Conjugation of different peptides is performed in four different reaction conditions, separately with BFCs mentioned below in detail.
Synthesis of p-SCN-Bn-NOTA conjugated Peptide
1. Calculate the amount of p-SCN-Bn-NOTA required to provide two-fold molar excess to the amount of peptide.
2. Weigh a minimum of 1 mg peptide in a plastic microtube.
! CAUTION Use of any metallic equipment should be completely avoided throughout the process as it can hinder radiolabeling.
3. Dissolve the peptide in a 10 μL volume of DMF and vortex for 1 min.
! CAUTION Volume of DMF should not be increased 0.2% v/v of final reaction.
4. Slowly add 80 - 100 μL of 0.1 M phosphate buffer to the peptide mixture, pH 7 followed by slow addition of 0.1 M sodium carbonate buffer to attain pH 9 (typically 50 – 70 μL).
! CAUTION Addition of phosphate buffer restrains the sudden shift of pH from acidic to basic which otherwise sometimes leads to precipitate formation.
5. Separately in another plastic microtube, dissolve the calculated amount of p-SCN-Bn-NOTA in 10 μL DMF and add 40 μL 0.1 M sodium bicarbonate buffer to attain pH 9.
! CAUTION Dissolve the chelating agents in weak acids to avoid hydrolysis.
6. Transfer the resulting p-SCN-Bn-NOTA volume to peptide mixture (Step 4) and vortex for 1 min.
! CAUTION Keep the reaction volume as minimum as possible for high conjugation yield. Peptide and chelating agents should be freshly dissolved.
! PAUSE POINT Incubate the conjugation reaction mixture at 4ºC for a period of 72 h ensuring the final reaction pH 9.
! CAUTION Conjugation yield increases with an increase in incubation time.
Synthesis of p-SCN-Bn-DOTA-GA conjugated peptide
The protocol for conjugation of the peptide with p-SCN-Bn-DOTA-GA is the same as that with p-SCN-Bn-NOTA with the following changes:
7. p-SCN-Bn-DOTA-GA can be dissolved in DMSO as well whereas p-SCN-Bn-NOTA gets precipitated in DMSO.
8. Requirement of the peptide to p-SCN-Bn-DOTA-GA molar ratio is relatively higher.
9. Requires approximately a relatively high volume of sodium bicarbonate buffer (100 -110 μL) for attaining pH 9 for the chelating agent mixture.
! CAUTION The experimental part involving radioactivity should be performed by a certified person as per the guidelines of local radiation safety norms.
10. Elute 68Ga from 68Ge-68Ga generator in a sterile glass vial using 4 mL 0.04M HCl as 68GaCl3.
11. Add 820-840 μL of 0.25 M sodium acetate buffer to attain pH 4.
! CRITICAL STEP The reaction pH should be strictly maintained at 4 for p-SCN-Bn-NOTA conjugated peptides and 4.5 for p-SCN-Bn-DOTAGA conjugated peptides.
12. From the crude conjugation reaction mixture, pipette the volume required so as to obtain 15μg of peptide (non-purified containing both conjugated and unconjugated peptide) and transfer to 68GaCl3 solution.
! CAUTION the amount of radioactivity typically ranges from 15-20 mCi for 15 μg of the non-purified conjugated peptide.
13. Incubate the radiolabeling reaction mixture at 95ºC for 10 min.
14. Pass 5 mL of 70% ethanol through a Sep-Pak C18 Plus light cartridge followed by 10 mL HPLC water in order to activate the cartridge.
! CRITICAL STEP Cartridge conditioning for wetting the silica bed is required in order to increase the surface area available trapping the analyte. The cartridge conditioning should be performed before starting the radiolabeling procedure to avoid radioactivity decay of Ga-68.
15. Allow trapping of radiolabeled analytes by slowly passing the radiolabeled mixture through the cartridge. The radiolabeled conjugated peptide and radiolabeled unconjugated chelating agent both get trapped in the cartridge while the free Ga-68 passes through the cartridge.
16. Slowly pass 10 mL of 15% ethanol through the cartridge in order to elute the radiolabeled unconjugated chelating agent and free Ga-68 if present.
! CRITICAL STEP Concentration of ethanol can vary from 12-15 % for eluting the radiolabeled BFC attributing to the variability in hydrophobic strength of peptide molecules analyzed.
17. In order to wash the cartridge, pass 2 mL of normal saline from the cartridge.
18. Elute the final product with 1 mL 50% ethanol in a sterile glass vial.
Measurement of radiolabeling efficiency by Radio-TLC
19. Transfer 0.5 M sodium citrate buffer in a 12 cm tall glass test tube until the 0.5 cm mark.
20. Place a 3 μL spot of the analyte at a 1 cm pencil mark of Whatman 3MM and TLC Silica gel strips.
21. Allow it to dry. Gently place the strips using a forcep in glass tubes with sodium citrate buffer and allow the solvent to run up to the mark of 9 cm.
! CAUTION Strips should be precisely placed in solvent without dipping the drop inside the mobile phase.
22. Remove the strip using a forcep and cover the dried strips with plastic tape in order to avoid radioactivity contamination on the Radio-TLC scanner plate.
23. Set the scanning time of up to 1 min to obtain the chromatogram and calculate the retention time of different radiolabeled components using software such as GINA Software.
24. Calculate retention factor for different possible radiolabeled components present in mixture using the formula:
Rf= Distance traveled by solvent front / Distance traveled by the solute front
25. The retention factor values of different possible radio-chemicals are:
1. Free unconjugated 68Ga- p-SCN-Bn-DOTAGA or 68Ga- p-SCN-Bn-NOTA has Rf value 1.00 using Whatman 3MM and sodium citrate as stationary and mobile phase, respectively.
2. Free Ga-68 has an Rf value of 1.00 using TLC Silica gel strips and sodium citrate as stationary and mobile phases, respectively.
3. Most of the 68Ga- p-SCN-Bn-DOTAGA-Peptide or 68Ga- p-SCN-Bn-NOTA-Peptide has Rf value 0.00 using both TLC Silica gel strips and Whatman 3MM as stationary phase and sodium citrate as mobile phase.
26. In absence of a Radio-TLC scanner simply cut the dried strips in two halves as the upper half of solvent front and the lower half of solute front.
27. Measure counts in well counter depending concentration of radioactivity and calculate % unconjugated chelating agent in the Whatman 3MM/sodium citrate and free Ga-68 in Whatman 3MM /TLC Silica gel strips combination as follows:
% unconjugated BFC or Ga-68=(Counts in the upper half of strip)/(Counts in both lower and upper half)×100
28. Similarly calculate % bound peptide as follows:
% bound peptide =(Counts in the lower half of strip)/(Counts in both lower and upper half)×100
In order to determine the conjugation yield, Radio-TLC chromatography of crude radiolabeled reaction mixture obtained before purification should be performed.