Saito H, Wen D R, Yamamoto S, Yamamoto T, Saxton R E, Cochran A J, Irie R F
Division of Surgical Oncology, UCLA School of Medicine 90024.
J Immunol Methods. 1990 Nov 6;134(1):121-8. doi: 10.1016/0022-1759(90)90120-k.
A new immunohistochemical assay was developed for the detection of human monoclonal antibody (HuMAb) bound to human biopsied tumor tissues. A murine anti-idiotype monoclonal antibody, alpha type, 18C6 (IgGl), was raised against an IgM HuMAb, L612, defining a tumor-associated ganglioside antigen (GM3) and used as a probe in a three step cell-binding assay (HuMAb + anti-id + biotinylated anti-mouse Ig). Anti-id 18C6 has an exclusive binding specificity for HuMAb L612, but does not interfere with the binding of L612 to antigen positive melanoma cell lines or to a purified antigen, GM3. The applicability of 18C6 in the three step cell-binding assay was tested first using a melanoma cell line, UCLASO-M12. L612 bound to M12 cells was specifically detected by 18C6 without any background reactivity in ELISA. When this assay was compared with the standard two-step cell-binding assay (HuMAb + peroxidase-conjugated anti-human IgM) using various cultured tumor cell lines, parallel reactivity was observed. The three-step cell-binding assay was then applied to various fresh-frozen human tumor sections. Positive reactivity was demonstrated on various histologic types of human tumor tissues: primary melanoma (10/10), metastatic melanoma (4/4), nevus (10/10), lung cancer (3/6), breast cancer (2/6), and colon cancer (1/1). Adjacent normal tissues were unstained. Control experiments included the cell-binding assay with L612 alone, 18C6 alone. L612 + unrelated mouse IgG, and unrelated IgM HuMAb (L72) + 18C6; but biotinylated anti-mouse IgG did not react with these control preparations. The results indicate that anti-id 18C6 is a highly specific probe to assess the expression of the ganglioside antigenic epitope recognized by the L612 HuMAb on biopsied human tumor tissues.
开发了一种新的免疫组织化学检测方法，用于检测与人类活检肿瘤组织结合的人单克隆抗体（HuMAb）。制备了一种抗独特型单克隆抗体，α型，18C6（IgG1），它针对一种IgM HuMAb，L612产生，L612可识别一种肿瘤相关神经节苷脂抗原（GM3），并在三步细胞结合检测（HuMAb + 抗独特型抗体 + 生物素化抗小鼠Ig）中用作探针。抗独特型抗体18C6对HuMAb L612具有独特的结合特异性，但不干扰L612与抗原阳性黑色素瘤细胞系或纯化抗原GM3的结合。首先使用黑色素瘤细胞系UCLASO-M12测试了18C6在三步细胞结合检测中的适用性。在ELISA中，18C6特异性检测到与M12细胞结合的L612，无任何背景反应性。当使用各种培养的肿瘤细胞系将该检测方法与标准的两步细胞结合检测（HuMAb + 过氧化物酶偶联抗人IgM）进行比较时，观察到了平行反应性。然后将三步细胞结合检测应用于各种新鲜冷冻的人类肿瘤切片。在各种组织学类型的人类肿瘤组织上均显示出阳性反应性：原发性黑色素瘤（10/10）、转移性黑色素瘤（4/4）、痣（10/10）、肺癌（3/6）、乳腺癌（2/6）和结肠癌（1/1）。相邻的正常组织未染色。对照实验包括单独使用L612、单独使用18C6、L612 + 无关小鼠IgG以及无关IgM HuMAb（L72）+ 18C6的细胞结合检测；但生物素化抗小鼠IgG与这些对照制剂无反应。结果表明，抗独特型抗体18C6是一种高度特异性的探针，可用于评估L612 HuMAb在人类活检肿瘤组织上识别的神经节苷脂抗原表位的表达。
