Barrio M M, Portela P, Mordoh J
Instituto de Investigaciones Bioquímicas Fundación Campomar, Capital Federal, Argentina.
Hybridoma. 1998 Dec;17(6):517-25. doi: 10.1089/hyb.1998.17.517.
Monoclonal antibody (MAb) FC-5.01, raised against the undifferentiated breast cancer cell line IIB-BR-G, has been recently shown to react with CD63. The antigen (Ag) recognized by MAb FC-5.01 is expressed in plasma membranes of IIB-BR-G and other neoplastic cells, as well as in activated platelets and endothelial cells, as detected by indirect immunofluorescence performed at 4 degrees C on live cells. In permeabilized cells, MAb FC-5.01 colocalizes with acridine orange in acidic vesicles (lysosomal/endosomal compartment). Scatchard plot analysis performed on IB-BR-G cells demonstrated a 1.4+/-0.4 x 10(7) M(-1) affinity constant and 2.1 x 10(6) antigenic sites per cell. MAb FC-5.01 is not able to mediate C fixation or ADCC toward CD63+ cells, but the FC-5.01-CD63 complex is efficiently internalized into cytoplasmic vesicles, as shown by an acid wash immunofluorescence assay. Cellular catabolism of the antibody bound by IIB-BR-G cells was studied using [125I]-FC-5.01. At 18 h, >70% of the radioactivity was present in the supernatant as degraded fragments (TCA-soluble). After internalization, rapid Ag re-expression could be demonstrated in IIB-BR-G cells. MAb FC-5.01 diminished migration of CD63+ cells in a Boyden chamber assay. Some of the above-mentioned properties would enable the use of MAb FC-5.01 as a vehicle to target different compounds inside CD63+ cells.
针对未分化乳腺癌细胞系IIB - BR - G产生的单克隆抗体（MAb）FC - 5.01，最近已证明它能与CD63发生反应。通过在4℃对活细胞进行间接免疫荧光检测发现，MAb FC - 5.01识别的抗原（Ag）在IIB - BR - G和其他肿瘤细胞的质膜中表达，同时也在活化的血小板和内皮细胞中表达。在通透化细胞中，MAb FC - 5.01与吖啶橙在酸性囊泡（溶酶体/内体区室）中共定位。对IB - BR - G细胞进行的Scatchard图分析显示，其亲和常数为1.4±0.4×10⁷ M⁻¹，每个细胞有2.1×10⁶个抗原位点。MAb FC - 5.01不能介导对CD63⁺细胞的补体固定或抗体依赖的细胞介导的细胞毒性作用（ADCC），但如酸洗免疫荧光测定所示，FC - 5.01 - CD63复合物能有效地内化到细胞质囊泡中。使用[¹²⁵I] - FC - 5.01研究了IIB - BR - G细胞结合的抗体的细胞分解代谢。在18小时时，>70%的放射性以降解片段（三氯乙酸可溶性）的形式存在于上清液中。内化后，可在IIB - BR - G细胞中证明抗原的快速重新表达。在Boyden小室试验中，MAb FC - 5.01减少了CD63⁺细胞的迁移。上述一些特性将使MAb FC - 5.01能够用作载体，将不同化合物靶向递送至CD63⁺细胞内。
