Karczmarczyk Urszula, Markiewicz Alina, Mikołajczak Renata, Lisiak Emil, Bilski Marek, Pietrzykowski Jacek, Michalik Joanna
Radioisotope Centre-POLATOM, Swierk, Poland.
Nucl Med Rev Cent East Eur. 2004;7(2):107-12.
Our goal was the efficient labelling of highly purified human gammaglobulin. This radioactive protein fraction can be used as a basic compound of radiopharmaceutical formulation for inflammation lesion diagnosis. This application was experimentally illustrated in animal models with artificially induced inflammatory lesions after turpentine oil injection into mouse leg muscle.
Hydrazine nicotinamine derivative of human gammaglobulin (IgG-HYNIC) was synthesized according to Abrams method. The radionuclide: technetium (99m)Tc has been introduced into protein molecules by indirect method incorporation in phosphate buffer, pH 7.4, in the presence of stannous chloride as a reducing agent for sodium pertechnetate, and EDTA as a coligand. Radiochemical purity was estimated by thin layer chromatography. The stability of labelled IgG-HYNIC derivative in human serum in presence of copper, cobalt, iron and manganum salts was analyzed by HPLC method (BioSEP SEC 4000, eluent: 0.1 mol/L phosphate). Inflammation lesions were induced in Balb/3 mice muscles by injection of 0.2 ml turpentine oil into the leg muscle. Five days later, inflammation lesions were visualized by hIgG-HYNIC- (99m)Tc injections. The tracer accumulation in tissue was evaluated by gamma camera at 1 to 24 hour intervals.
Efficiency of technetium99m Tc human gammaglobulin labelling (pH 7.4, temp. 37 degrees C) was strictly dependant on ligand and coligand presence in the reaction mixture. Labelling of IgG molecules without any supplements resulted in very low efficiency, never exceeding the range of 5%. Presence of EDTA or hydrazine nicotinamide (HYNIC) conjugated with IgG increased radiolabelling efficiency to 50%. IgG-HYNIC derivative in EDTA presence enables us to reach value above 95% radiochemical purity. Stability of IgG-HYNIC derivative labelled with technetium (99m) Tc decreased rapidly in serum in time--up to 70% of initial value in 30 minutes and only 20% during further 4 hr incubation. This means that as much as 80% of radiotracer present in IgG molecules has been dissociated during incubation with serum. This forced us to find proper conditions for improving the stability of radioactive IgG-HYNIC conjugate in circulating serum for at least six hours. It was achieved by using a reaction medium supplement with divalent metal cations in the following compounds: MgCl2, CoSO4, Cu (NO3)2 and FeCl2 in equimolar ratio to EDTA. Scintigraphy of (99m)Tc gammaglobulin in artificially induced inflammatory lesions of mouse thigh muscle showed a 4 times higher accumulation of the tracer after 6 hours post injection, and 6 times higher after 24 hours.
A human gammaglobulin derivative (hIgG-HYNIC) labelled with technetium (99m)Tc by indirect method with high radiochemical purity can be a basic compound of formulation for infection/inflammation scintigraphy.
我们的目标是高效标记高纯度人γ球蛋白。这种放射性蛋白组分可作为用于炎症病变诊断的放射性药物制剂的基础化合物。在将松节油注射到小鼠腿部肌肉中人工诱导炎症病变的动物模型中，对该应用进行了实验验证。
根据艾布拉姆斯方法合成人γ球蛋白的肼烟酰胺衍生物（IgG-HYNIC）。通过间接法将放射性核素：锝（99m）Tc引入蛋白质分子中，即在pH 7.4的磷酸盐缓冲液中，在氯化亚锡作为高锝酸钠的还原剂以及乙二胺四乙酸作为共配体的存在下进行。通过薄层色谱法估算放射化学纯度。采用高效液相色谱法（BioSEP SEC 4000，洗脱液：0.1 mol/L磷酸盐）分析在铜、钴、铁和锰盐存在下标记的IgG-HYNIC衍生物在人血清中的稳定性。通过向腿部肌肉注射0.2 ml松节油在Balb/3小鼠肌肉中诱导炎症病变。五天后，通过注射hIgG-HYNIC-（99m）Tc使炎症病变显影。在1至24小时间隔内通过γ相机评估示踪剂在组织中的蓄积情况。
锝99m Tc标记人γ球蛋白的效率（pH 7.4，温度37℃）严格取决于反应混合物中配体和共配体的存在。在没有任何添加剂的情况下标记IgG分子效率非常低，从未超过5%的范围。与IgG共轭的乙二胺四乙酸或肼烟酰胺（HYNIC）的存在将放射性标记效率提高到50%。在乙二胺四乙酸存在下的IgG-HYNIC衍生物使我们能够达到高于95%的放射化学纯度。用锝（99m）Tc标记IgG-HYNIC衍生物在血清中的稳定性随时间迅速降低——30分钟内降至初始值的70%，在接下来的4小时孵育期间仅为20%。这意味着在与血清孵育期间，IgG分子中存在的多达80%的放射性示踪剂已解离。这迫使我们寻找合适的条件以将放射性IgG-HYNIC共轭物在循环血清中的稳定性提高至少6小时。通过在反应介质中添加与乙二胺四乙酸等摩尔比的以下化合物中的二价金属阳离子来实现：氯化镁、硫酸钴、硝酸铜和氯化铁。对小鼠大腿肌肉人工诱导炎症病变中（99m）Tcγ球蛋白的闪烁扫描显示，注射后6小时示踪剂的蓄积量高4倍，24小时后高6倍。
通过间接法用高放射化学纯度的锝（99m）Tc标记的人γ球蛋白衍生物（hIgG-HYNIC）可以是用于感染/炎症闪烁扫描制剂的基础化合物。
