對稱性二甲基精胺酸多株抗體及核酸適體之製備並將其分別應用於酵素連結免疫吸附分析法和核酸適體分析法之開發

Production of Polyclonal Antibody and Aptamer and Their Application to Enzyme-linked Immunosorbent Assay and Aptamer-based Assay for Symmetric dimethylarginine

林家君(Jia-Jyun Lin)

指導教授 ： 余豐益

中山醫學大學/醫學科技學院/生物醫學科學學系/碩士(2022年)

https://doi.org/10.6834/csmu202200172

摘要

腎臟疾病是國人十大死因之一，也是伴侶動物疾病中的隱藏殺手。對稱性二甲基精胺酸 (Symmetric dimethylarginine, SDMA)，是由 L-精胺酸經由蛋白質精胺酸甲基轉移酶轉化而來的小分子化合物，僅能藉由腎臟來代謝排除，而且不會受到個體生理狀態影響，在腎功能受損初期時，SDMA 的含量也會相對升高，因此被認為是一種腎功能衰竭之生物標記物。本研究期望能製備對 SDMA 具有專一性的多株抗體和核酸適體，並且利用多株抗體與核酸適體分別建立一套快速且靈敏的分析法，用以檢測 SDMA 的含量，以期能為民眾與動物健康問題來進行早期的預防篩檢。由於 SDMA 是一種小分子化合物，因此需要與載體蛋白質接合後，進行實驗動物的免疫，藉此製備出對 SDMA 具有專一性的抗體，並以此抗體來建立免疫分析法。此外，為了克服製備多株抗體時效價不穩定及減少實驗動物用量等問題，本研究也利用配體指數擴增系統進化技術 (Systematic Evolution of Ligands by Exponential enrichment, SELEX) 來篩選出對 SDMA 具有專一性的適體 (Aptamer)，並以此適體建立適體分析法來檢測 SDMA。結果顯示，在抗體製備部分，本研究雖然嘗試多種接合載體蛋白質的方式，並以上述接合物進行實驗動物的免疫，但結果顯示無 SDMA 專一性抗體產生。另一方面在適體開發上，本研究成功找出對 SDMA 具有專一性的適體 (Apt #8)，以適體為基礎的西方點墨法裡，發現 Apt #8 不會與載體蛋白質或其他小分子-載體蛋白質接合物產生交叉反應，而且其檢測極限可達 0.5 μg 的 SDMA-SH-OVA。另外，利用適體建立的連結吸附分析法，其競爭效果不符合預期。本研究成功篩選對 SDMA 具有專一性的適體，並且建立一種適體為基礎的西方點墨法，但在多株抗體的生產上尚須改善 SDMA 接合載體蛋白質的方法，以期提升抗體專一性與敏感度。

關鍵字

對稱性二甲基精胺酸 ； 多株抗體 ； 核酸適體 ； 酵素連結免疫吸附分析法 ； 核酸適體分析法

並列摘要

Renal disease is one of ten leading causes of death in Taiwan as well as the deadly disease of companion animals. However, chronic kidney failure is an irreversible disease progression. Symmetrical dimethylarginine (SDMA), derived from L-arginine by protein-arginine methyltransferase, is strictly excreted by the kidney, and the early appearance of SDMA during renal failure makes it a potential biomarker for detecting renal function in animals. Therefore, to protect human and animal health, high sensitivity and rapid detection methods such as enzyme-linked immunosorbent assay (ELISA) and aptamer-based assay are developed herein to examine the SDMA level. SDMA belongs to a small molecular weight compound, which needs to conjugate with carrier proteins to render their immunogenicity. SDMA-protein conjugates as antigens were immunized to the animal and produced the antibody against SDMA for the establishment of the ELISA methods. To prevent the poor reproducibility of antibody production and reduce the use of the animals, an alternative choice such as aptamer was used to develop the aptamer-based assay. Although several methods for antigen preparation had been carried out in the antibody section, there was no specific antibody produced for SDMA after immunization. In the aptamer section, selected aptamer #8 (Apt #8) showed the highest specificity against SDMA in the aptamer-based Western blotting; additionally, Apt #8 did not cross-react with other proteins or conjugates, indicating its high specificity. A dose-dependent manner was successfully developed with the detection limit of 0.5 μg SDMA-SH-OVA. Apt #8 also applied to aptamer-linked immobilized sorbent assay (ALISA), but the ideal didn’t fulfill by the experimental results. In this study, we have successfully established the aptamer-based assay for detecting SDMA. However, currently produced polyclonal antibodies still need to improve the method for SDMA and carrier proteins conjugation to make it more specific and sensitive for detecting the SDMA level.

並列關鍵字

Symmetric dimethylarginine ； Polyclonal Antibody ； Aptamer ； Enzyme-linked Immunosorbent Assay ； Aptamer-based Assay

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