Deby-Dupont G, Reuter A M, Joris J, Vrindts-Gevaert Y, Franchimont P
C R Seances Soc Biol Fil. 1983;177(2):259-68.
5 micrograms of human beta-endorphin were labelled with 2 mCi 125I by the chloramine T technique. After two gel filtrations on Sephadex G-15 and on Sephadex G-50 in phosphate buffer with EDTA, Trasylol and mercapto-ethanol, a pure tracer was obtained with a specific activity about 150 microCi/ug. Kept at + 4 degrees C, the tracer remained utilizable for 30 days without loss of immunoreactivity. The labelling with lactoperoxydase and the use of another gel filtration method (filtration on Aca 202) gave a 125I beta-END tracer with the same immunoreactivity. The binding of this tracer to the antibody of an anti-beta-END antiserum diluted at 1/8000 was 32% with a non specific binding of 2%. 5 micrograms of human beta-lipotropin were labelled with 0.5 mCi 125I by the lactoperoxydase method. After two gel filtrations on Sephadex G-25 and on Sephadex G-75 in phosphate buffer with EDTA, Trasylol and mercapto-ethanol, a pure tracer with a specific activity of 140 microCi/micrograms was obtained. It remained utilizable for 30 days when kept at + 4 degrees C. Gel filtration on Aca 202 did not give good purification, while gel filtration on Aca 54 was good but slower than on Sephadex G-75. The binding to antibody in absence of unlabelled beta-LPH was 32% for an anti-beta-LPH antiserum diluted at 1/4000. The non specific binding was 2.5%.
采用氯胺T法用2毫居里125I标记5微克人β-内啡肽。在含有乙二胺四乙酸(EDTA)、抑肽酶和巯基乙醇的磷酸盐缓冲液中,先后通过Sephadex G - 15和Sephadex G - 50进行两次凝胶过滤后,得到了比活度约为150微居里/微克的纯示踪剂。该示踪剂在4℃保存时,30天内仍可使用且免疫反应性无损失。用过氧化物酶标记以及采用另一种凝胶过滤方法(在Aca 202上过滤)得到的125I-β-内啡肽示踪剂具有相同的免疫反应性。这种示踪剂与稀释至1/8000的抗β-内啡肽抗血清抗体的结合率为32%,非特异性结合率为2%。采用过氧化物酶法用0.5毫居里125I标记5微克人β-促脂素。在含有EDTA、抑肽酶和巯基乙醇的磷酸盐缓冲液中,先后通过Sephadex G - 25和Sephadex G - 75进行两次凝胶过滤后,得到了比活度为140微居里/微克的纯示踪剂。在4℃保存时,30天内仍可使用。在Aca 202上进行凝胶过滤纯化效果不佳,而在Aca 54上进行凝胶过滤效果良好,但比在Sephadex G - 75上慢。对于稀释至1/4000的抗β-促脂素抗血清,在无未标记β-促脂素的情况下与抗体的结合率为32%。非特异性结合率为2.5%。