| 摘要: | 人類血液是最常被使用於臨床診斷的體液樣本。某些存在於血液中與疾病相關的特定蛋白質，其濃度變化或是結構上的改變，被視為疾病發展的重要指標。由於疾病發生機制和發展過程十分複雜，單一的蛋白質檢測法通常無法提供足夠的檢驗特異度（specificity）。發展多樣性檢測方法（multiplexed assay）除了可以提高檢測的特異度外，同時也縮短了分析時間、減少試劑的使用量，並且降低樣品的需求量。然而，血液中蛋白質的廣大濃度分布範圍，造成了發展多樣性分析上的困難。 在本篇論文中，我們發展了結合磁性奈米粒子和質譜分析技術（NBAMS）的多樣性免疫分析方法，利用表面結合有抗體的磁性奈米粒子自人類血漿中同時萃取並濃縮三種疾病相關蛋白質：C-反應蛋白質（C-reactive protein, CRP）、血清澱粉蛋白P（serum amyloid P component, SAP）和血清澱粉蛋白A（serum amyloid A, SAA）,後，再結合具有高靈敏度的基質輔助雷射脫附游離飛行時間質譜儀進行樣品偵測。實驗結果顯示，此多樣性免疫分析方法能夠同時從血漿中純化出標的蛋白質，且能夠同時對多樣的標的蛋白質進行定量分析。以正常人、進行心導管手術或胃癌病人血清為樣品的實驗結果，顯示可根據所測得的相對蛋白質含量，對上述三類不同人體生理狀況進行區分。 藉著在奈米粒子表面可容易修飾不同性質的官能基或分子、低成本和操作上的簡易性並結合高靈敏度的質譜分析技術，這個多樣性免疫分析方法極有潛力可應用於臨床蛋白質體診斷上。 Human plasma is the most used specimen for diagnosis where the level and/or modification of some specific proteins are associated with the progression of disease. Because of the complex nature of disease, diagnosis using single protein assay often results in insufficient detection specificity. The development and application of a multiplexed assay provide advantages of reduced analytical time, lower cost of reagents and smaller sampling quantity. Unfortunately, the high protein content of plasma is present across an extraordinary dynamic range of concentration that is likely to span more than 1012, which result in analytical challenge. Recent success of mass spectrometry in clinic proteomics has shown the ability to screen and discover multiple biomarkers simultaneously that increases the sensitivity and specificity of disease diagnosis. We have developed a multiplexed immunoassay based on nanoprobe-based affinity mass spectrometry (NBAMS) to specifically isolate, preconcentrate, and characterize several disease-associated antigens in plasma simultaneously. To demonstrate the availability of the approach, three serum proteins, C-reactive protein (CRP), serum amyloid P component (SAP), and serum amyloid A (SAA) with different concentrations are chosen as model system. Based on the antibody-antigen interaction, antibody-conjugated magnetic nanoparticles (MNPs), a-CRP MNPs, a-SAP MNPs, and a-SAA MNPs, can be used as the affinity probe to specifically extract antigens from plasma and quantitativly analyze multiple proteins. When applying this multiplex assay to human plasma analysis, distinct pattern of the protein profile can be used to differentiate healthy controls, patients with surgical operation of cardiac catheter, and gastric cancer patients. Given the flexibility of manipulating functional groups on the nanoparticles, their low cost, and simplicity of the assay, this technique is capable for clinical proteomic diagnosis. |
