Generation and characterization of hamster monoclonal antibodies that neutralize murine tumor necrosis factors.
Generation and characterization of hamster monoclonal antibodies that neutralize murine tumor necrosis factors.
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DOI：
10.4049/jimmunol.142.11.3884
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发表时间：
1989-06
影响因子：
4.4
通讯作者：
K. Sheehan;N. Ruddle;R. Schreiber
中科院分区：
文献类型：
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作者：
K. Sheehan;N. Ruddle;R. Schreiber
使用纯化的、源自大肠杆菌的 rMuTNF-α 免疫亚美尼亚仓鼠后，产生针对鼠 TNF (MuTNF) 的单克隆抗体。从克隆 TN3-19.12 产生的抗体经过纯化，发现在 25 ng/U 的抗体输入量下可 100% 抑制重组或天然 MuTNF-α 的裂解活性。 TN3-19.12 还抑制多种 T 细胞来源的培养物上清液中的所有裂解活性，包括活化的 T 细胞克隆和 T 细胞杂交瘤（所有这些细胞均表达高水平的 TNF-α 和 TNF-β（淋巴毒素，LT）mRNA）。使用蛋白质印迹分析来记录 TN3-19.12 识别的 MuTNF 的物理形式。重组和巨噬细胞来源的 TNF 显示出与 Mr 17 kDa 相同的单条带模式。相比之下，T 细胞培养上清液表现出由 Mr 17 和 24.7 kDa 两条带组成的模式。分子量越高根据其对 n-聚糖酶的敏感性，该形式被糖基化，并显示出分子量。与 TNF-β (LT) 一致。这些数据表明 TN3-19.12 可识别 MuTNF-α 和 MuTNF-β (LT)。使用单克隆 TN3-19.12 和多价兔抗 rTNF 建立 MuTNF 特异性 ELISA，能够检测皮克量的重组或天然 TNF。该测定用于检测用致死剂量的 LPS 攻击的小鼠血清中的 TNF。腹腔注射后 90 分钟，在这些动物中观察到 TNF 血清峰值水平为 11 ng/ml。 LPS 施用然后在 3 小时内迅速下降至接近基线水平。这些值通过定量同一样品中 TNF 功能活性水平得到证实。随后将 TN3-19.12 注射到接受 LPS 处理的小鼠体内，可防止通过任一检测方法检测到循环中的 TNF，并保护小鼠免受内毒素休克的致命影响。因此，TN3-19.12 可有效中和体内内源性产生的 TNF。
mAb to murine TNF (MuTNF) were produced after immunization of Armenian hamsters with purified, Escherichia coli-derived rMuTNF-alpha. Antibody produced from clone TN3-19.12, was purified and was found to inhibit 100% of the lytic activity of either recombinant or natural MuTNF-alpha at an antibody input of 25 ng/U. TN3-19.12 also inhibited all the lytic activity in culture supernatants from a variety of T cell sources, including activated T cell clones and T cell hybridomas (all of which expressed high levels of TNF-alpha and TNF-beta (lymphotoxin, LT) mRNA). Western blot analysis was used to document the physical form(s) of MuTNF recognized by TN3-19.12. Recombinant and macrophage-derived TNF displayed identical patterns of a single band with Mr 17 kDa. In contrast, T cell culture supernatants exhibited patterns consisting of two bands with Mr 17 and 24.7 kDa. The higher m.w. form was glycosylated based on its sensitivity to n-glycanase and displayed a m.w. consistent with that of TNF-beta (LT). These data suggest that TN3-19.12 recognizes both MuTNF-alpha and MuTNF-beta (LT). Monoclonal TN3-19.12 and polyvalent rabbit anti-rTNF were used to establish a MuTNF-specific ELISA capable of detecting picogram quantities of recombinant or natural TNF. This assay was used to detect TNF in the sera of mice challenged with a lethal dose of LPS. Peak TNF serum levels of 11 ng/ml were observed in these animals 90 min after i.p. LPS administration and then rapidly declined to near base line levels by 3 h. These values were confirmed by quantitating levels of TNF functional activity in the same samples. TN3-19.12 injected into mice subsequently treated with LPS prevented the detection of TNF in the circulation by either assay and protected mice from the lethal effects of endotoxin shock. Thus, TN3-19.12 effectively neutralizes endogenously produced TNF in vivo.