DePalatis L R, Johnson K A, Kaplan D A
Bioproducts Laboratory Dow Chemical Company, Midland, Michigan.
Lab Invest. 1991 Jul;65(1):111-20.
A study was conducted to establish optimal conditions which would allow for the simultaneous localization of a carcinoma antigen and its complementary radiolabeled antibody. Immunoperoxidase staining was used to identify the tumor distribution of antigen, while tissue localization of the radiolabeled antibody was identified by autoradiography. The tumor associated glycoprotein-72 (TAG-72) antigen and the high affinity murine monoclonal antibody, CC49 IgG were used as the model antigen/antibody pair. Athymic female mice bearing either CX-1 or LS-174T human colorectal adenocarcinoma xenografts were used as animal/tumor test systems. Experimental mice each received a bolus intravenous injection of the CC49 antibody which was labeled with 125I (specific activity, 0.17 to 0.26 microCi/microgram). Control mice were given a bolus injection of MOPC-21 IgG monoclonal antibody (tumor irrelevant antibody) which was also radiolabeled with 125I (specific activity, 0.24 to 0.35 microCi/microgram). At 24 hours postinjection, all tumors removed, counted for radioactivity, and fixed in formalin. The avidin/biotin immunoperoxidase complex technique was used to identify TAG-72 antigenic sites on slide-mounted tissue sections. Nonradiolabeled CC49 IgG (0.5 micrograms/ml) was used as the specific antigen binding primary antibody in the immunostaining procedures. Nonradiolabeled MOPC-21 IgG (0.5 micrograms/ml) served as the negative control. Immunohistochemically stained tissue sections were coated with photographic emulsion and processed for autoradiographic localization of 125I-CC49 or 125I-MOPC-21. After an optimal exposure time of 6 days, slides were processed and examined under a light microscope. Results of the biolocalization experiment revealed that the % of injected dose/gram of 125I-CC49 in both LS-174T and CX-1 tumors (30.4 +/- 5.2% and 20.6 +/- 5.4%, respectively) were significantly greater (p greater than 0.01) than those for 125I-MOPC-21 (4.9 +/- 0.5% and 5.1 +/- 0.7%, respectively). In both tumor lines from mice injected with 125I-CC49, dense clusters of silver grains were found over those regions which were positive for TAG-72 immunoreactivity. These dual-labeled structures were also found in contact with, or in close proximity to the microvasculature. Tumors from mice which were injected with the control radioconjugate showed a random distribution of silver grains within stromal tissue but no specific localization to TAG-72 positive regions. We conclude that intravenously administered 125I-CC49 IgG localizes specifically to antigen-containing sites in the LS-174T and CX-1 tumor models. The methods described herein should serve as useful tools for the direct study of antigen-antibody interactions in tumor biology.
进行了一项研究以建立最佳条件，从而能够同时定位癌抗原及其互补的放射性标记抗体。免疫过氧化物酶染色用于识别抗原的肿瘤分布，而放射性标记抗体的组织定位则通过放射自显影来确定。肿瘤相关糖蛋白-72（TAG-72）抗原和高亲和力鼠单克隆抗体CC49 IgG被用作模型抗原/抗体对。携带CX-1或LS-174T人结肠直肠腺癌异种移植物的无胸腺雌性小鼠被用作动物/肿瘤测试系统。实验小鼠每只接受一次静脉推注用125I标记的CC49抗体（比活度，0.17至0.26微居里/微克）。对照小鼠接受一次静脉推注同样用125I标记的MOPC-21 IgG单克隆抗体（肿瘤无关抗体）（比活度，0.24至0.35微居里/微克）。注射后24小时，切除所有肿瘤，计数放射性，并固定在福尔马林中。抗生物素蛋白/生物素免疫过氧化物酶复合物技术用于识别载玻片上组织切片上的TAG-72抗原位点。未标记的CC49 IgG（0.5微克/毫升）在免疫染色过程中用作特异性抗原结合一抗。未标记的MOPC-21 IgG（0.5微克/毫升）用作阴性对照。免疫组织化学染色的组织切片涂覆有照相乳剂，并进行处理以对125I-CC49或125I-MOPC-21进行放射自显影定位。在最佳曝光时间6天后，处理玻片并在光学显微镜下检查。生物定位实验结果显示，在LS-174T和CX-1肿瘤中，125I-CC49的每克注射剂量百分比（分别为30.4±5.2%和20.6±5.4%）显著高于125I-MOPC-21（分别为4.9±0.5%和5.1±0.7%）（p>0.01）。在注射了125I-CC49的小鼠的两种肿瘤系中，在那些对TAG-72免疫反应呈阳性的区域发现了密集的银颗粒簇。这些双标记结构也被发现与微血管接触或紧邻微血管。注射了对照放射性缀合物的小鼠肿瘤在基质组织内银颗粒呈随机分布，但未特异性定位于TAG-72阳性区域。我们得出结论，静脉内给予的125I-CC49 IgG在LS-174T和CX-1肿瘤模型中特异性定位于含抗原的位点。本文所述方法应可作为直接研究肿瘤生物学中抗原-抗体相互作用的有用工具。
