Abstract
Apoptosis, also known as programmed cell death, is an dispensable component of living body growth and development, immunoregulation, homeostasis. The early stage of many disease has appeared apoptosis symptom; therefore, we can detect apoptosis to diagnose disease and confirm if therapeutic is success or failure. The distinct feature of apoptosis is that phosphatidylserine (PS) expressed on the outer leaflet of cell membrane, so the apoptosis is detected by specific binding of Annexin V to PS. Annexin V l led with fluorescein dye, has been used to detect apoptosis in vitro, while the annexin V l l with radioisotope is appropriate for in vivo diagnosis in nuclear medicine. For developing annexin V l led with 99mTc, this study evaluates (1) the conjugation reaction of Annexin V with HYNIC, and the optimization dialysis to purify HYNIC-Annexin V; (2) formulation optimization of HYNIC-Annexin V kit for l ling with 99mTc, including: the buffer effect、the pH effect、the volume effect and the stability for analysis for 99mTc-HYNIC-Annexin V; (3) bioactivity assessment of 99mTc-HYNIC-Annexin V Using PS expressed E. coli/EH150 strain. This study revealed that the phosphate buffer was superior to the sodium citrate in improving the yield of HYNIC-Annexin V from 31% to 62% preventing protein from precipitation during dialysis procedure, The 99mTc l ling efficiency of neutral formulation buffered with phosphate salts was much better than that of acidic composition buffered with sodium citrate (60% vs 8%). An optimal lyophilized kit formulation of HYNIC-Annexin V was explored in this study, each kit contains HYNIC-Annexin V 100 μg、SnCl2．2H2O、tricine 716 μg、Na2HPO4 0.479 mg、NaH2PO4 0.431 mg、NaCl 6.995 mg, pH 7.3; with the optimal volume (0.5 ml) and the radioactivity up to 15 mCi, the l ling efficiency was greater than 90%, which can be lasted for at least 6hr after preparation. The strain of E. coli/EH150 which expresses PS on the outer leaflet of cell membrane was explored to confirm the characteristic binding of 99mTc-HYNIC-Annexin V to PS, which was competed with native Annexin V protein. The optimal quantity of E. coli/EH150 for 99mTc-HYNIC-Annexin V bioactivity assessment was 1 109 cells. By using size-exclusion HPLC to analyze species based on molecular size, we found that the radiochemical purity of 99mTc-HYNIC-Annexin V was about 60%, the most part of impurity maybe come from the complex of HYNIC due to incomplete removal of unconjugated HYNIC molecules, it is necessary to improve to purify HYNIC-Annexin V.