Yang H W, Ikeda H, Kato K, Nakagawara A, Choi S H, Hayashi Y, Obana K, Hemmi H, Tsuchida Y
Department of Pediatric Surgery, University of Tokyo, Japan.
J Pediatr Surg. 1999 Mar;34(3):454-60. doi: 10.1016/s0022-3468(99)90498-0.
BACKGROUND/PURPOSE: The importance of determining N-myc oncoprotein rather than genomic N-myc amplification has been emphasized in neuroblastoma, especially in an international project to register biological risk factors in all neuroblastomas. A method to raise a specific polyclonal antibody against the N-myc oncoprotein in large quantities was sought using the synthetic antigen peptide and the multiple antigen peptide (MAP) method.
Two sets of peptides, HGRGPPTAGSTAQSPG and GVAPPRPGGRQTSGGDH, conserved in the N-myc oncoprotein were synthesized. The hemocyanin-conjugated peptides and the lysine core-conjugated (multiple antigen peptide method) peptides were injected into rabbits with adjuvant. IgG fractions precipitated from the sera were purified on an affinity column coupled with these peptides, and the potency and specificity of the purified IgGs were examined by immunoblotting and immunohistochemistry in small cell lung cancer cell lines with known positivity-negativity of amplification and expression of N-myc, c-myc, and L-myc.
Peptides conjugated to the lysine core raised more potent antibodies than those conjugated to hemocyanin. Purified IgG against GVAPPRPGGRQTSGGDH reacted positively with an N-myc-amplified lung cancer cell line, but not with N-myc-unamplified and c-myc/L-myc-amplified cell lines on either immunoblotting or immunostaining. This IgG strongly stained the nuclei of cells in a series of surgical specimens and cell lines of neuroblastoma with N-myc amplification.
A polyclonal antibody specific for a synthetic peptide from the N-myc oncoprotein was thus obtained and will find wide international use.
背景/目的：在神经母细胞瘤中，尤其是在一项登记所有神经母细胞瘤生物学危险因素的国际项目中，已强调确定N - myc癌蛋白而非基因组N - myc扩增的重要性。人们试图使用合成抗原肽和多抗原肽（MAP）方法大量制备针对N - myc癌蛋白的特异性多克隆抗体。
合成了两组在N - myc癌蛋白中保守的肽，即HGRGPPTAGSTAQSPG和GVAPPRPGGRQTSGGDH。将与血蓝蛋白偶联的肽和与赖氨酸核心偶联的（多抗原肽方法）肽与佐剂一起注射到兔子体内。从血清中沉淀出的IgG级分在与这些肽偶联的亲和柱上进行纯化，并通过免疫印迹和免疫组织化学在已知N - myc、c - myc和L - myc扩增及表达阳性或阴性的小细胞肺癌细胞系中检测纯化IgG的效力和特异性。
与赖氨酸核心偶联的肽比与血蓝蛋白偶联的肽产生更强效的抗体。针对GVAPPRPGGRQTSGGDH的纯化IgG在免疫印迹或免疫染色中与N - myc扩增的肺癌细胞系呈阳性反应，但与N - myc未扩增以及c - myc/L - myc扩增的细胞系无反应。该IgG强烈染色一系列具有N - myc扩增的神经母细胞瘤手术标本和细胞系中的细胞核。
由此获得了一种针对来自N - myc癌蛋白的合成肽的多克隆抗体，它将在国际上得到广泛应用。
