Hwang S Y, Greenway A L, McMullen G L, Hertzog P J
Centre for Molecular Biology and Medicine, Monash University, Clayton, Victoria, Australia.
Immunol Cell Biol. 1994 Jun;72(3):234-40. doi: 10.1038/icb.1994.35.
The purpose of this study was to produce antibodies which could be used to investigate the expression of murine (Mu)IFN-alpha. Rabbits were immunized with a peptide, corresponding to the 15 COOH-terminal amino acids of MuIFN-alpha-1, conjugated to keyhole limpet haemocyanin (KLH), and the resulting antipeptide antibodies were identified by indirect ELISA. Antipeptide antibodies were purified from rabbit immune sera by immunoadsorption to peptide immobilized on nitrocellulose and any remaining antibodies to KLH removed by immunoadsorption to KLH-Sepharose. The characterization of the antipeptide antibodies by ELISA, immunoprecipitation, affinity chromatography and immunofluorescence demonstrated that the antibodies recognize the peptide immunogen and the native IFN-alpha molecule. Using these antibodies for immunofluorescence staining and flow cytometric analyses of stained cells, we have shown that unstimulated murine spleen cells produce IFN-alpha. This finding is in agreement with the recent demonstration of constitutive IFN-alpha production by unstimulated human leucocytes and has important implications for the functions of interferons. The production, characterization and use of antipeptide antibodies as described herein may also have broader application for studies of the expression of other cytokines.
本研究的目的是制备可用于研究小鼠（Mu）干扰素-α表达的抗体。用与Mu干扰素-α-1的15个羧基末端氨基酸对应的肽与钥孔血蓝蛋白（KLH）偶联后免疫兔子，通过间接酶联免疫吸附测定（ELISA）鉴定产生的抗肽抗体。抗肽抗体通过免疫吸附固定在硝酸纤维素上的肽从兔免疫血清中纯化，任何残留的抗KLH抗体通过免疫吸附到KLH-琼脂糖上除去。通过ELISA、免疫沉淀、亲和层析和免疫荧光对抗肽抗体进行表征，证明这些抗体识别肽免疫原和天然干扰素-α分子。使用这些抗体对染色细胞进行免疫荧光染色和流式细胞术分析，我们发现未刺激的小鼠脾细胞产生干扰素-α。这一发现与最近关于未刺激的人白细胞组成性产生干扰素-α的证明一致，并且对干扰素的功能具有重要意义。本文所述抗肽抗体的制备、表征和使用对于其他细胞因子表达的研究可能也具有更广泛的应用。
