Soule H R, Lanford R E, Butel J S
Int J Cancer. 1982 Mar 15;29(3):337-44. doi: 10.1002/ijc.2910290318.
To facilitate detection of SV40 surface-associated tumor antigen (T-ag), conditions were established to surface label T-ag on intact cells by lactoperoxidase-catalyzed radioiodination (125I/LPO). SDS-PAGE analysis of anti-T immunoprecipitates of SV40-transformed and -infected cells labelled with 125I/LPO revealed the presence of iodinated T-ag. Several types of control experiments were employed to guarantee the surface specificity of the 125I/LPO labelling technique. When SV40-transformed mouse cells were surface labelled with lactoperoxidase and glucose oxidase immobilized on insoluble beads, a preparation less readily internalized than soluble enzymes, T-ag was iodinated. Selective immunoprecipitation of surface antigens demonstrated that lactoperoxidase did not iodinate internally localized T-ag. A reconstruction experiment in which an extract of SV40-infected cells was added to uninfected cells prior to surface labelling suggested that T-ag released from lysed cells did not adhere significantly to monolayer surfaces and become iodinated. Finally, systematic omission of reactants from the iodination reaction revealed that exogenous addition of lactoperoxidase and H2O2 was necessary to generate an iodinated T-ag, indicating that endogenous host cell reactants do not contribute significantly to the iodination of T-ag. 125I-labelled T-ag was detectable on the surface of SV40 tsA-infected cells at the nonpermissive temperature 24 h post infection, indicating that the tsA lesion does not prevent the interaction of T-ag with the cell surface. When 125I/LPO-labelled transformed or infected cells were chased for 2.5 h after labelling, iodinated T-ag was no longer associated with the cell monolayer but was immunoprecipitable from culture supernatants. Cultures from which labelled T-ag had been shed could then be relabelled with 125I/LPO and surface-associated T-ag was again detectable. These data suggest that surface-associated T-ag is continuously shed from the cell surface and is rapidly replaced in the membrane by intracellular T-ag.
为便于检测SV40表面相关肿瘤抗原（T抗原），建立了通过乳过氧化物酶催化放射性碘化（125I/LPO）对完整细胞表面的T抗原进行标记的条件。用125I/LPO标记的SV40转化细胞和感染细胞的抗T免疫沉淀物的SDS-PAGE分析显示存在碘化T抗原。采用了几种类型的对照实验来确保125I/LPO标记技术的表面特异性。当用固定在不溶性珠子上的乳过氧化物酶和葡萄糖氧化酶对SV40转化的小鼠细胞进行表面标记时（这种制剂比可溶性酶更不易内化），T抗原被碘化。表面抗原的选择性免疫沉淀表明乳过氧化物酶不会碘化细胞内定位的T抗原。一项重建实验，即在表面标记之前将SV40感染细胞的提取物添加到未感染细胞中，结果表明从裂解细胞中释放的T抗原不会显著粘附于单层表面并被碘化。最后，在碘化反应中系统地省略反应物表明，外源添加乳过氧化物酶和H2O2对于产生碘化T抗原是必要的，这表明内源性宿主细胞反应物对T抗原的碘化作用不显著。在感染后24小时的非允许温度下，可在SV40 tsA感染细胞的表面检测到125I标记的T抗原，这表明tsA损伤不会阻止T抗原与细胞表面的相互作用。当用125I/LPO标记的转化细胞或感染细胞在标记后追踪2.5小时时，碘化T抗原不再与细胞单层相关，但可从培养上清液中进行免疫沉淀。然后可以用125I/LPO对已释放标记T抗原的培养物进行重新标记，并且再次可检测到表面相关的T抗原。这些数据表明，表面相关的T抗原不断从细胞表面脱落，并被细胞内的T抗原迅速取代。
