Surman D R, Irvine K R, Shulman E P, Allweis T M, Rosenberg S A, Restifo N P
Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
J Immunol Methods. 1998 May 1;214(1-2):51-62. doi: 10.1016/s0022-1759(98)00036-2.
Lymphocytes from patients with melanoma have been used to clone melanoma associated antigens which are, for the most part, nonmutated melanocyte tissue differentiation antigens. To establish a mouse model for the use of these 'self' antigens as targets for anti-tumor immune responses, we have employed the mouse homologues of the human melanoma antigens Tyrosinase, Tyrosinase Related Protein-1 (TRP-1), gp100, and MART-1. We sought to generate antisera against these proteins for use in the construction of experimental recombinant and synthetic anti-cancer vaccines, and for use in biologic studies. Using genes cloned from the B16 mouse melanoma or from murine melanocytes, we immunized rabbits with plasmid DNAs coated onto microscopic gold beads that were then delivered using a hand-held, helium-driven 'gene gun'. This strategy enabled us to generate polyclonal rabbit sera containing antibodies that specifically recognized each antigen, as measured by immunostaining of vaccinia virus infected cells. The sera that we generated specifically for TRP-1, gp100, and MART-1 recognized extracts of the spontaneous murine melanoma, B16. The identities of the recognized proteins was confirmed by Western blot analysis. The titers and specificities of these antisera were determined using ELISA. Interestingly, serum samples generated against murine MART-1 and gp100 developed antibodies that were cross-reactive with the corresponding human homologues. Recognition of human gp100 and murine Tyrosinase appeared to be dependent upon conformational epitopes since specificity was lost upon denaturation of the antigens. These antisera may be useful in the detection, purification and characterization of the mouse homologues of recently cloned human tumor associated antigens and may enable the establishment of an animal model of the immune consequences of vaccination against 'self antigens.
来自黑色素瘤患者的淋巴细胞已被用于克隆黑色素瘤相关抗原，这些抗原在很大程度上是未突变的黑素细胞组织分化抗原。为了建立一个将这些“自身”抗原用作抗肿瘤免疫反应靶点的小鼠模型，我们采用了人类黑色素瘤抗原酪氨酸酶、酪氨酸酶相关蛋白-1（TRP-1）、gp100和MART-1的小鼠同源物。我们试图产生针对这些蛋白质的抗血清，用于构建实验性重组和合成抗癌疫苗，以及用于生物学研究。利用从B16小鼠黑色素瘤或小鼠黑素细胞中克隆的基因，我们用包被在微小金珠上的质粒DNA免疫兔子，然后使用手持式氦驱动“基因枪”进行递送。这一策略使我们能够产生含有特异性识别每种抗原的抗体的多克隆兔血清，通过对痘苗病毒感染细胞的免疫染色来测定。我们专门针对TRP-1、gp100和MART-1产生的血清能够识别自发性小鼠黑色素瘤B16的提取物。通过蛋白质印迹分析证实了所识别蛋白质的身份。使用酶联免疫吸附测定法（ELISA）测定了这些抗血清的效价和特异性。有趣的是，针对小鼠MART-1和gp100产生的血清样本产生了与相应人类同源物交叉反应的抗体。对人类gp100和小鼠酪氨酸酶的识别似乎依赖于构象表位，因为抗原变性后特异性丧失。这些抗血清可能有助于检测、纯化和鉴定最近克隆的人类肿瘤相关抗原的小鼠同源物，并可能有助于建立针对“自身”抗原接种疫苗的免疫后果的动物模型。
