Protein-based Open Sandwich Immuno-PCR for Sensitive Detection of Small Biomarkers
Protein-based Open Sandwich Immuno-PCR for Sensitive Detection of Small Biomarkers
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DOI：
10.2116/analsci.29.871
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发表时间：
2013-09
影响因子：
1.6
通讯作者：
Sharif Hasan;Jinhua Dong;Y. Hara;Y. Morizane;F. Shibasaki;H. Ueda
中科院分区：
文献类型：
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作者：
Sharif Hasan;Jinhua Dong;Y. Hara;Y. Morizane;F. Shibasaki;H. Ueda
开放夹心（OS）免疫分析法利用抗体可变区的抗原依赖性稳定化来量化各种抗原，从而实现具有广泛工作范围的小分子非竞争性检测。为了进一步提高其敏感性，我们尝试用重组融合蛋白进行OS免疫pcr。将识别骨相关疾病生物标志物人骨钙素（bone Gla protein， BGP） c端片段的抗体的麦麦糖结合蛋白融合重链可变区（MBP-VH）固定在微孔板上，并将抗原与同一抗体的链亲和素（SA）融合轻链可变区（SA- v_l）加在一起孵育。固定化SA-V_L的数量通过拴链生物素化DNA进行定量，用于实时PCR估计抗原的数量。当检测BGP c端肽时，检测限为100 fg/mL，优于我们之前报道的基于噬菌体的OS immune - pcr。所开发的OS免疫pcr系统将有助于检测疾病预防的小分子生物标志物。
Open sandwich (OS) immunoassay utilizes antigen-dependent stabilization of an antibody variable region to quantify various antigens, enabling noncompetitive detection of small molecules with a broad working range. For further improvement of its sensitivity, OS Immuno-PCR was attempted with recombinant fusion proteins. The maltose binding protein-fused heavy chain variable region (MBP-VH) of an antibody that recognizes the C-terminal fragment of human osteocalcin (bone Gla protein, BGP), a biomarker for bone-related diseases, was immobilized onto microplate wells, and the antigen together with streptavidin (SA)-fused light chain variable region of the same antibody (SA-V_L) was added and incubated. The amount of immobilized SA-V_L was quantified by tethered biotinylated DNA, which was used to estimate the amount of antigen by realtime PCR. When BGP C-terminal peptide was detected, the limit of detection was 100 fg/mL, which was superior than that of our previously reported phage-based OS Immuno-PCR. The developed OS Immuno-PCR system will be useful for the detection of small molecule biomarkers for disease prevention.