Zr-89 Radiolabeling protocol for mAb conjugates

  1. Prepare a solution of 0.5-2.0 mg of mAb-DFO in 200 μL of chelexed PBS buffer, pH 7.5
  2. Pipette a volume of the 89Zr4+ stock solution (typically supplied in 1.0 M oxalic acid) corresponding to 1.0-6.0 mCi (37-222 MBq) into a 2 mL microcentrifuge tube.
  3. Adjust the pH of the 89Zr4+ solution to 6.8-7.5 using 1.0 M Na2CO3. Begin by adding 100 μL of 1.0 M Na2CO3 to the 89Zr4+ solution and subsequently add smaller (<10 μL) aliquots of base to achieve the desired pH.
  4. Add the desired amount of pH-adjusted 89Zr4+solution to the mAb-DFO solution prepared in Step 1.
  5. Check the pH of the radiolabeling reaction mixture to ensure that it falls within the desired range of 6.8-7.5.
  6. Incubate the radiolabeling reaction for 60 – 120 min at RT on the rotisserie.
  7. After 60 min of incubation, measure the radiolabeling yield of the reaction using radio-TLC.
  1. To this end, spot 1 μCi of the radiolabeling reaction mixture (or ~1 μL of reaction mixture) on a silica-impregnated TLC strip. Allow the aliquot to dry, run the TLC using an eluent of 50 mM DTPA (pH 5.5). Seal the TLC strip with clear tape and analyze the TLC strip using a radio-TLC scanner. 89Zr4+ bound to the mAb-DFO construct will appear at the origin (Rf<0.1), while free 89Zr4+ cations will be chelated by DTPA and will elute with the solvent front (Rf>0.9).
  2. Calculate the radiolabeling yield of the reaction by integrating the radiochromatogram, dividing the area under the curve from Rf 0.0-0.1 by the total area unde the curve, and multiplying by 100.
  1. If the radiolabeling yield is sufficient (typically a theoretical specific activity of >2 mCi/mg), quench the reaction with 5 μL of 50 mM DTPA, pH 5.5.
  2. Purify the resulting immunoconjugate using a pre-packed disposable size exclusion desalting column with a 50,000 molecular weight cut-off using an eluent of either 0.9% sterile saline with 5 mg/mL gentisic acid or 0.25 M sodium acetate (pH 5.5) with 5 mg/mL gentisic acid (for Tz-DFO, chelexed PBS can be used instead).
  3. After purification, verify the radiochemical purity of the 89Zr4+-DFO-mAb using radio-TLC as described in step 7. SGS PAGE and phosphor imaging can be performed for additional analysis.