Dear Raghuvir,

I have copied an important section from the attached file that might help you in your calculations:

ran J Pharm Res. 2015 Winter; 14(1): 97–110.

PMCID: PMC4277623

Synthesis and Preliminary Evaluation of a New 99mTc Labeled Substance P Analogue as a Potential Tumor Imaging Agent

Saturation binding experiment (Kd and Bmax)

For saturation binding experiment, U373MG cells were incubated with increasing concentrations of 99mTc labeled HYNIC-Tyr8-Met(O)11-SP in the presence and absence of excess unlabeled SP to determine nonspecific (NSB) and total binding (T), respectively. In brief, newly detached U373MG cells were suspended in fresh RPMI medium and aliquots of 800000 cell/1mL were prepared in 5 mL test tubes. In a series of tubes, various concentrations of radiolabeled peptide (0.01-10 nM) in triplicate were the only added but in the other series the radiolabeled peptide was added as well with a fixed amount of excess unlabeled SP. The tubes were incubated for 90 min with a slight shaking every 5 min and were centrifuged at 200 g. Then the supernatants were removed and the surface of cell pellets were washed with cold PBS 1x slightly. The radioactivity of the supernatants was counted to determine the free radiopeptide amounts. The radioactivity of the pellets was counted to measure the NSB and T. The affinity for the radio ligand (Kd) and the maximal number of receptor binding sites (Bmax) were calculated from specific binding (SB) curve using the nonlinear regression analysis of Graphpad Prism 5th (SB = T - NSB).

Hoping this will be helpful,

Rafik

Dear Raghuvir,

I have copied an important section from the attached file that might help you in your calculations:

ran J Pharm Res. 2015 Winter; 14(1): 97–110.

PMCID: PMC4277623

Synthesis and Preliminary Evaluation of a New 99mTc Labeled Substance P Analogue as a Potential Tumor Imaging Agent

Saturation binding experiment (Kd and Bmax)

For saturation binding experiment, U373MG cells were incubated with increasing concentrations of 99mTc labeled HYNIC-Tyr8-Met(O)11-SP in the presence and absence of excess unlabeled SP to determine nonspecific (NSB) and total binding (T), respectively. In brief, newly detached U373MG cells were suspended in fresh RPMI medium and aliquots of 800000 cell/1mL were prepared in 5 mL test tubes. In a series of tubes, various concentrations of radiolabeled peptide (0.01-10 nM) in triplicate were the only added but in the other series the radiolabeled peptide was added as well with a fixed amount of excess unlabeled SP. The tubes were incubated for 90 min with a slight shaking every 5 min and were centrifuged at 200 g. Then the supernatants were removed and the surface of cell pellets were washed with cold PBS 1x slightly. The radioactivity of the supernatants was counted to determine the free radiopeptide amounts. The radioactivity of the pellets was counted to measure the NSB and T. The affinity for the radio ligand (Kd) and the maximal number of receptor binding sites (Bmax) were calculated from specific binding (SB) curve using the nonlinear regression analysis of Graphpad Prism 5th (SB = T - NSB).

Hoping this will be helpful,

Rafik

 Thank you so much dear rafik for your guidance.

99mTc at high specific activities (84-112GBq/µmol)

i want to know how the above value is calculated i have taken it from the same paper which you have attached. need your help

Dear Raghuvir,

The calculated number was obtained using the following:

The radioactivity of the pellets was counted to measure the NSB and T. The affinity

for the radio ligand (Kd) and the maximal number of receptor binding sites (Bmax) were calculated from specific binding (SB) curve using the nonlinear regression analysis of Graphpad Prism 5th (SB = T - NSB).

For more details see page 100 of the attached paper.

Rafik

I am confused!!!

Raghuvir H. Gaonkar is asking about Specific activity but Rafik Karaman is answering  specific binding.

To calculate specific activity, use this formula

SA( in Bq/g) = (Avogadro's Constant)(ln[2]) / (Am)(t½ in sec)

where A's # = 6.02x10^23, Ln[2] = 0.693, Am = Atomic Mass (roughly 99 for Tc99m) and T1/2 = 6 hours for Tc99m... but be sure to convert 6hr to seconds first.

If you want your answer in Curies/gram, the formula can actually be modified as follows (note: here the half-life is in hours):

=(Avagadro's #)(.693) (2.7x10^-11)/(3600 sec in a hour)/(Am)(t½ in minutes)

where 2.7x10^-11 converts from Bq to Curies, and 3600 is the number of seconds in an hour.