Onda Masanori, Nagata Satoshi, Ho Mitchell, Bera Tapan K, Hassan Raffit, Alexander Richard H, Pastan Ira
Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892-4264, USA.
Clin Cancer Res. 2006 Jul 15;12(14 Pt 1):4225-31. doi: 10.1158/1078-0432.CCR-06-0472.
To establish monoclonal antibodies (mAb) against megakaryocyte potentiation factor (MPF) and detect MPF in the blood of patients with mesothelioma.
Mice were immunized with a purified recombinant human MPF-rabbit-Fc fusion protein and with MPF. Several hybridomas producing mAbs to MPF were established. A double-determinant (sandwich) ELISA was constructed using mAbs to two different epitopes and used to determine if MPF is present in the serum of patients with mesothelioma.
We established seven anti-MPF mAbs whose topographical epitopes were classified into three nonoverlapping groups. All the mAbs reacted with recombinant MPF protein by ELISA. One of the mAbs detected MPF and the mesothelin precursor protein containing MPF in cell lysates on Western blotting. A sandwich ELISA using mAbs to two different epitopes was constructed and used to measure the presence of MPF in the media of various mesothelin-expressing cancer cell lines and in human serum. The ELISA showed that MPF levels were elevated in 91% (51 of 56) of patients with mesothelioma compared with healthy controls. Furthermore, serum MPF fell to normal levels in two patients after surgery for their peritoneal mesothelioma.
Using new mAbs to MPF, we showed that MPF is secreted by several mesothelioma cell lines and is frequently elevated in the blood of patients with mesothelioma. Measurement of MPF may be useful in following the response of mesothelioma to treatment.
制备抗巨核细胞增强因子（MPF）的单克隆抗体（mAb），并检测间皮瘤患者血液中的MPF。
用纯化的重组人MPF-兔-Fc融合蛋白和MPF免疫小鼠。建立了几种产生抗MPF单克隆抗体的杂交瘤细胞系。使用针对两个不同表位的单克隆抗体构建双抗原夹心法ELISA，用于检测间皮瘤患者血清中是否存在MPF。
我们制备了7种抗MPF单克隆抗体，其拓扑表位分为3个不重叠的组。所有单克隆抗体通过ELISA与重组MPF蛋白反应。其中一种单克隆抗体在蛋白质印迹法中检测到细胞裂解物中的MPF和含MPF的间皮素前体蛋白。构建了使用针对两个不同表位的单克隆抗体的夹心ELISA，用于检测各种表达间皮素的癌细胞系培养基和人血清中MPF的存在。ELISA结果显示，与健康对照相比，91%（56例中的51例）间皮瘤患者的MPF水平升高。此外，两名腹膜间皮瘤患者术后血清MPF降至正常水平。
利用针对MPF的新型单克隆抗体，我们发现MPF由几种间皮瘤细胞系分泌，且在间皮瘤患者血液中经常升高。检测MPF可能有助于监测间皮瘤的治疗反应。
