Tc99. In that case, by multiplying molar fraction of Tc99m (depending upon the elution time of generator, mentioned in literature) with non-carrier specific activity, we can easily find out the specific activity of Tc99m.
My question is
How specific activity of 25 mCi of Tc99m can be find out, or we can use the above-mentioned specific activity of Tc99m?
Is there any procedure for calculating the specific activity for Tc99m.
It is not clear that you need the specific activity. You have added 30 uCi of Tc99m labeled precursor. There will be some Tc99 at all times and the amount will increase with time. The precursor will be labeled with Tc99 and Tc99m. You trace the Tc99m activity not the specific activity. You should decay correct the Tc99m activity.
@Joseph thanks for your reply, To find out the concentration of Tc99m-labeled compound (nM), I need specific activity of Tc99m.
My concern is how to calculate the specific activity of 30 uCi of Tc99m labeled compound in a cell binding.
Or above mentioned specific activity can be used to find out the concentration of Tc99m-labeled compound.
@Wagas
The specific activity of Tc99m does not change with addition of Tc99. The concentration of Tc99m does not change with addition of Tc99.
Use the specific activity of Tc99m.
There are no general formula to calculate the part of the Tc99 amount unless you know the exact time of the elution you use and the previous one, suppose that the elution efficiency is 100 %. Then at the next elution there will be Tc-99, and Tc99m on the column and in the eluate. The Mo decays with 12,5 % directly to Tc-99, the rest to Tc-99m. Then the Tc-99m decays to Tc-99. Such a way usig the decay equations you can calculate the composition of your Tc isotopes mixture. Then you have to add a calculation of decay between the elution and the experiments. Not a simple calculation, but doable.
As stated above, you need to know the elution history of the generator, as well as the Mo-99 content at a reference time, but since every Mo-99 atom that decays will produce an atom of Tc-99 (or Tc-99m), the total Tc content of your generator eluate (in atoms) should be equal to the number of Mo-99 atoms that decayed between elutions. Thus, the number of Tc-99 atoms in the eluate equals the difference between how may atoms of Mo-99 were on the generator at the end of the previous elution, and the number of atoms of Mo-99 that are present on the generator at the beginning of the current elution.
So, calculate the Mo-99 content of the generator (in Bq) at each of the elution times. Then use the relationship: Decay rate, atoms/sec or Bq = (decay constant)(number of atoms of the radionuclide), where decay constant = ln2/half-life (entering the half-life of Mo-99 in seconds) to define how many atoms of Mo-99 were present on the generator at each elution time. The difference will be the sum of Tc-99 + Tc-99m atoms in your eluate, assuming 100% elution yield. Convert the number of Tc atoms to moles or grams and you can calculate molar or specific activity from the (independently) measured quantity of Tc-99m present.
As also indicated above, one may also need to know whether the excess ligand in the formulated product (from making the Tc-99m radiopharmaceutical) is also competing for binding sites, effectively lowering your specific activity. One may also need to worry about the ligand's ability to also chelate other trace-level transition metals in the solution, with the resulting complexes similarly competing for binding sites.
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