Reconstruction of a robust glycodiagnostic agent supported by multiple lectin-assisted glycan profiling
Reconstruction of a robust glycodiagnostic agent supported by multiple lectin-assisted glycan profiling
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DOI：
10.1002/prca.201300010
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发表时间：
2013-10-01
影响因子：
2
通讯作者：
Narimatsu, Hisashi
中科院分区：
文献类型：
--
作者：
Kuno, Atsushi;Sato, Takashi;Narimatsu, Hisashi
目的用全自动凝集素-抗体夹心免疫分析法证实花紫藤凝集素阳性人Mac-2结合蛋白（WFA（+）-hM 2BP）是肝纤维化的糖标志物。本研究以重组WFA（+）-hM 2BP为标准糖蛋白和抗体，以提高WFA（+）-hM 2BP定量的稳定性。实验设计利用凝集素微阵列技术对重组WFA（+）-hM 2BP进行细胞糖组学分析，筛选重组WFA（+）-hM 2BP的最佳制备条件。使用抗体覆盖凝集素微阵列测定重组WFA（+）-hM 2BP的批间变异性。结果凝集素微阵列分析显示，人胚肾293细胞在含10%FCS的DMEM培养基中能高效稳定地产生WFA（+）-hM 2BP，且M2 BP的糖基化水平无任何变化。重组WFA（+）-hM 2BP的加标实验对于抗体筛选最有效。重组夹心免疫法可用于连续定量检测血清WFA（+）-hM 2BP的表达，并可作为临界指数。结论与临床意义凝集素辅助聚糖谱的多重应用使我们能够构建一种可靠的检测病毒性肝炎患者肝纤维化的夹心检测试剂盒。这将有助于其他糖诊断剂的开发管道。
PurposeWisteria 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.Experimental designThe 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.ResultsThe 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.Conclusions and clinical relevanceThe 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.