Kuno Atsushi, Sato Takashi, Shimazaki Hiroko, Unno Sachiko, Saitou Kozue, Kiyohara Katsue, Sogabe Maki, Tsuruno Chikayuki, Takahama Youichi, Ikehara Yuzuru, Narimatsu Hisashi
Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan.
Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
Proteomics Clin Appl. 2013 Oct;7(9-10):642-7. doi: 10.1002/prca.201300010. Epub 2013 Aug 7.
Wisteria floribunda agglutinin positive human Mac-2-binding protein (WFA(+)-hM2BP) was recently validated as a liver fibrosis glycobiomarker with a fully automated lectin-antibody sandwich immunoassay. In this study, we supplied recombinant WFA(+)-hM2BP as the standard glycoprotein and the overlaid antibody to enhance the robustness of WFA(+)-hM2BP quantification.
The optimum conditions for producing recombinant WFA(+)-hM2BP were selected by cell glycome analysis based on a lectin microarray. Interlot variability of recombinant WFA(+)-hM2BP was determined using an antibody-overlay lectin microarray. Screening of anti-M2BP mAb was completed by incorporating a WFA-antibody sandwich ELISA and an antibody-overlay lectin microarray.
The lectin microarray analysis revealed that human embryonic kidney 293 cells efficiently and stably produced WFA(+)-hM2BP in DMEM containing 10% FCS without any variation in the M2BP glycosylation level. A spiking experiment with recombinant WFA(+)-hM2BP was mostly effective for antibody screening. The reconstituted sandwich immunoassay was useful for the continuous quantification and cutoff index expression of serum WFA(+)-hM2BP.
The multiple use of lectin-assisted glycan profiling enabled us to construct a reliable sandwich assay kit for monitoring liver fibrosis in patients with viral hepatitis. This will assist in the development pipeline for other glycodiagnostic agents.
紫藤凝集素阳性的人Mac-2结合蛋白（WFA(+)-hM2BP）最近已通过全自动凝集素-抗体夹心免疫测定法验证为肝纤维化糖生物标志物。在本研究中，我们提供重组WFA(+)-hM2BP作为标准糖蛋白和包被抗体，以增强WFA(+)-hM2BP定量的稳健性。
基于凝集素微阵列的细胞糖组分析选择生产重组WFA(+)-hM2BP的最佳条件。使用抗体包被的凝集素微阵列测定重组WFA(+)-hM2BP的批次间变异性。通过结合WFA-抗体夹心ELISA和抗体包被的凝集素微阵列完成抗M2BP单克隆抗体的筛选。
凝集素微阵列分析显示，人胚肾293细胞在含有10%胎牛血清的DMEM中高效稳定地产生WFA(+)-hM2BP，且M2BP糖基化水平无任何变化。重组WFA(+)-hM2BP的加标实验对抗体筛选大多有效。重构的夹心免疫测定法可用于血清WFA(+)-hM2BP的连续定量和临界指数表达。
凝集素辅助聚糖分析的多次使用使我们能够构建一种可靠的夹心测定试剂盒，用于监测病毒性肝炎患者的肝纤维化。这将有助于其他糖诊断试剂的开发流程。
