以田口法進行三明治酵素免疫分析之最佳化 於早期糖尿病腎病變篩檢晶片開發

Abstract

本研究目的在於最佳化以微陣列晶片形式之三明治酵素免疫分析的參數設計，對象為TNFα、α1-Antitrypsin、Cystatin C、E-Cadherin 這四種被認為可能與早期糖尿病腎病變相關的生物標記。具體研究目標分為以下兩點: 1. 使用田口法(Taguchi Method)最佳化抗體微陣列參數 2. 評估搭配訊號放大(Tyramide Signal Amplification, TSA)系統於抗體微陣列晶片的效用。 總共進行兩階段田口法，每階段皆包含無訊號放大組(without TSA)及訊號放大組 (with TSA)；第一階段無訊號放大組，我們探討四種因子(factor)的濃度，包含捕獲抗體 (capture antibody)、分析物(analyte)、偵測抗體(detection antibody)及streptavidin-conjugated Cy5 (SA-Cy5)，每因子有三個水準(level)，而訊號放大組則是增加了streptavidin-conjugated horseradish peroxidase (SA-HRP)；第二階段無訊號放大組探討五種因子，其中包含四種因子的培養時間(blocking、分析物、偵測抗體及SA-Cy5)及SA-Cy5的濃度，每因子有兩個水準，而訊號放大組則是增加tyramide的培養時間進行探討。 我們利用因子反應表選擇最高S/N比的因子水準當作最佳參數組合，經由田口法優化後，四種不同的蛋白質於無訊號放大組及訊號放大組的偵測極限降低幅最好可達到89.7 %和79.0 %。我們的結果也驗證了搭配訊號放大系統能夠改善偵測極限，對於偵測低濃度分析物是有幫助的。

The goal of this research is to optimize sandwich enzyme-linked immunosorbent assay (ELISA) in microarray format with 4 different proteins (TNFα, α1-Antitrypsin, Cystatin C and E-Cadherin), which can potentially be used as biomarkers for screening early stage of diabetic nephropathy (DN). The specific objectives are to: 1. optimize assay parameters with Taguchi Method and 2. systematically evaluate the effectiveness of incorporating Tyramide Signal Amplification (TSA) in an antibody microarray. Two optimization rounds based on Taguchi method were performed in both assay with and without TSA. In the first round, for assay without TSA, we tested four different factors including capture antibody (CapAb), analyte, detection antibody (DetAb) and streptavidin-conjugated Cy5 (SA-Cy5) at three different concentrations. For assay with TSA, we tested one additional factor, streptavidin-conjugated horseradish peroxidase (SA-HRP). In the second round, for assay without TSA, we tested five factors including four different incubation steps (blocking, analyte, DetAb, SA-Cy5) and SA-Cy5 concentration at two different levels. For assay with TSA, additional tyramide incubation time was also tested. For each factor, the level with highest S/N ratio in response table was selected as the optimum condition. After Taguchi optimization, the limit of detection (LOD) for 4 different analytes were improved by 16.9% - 89.7% and 24.1% - 79.0% for assay without and with TSA respectively. Our result also verifies the effectiveness of incorporating TSA to improve the LOD, which will be beneficial for detection of low concentration analytes.