Sado Y, Kagawa M, Kishiro Y, Sugihara K, Naito I, Seyer J M, Sugimoto M, Oohashi T, Ninomiya Y
Division of Immunology, Shigei Medical Research Institute, Okayama, Japan.
Histochem Cell Biol. 1995 Oct;104(4):267-75. doi: 10.1007/BF01464322.
A group of rat monoclonal antibodies recognizing the six different alpha chains of human type IV collagen have been established by our novel method. The method is designated the rat lymph node method in which enlarged medial iliac lymph nodes of a rat injected with an antigen emulsion via hind footpads are used as a source of B cells for cell fusion to produce hybridomas. The immunogens used were synthetic peptides having non-consensus amino acid sequences near the carboxyl termini of type IV collagen alpha chains. Hybridomas were screened both by ELISA with synthetic peptides and by indirect immunofluorescence with cryostat sections of human kidneys. Because the epitopes of all antibodies were determined by multipin-peptide scanning, they were confirmed to be isoform-specific. They are useful for identification of alpha chains of type IV collagen at the protein level in normal and abnormal conditions. The combined use of synthetic peptides as immunogens, the rat lymph node method as making monoclonal antibodies, and the multipin-peptide scanning as epitope mapping is found to be a strong tool for identification of peptides and proteins whose amino acid sequences are known or have been deduced.
通过我们的新方法已制备出一组可识别人类IV型胶原六种不同α链的大鼠单克隆抗体。该方法被称为大鼠淋巴结法，即通过后足垫向大鼠注射抗原乳剂，利用其肿大的髂内侧淋巴结作为B细胞来源进行细胞融合以产生杂交瘤。所用免疫原是在IV型胶原α链羧基末端附近具有非共有氨基酸序列的合成肽。杂交瘤通过用合成肽进行ELISA筛选以及用人肾冰冻切片进行间接免疫荧光筛选。由于所有抗体的表位均通过多针肽扫描确定，因此证实它们具有异构体特异性。它们可用于在正常和异常情况下在蛋白质水平鉴定IV型胶原的α链。发现将合成肽用作免疫原、将大鼠淋巴结法用于制备单克隆抗体以及将多针肽扫描用作表位作图相结合，是鉴定氨基酸序列已知或已推导的肽和蛋白质的有力工具。
