Production, characterisation and applicability of monoclonal antibodies to immunoglobulin of Japanese flounder (Paralichthys olivaceus)
Production, characterisation and applicability of monoclonal antibodies to immunoglobulin of Japanese flounder (Paralichthys olivaceus)
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DOI：
10.1016/j.fsi.2007.03.008
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发表时间：
2007-11-01
影响因子：
4.7
通讯作者：
Sheng, Xiuzhen
中科院分区：
文献类型：
--
作者：
Li, Qiang;Zhan, Wenbin;Sheng, Xiuzhen
采用盐析和DEAE Sepharose柱层析相结合的方法，对牙鲆免疫球蛋白(Ig)进行纯化。纯化的免疫球蛋白在SDS-PAGE中的表观分子量分别为74 kDa(重链)和24 kDa(轻链)。用纯化的免疫球蛋白(Ig)免疫Balb/C小鼠，经双间接酶联免疫吸附试验(D-ELISA法)筛选，获得18株分泌抗牙鲆12单抗的杂交瘤细胞。用有限稀释法克隆了2个新基因2D8和2H1，并进行了Western blotting、间接免疫荧光试验(IIFAT)和荧光激活细胞分类(FACS)分析。在免疫印迹的还原条件下，用免疫球蛋白单抗2D8和单抗2H1对牙鲆重链进行了鉴定。用流式细胞术检测了健康牙鲨外周血、脾和前肾的表面免疫球蛋白阳性淋巴细胞。FACS分析显示，40.48%的外周血淋巴细胞、17.32%的脾淋巴细胞和9.67%的前肾淋巴细胞对2D8呈阳性反应。(C)2007爱思唯尔有限公司。保留所有权利。
Immunoglobulin (Ig) of Japanese flounder (Paralichthys olivaceus) was purified by a combination of salting-out and DEAE Sepharose Column chromatography. The purified immunoglobulin had an apparent molecular weight of 74 kDa (heavy chain) and 24 kDa (light chain) in SDS-PAGE. Eighteen hybridomas secreting monoclonal antibodies (MAbs) against Japanese flounder 12 were obtained by immunisation of Balb/C mice with purified Ig preparations, which were selected on the basis of the double indirect enzyme-linked immunosorbent assay (D-ELISA). Two of them designated as 2D8 and 2H1 were cloned by limiting dilution and characterised with western blotting, indirect immunofluorescence assay test (IIFAT) and fluorescence-activated cell sorter (FACS) analysis. Under reducing conditions in western blotting, both MAb 2D8 and MAb 2H1 were specific for the heavy chain of Japanese flounder Ig MAb 2D8 was used to identify surface Ig-positive lymphocytes in the peripheral blood, spleen and pronephros of healthy Japanese flounder by flow cytornetry. FACS analysis revealed that 40.48% of lymphocytes in the peripheral blood, 17.32% in the spleen and 9.67% in the pronephros were reactive to 2D8. (C) 2007 Elsevier Ltd. All rights reserved.