The Bioconjugation and Radiosynthesis of ⁸⁹Zr-DFO-labeled Antibodies

The overall goal of this procedure is to synthesize a zirconium 89 DFO labeled radio immunoconjugate and evaluate its performance in vivo. This is accomplished by first attaching the chelator deferoxamine to the antibody. The second step is to radio label the antibody DFO conjugate with zirconium 89.

Next, the radio immunoconjugate is purified using size exclusion chromatography. The final step is to perform small animal positron emission tomography or PET imaging in a mouse model of cancer. Ultimately, PET imaging is used to acquire images of the in vivo distribution of the zirconium 89 labeled radio immunoconjugate.

Generally, individuals new to this method may struggle because the adjustment of the zirconium 89 oxalate solution pH can be a little tricky. I'll be demonstrating this procedure along with Dalia Abdi, a technician in our laboratory. First, prepare a two to five milligram per milliliter solution of J 5 91 in one milliliter of 0.5 molar HEPs buffer in a 1.7 milliliter micro centrifuge tube, dissolve D-F-O-N-C-S in dry DMSO at a concentration between five and 10 millimolar sonicate and vortex the solution thoroughly in order to facilitate complete dissolution.

Next, adjust the pH of the J 5 9 1 solution to 8.8 to nine by adding small aliquots of 0.1 molar sodium carbonate. Once the antibody solution is at the correct pH, add a volume of the D-F-O-N-C-S solution corresponding to a three to fourfold molar excess of the bifunctional chelator. Then incubate the reaction for 30 minutes at 37 degrees Celsius on an agitating heating block.

At 350 revolutions per minute after one hour at 37 degrees Celsius, purify the remaining immunoconjugate using a pre-packed disposable size exclusion desalting column with a 50, 000 molecular weight cutoff and 0.5 molar Hess buffer. As the EENT following purification. Measure the concentration of the J 5 91 DFO construct on a UV vis spectrophotometer Store the solution of the completed J 5 91 DFO immunoconjugate at negative 20 degrees Celsius in the dark.

At this point, prepare a solution of 0.5 to two milligrams of J 5 91 DFO in 200 microliters of 0.5 molar HEPs buffer PIPEE volume of the zirconium 89 stock solution, corresponding to one to six millicuries into a two milliliter plastic screw cap micro centrifuge tube. Adjust the volume of this solution to a total of 300 microliters using one molar oxalic acid. Next, adjust the pH of the zirconium 89 solution to 6.8 to 7.5 using 250 microliters of one molar sodium carbonate.

Subsequently adding smaller aliquots of base to achieve the desired pH. Add the desired amount of pH adjusted zirconium 89 solution to the J 5 91 DFO solution. Then check the pH of the radio labeling reaction mixture to ensure that it falls within the desired range of 6.8 to 7.5.

Following this, incubate the radio labeling reaction for 60 minutes at room temperature on an agitating heating block at 350 revolutions per minute. After 60 minutes of incubation, measure the radio labeling yield of the reaction using radio TLC by spotting one micro curry of the radio labeling reaction mixture on a silica impregnated TLC strip. After running the TLC using an lent of 50 millimolar DTPA, analyze the TLC strip using a radio TLC scanner.

Calculate the radio labeling yield of the reaction by integrating the radio chromatogram dividing the area under the curve from retention factor zero to 0.1 by the total area under the curve and multiplying by 100. If the radio labeling yield is sufficient, quench the reaction with five microliters of 50 millimolar DTPA purified the resulting immunoconjugate using a pre-packed disposable size exclusion desalting column with a 50, 000 molecular weight cutoff using an EENT of 0.9%sterile saline with five milligrams per milliliter gent acid after purification. Verify the radio chemical purity of the zirconium 89 DFOJ 5 91 radio immunoconjugate.

Using radio TLC as previously described, calculate the overall radio labeling yield of the reaction by dividing the amount of activity initially added to the antibody solution by the amount of radioactivity isolated with the purified zirconium 89 DFOJ 5 91 radio Immunoconjugate Then calculate the final specific activity by dividing the amount of activity isolated with the purified zirconium. 89 DFOJ 5 91 radio immunoconjugate by the initial mass of DFOJ 5 91 in the radio labeling reaction in male, a thymic nude mice subcutaneously implant five times 10 to the six ln cap prostate cancer cells allowing these to grow to a 100 to 150 millimeters cubed xenograft. Three to four weeks after inoculation, the tumors should be ready for imaging.

Dilute the zirconium 89 DFOJ 5 91 radio immunoconjugate to a concentration of one milli curie per milliliter in 0.9%sterile saline lean. Next, inject 200 microliters of the zirconium 89 DFOJ 5 91 solution into the lateral tail vein of the xenograft bearing mice at the desired imaging time point. Anesthetize the mouse with a 2%ISO fluorine oxygen gas mixture.

Place the mouse on a small animal pet scanner bed. Verify anesthesia using the toe pinch method and apply veterinary ointment to the eyes of the mouse to prevent drying during anesthesia. Maintain anesthesia during the scan using a 1%ISO fluorine oxygen gas mixture.

Following this, acquire the pet data for the mouse via a static scan with a minimum of 40 million coincident events using an energy window of 350 to 700 kilo electron volts and a coincidence timing window of six nanoseconds. The radio TLC chromatogram of the crude radio labeling mixture reveals some DTPA bound zirconium 89 that eludes at the solvent front. However, after quenching the reaction and purifying the zirconium 89 D-F-O-M-A-B construct, the radio chemical purity of the purified isolated zirconium.

89 D-F-O-M-A-B conjugate is greater than 95%Both acute biodistribution and PET imaging experiments revealed that zirconium 89 DFOJ 5 91 clearly delineates the prostate cancer xenografts with excellent image contrast and high tumor to background activity ratios. The uptake of the radio immunoconjugate in the tumor is evident as early as 24 hours, and the activity concentration in the tumor increases to a maximum of 57.5 plus or minus five 3%injected dose per gram at 96 hours post injection. Don't forget that working with radioactivity can be hazardous, so precautions such as wearing appropriate PPE and making sure the radiation is shielded at all times should be taken during the procedure.