| 摘要: | 在台灣，心臟病患最主要的死因是由於急性心肌梗塞 ( acute myocardial infarction, AMI )，其因冠狀動脈發生突發性的阻塞，造成部分心肌細胞壞死。在臨床研究中，人類心臟型脂肪酸結合蛋白 ( heart-type fatty acid-binding protein, H-FABP ) 可作為急性心肌梗塞早期檢測的高靈敏度心臟標記。
本研究之目的為構築一自製式表面電漿共振 ( surface plasmon resonance, SPR ) 光學免疫感測系統用以檢測H-FABP，並比較不同的固定方法修飾晶片表面以固定H-FABP之單株抗體 ( anti-H-FABP monoclonal antibodies; anti- H-FABP MAbs )。在固定化技術方面，採用包括直接吸附、Protein A、cystamine、N-succinimidyl-3-(2-pyridyldithio)propionate ( SPDP ) - Protein A、cystamine-glutaraldehyde-Protein A ( CGP ) 法。目前研究結果顯示使用cystamine法將抗體固定於感測晶片上之再使用性最佳，再生緩衝液為0.1 M glycine-HCl buffer，可重複10次檢測，小鼠血清非專一性結合蛋白的清洗以10 M NaOH 效果最佳。食鹽磷酸緩衝液 ( phosphate buffered saline, PBS ) 和小鼠血清中H-FABP之檢測，於濃度0.2 - 100 ng/mL間 SPR角度移動與抗原濃度之log值呈現一良好之線性關係。在操作穩定性上，進行10天檢測後之相對角度移動與第一天相比皆在95% 以上。對於H-FABP之檢測相關性方面，SPR系統和ELISA系統之相關係數達0.9986。
本研究所構築之SPR光學免疫感測系統不僅具有固定化步驟簡單、所需樣品量少、樣品不需前處理且不需標識、高靈敏度、高專一性、再使用性高等優點，也可應用於早期心肌梗塞之居家照護 ( homecare )、臨床檢測、護理站 ( point-of-care ) 檢測及預防醫學之研究上。
Acute myocardial infarction ( AMI ) is the result of a sudden occlusion that cause a portion of myocardium cell death, and accounted for the greatest percentages of deaths from heart diseases in Taiwan. In clinical research, human heart-type fatty acid binding protein ( H-FABP ) is a sensitive cardiac marker useful for early diagnosis of AMI.
In this study, we establish a self-assembled surface plasmon resonance ( SPR ) optical immunosensor system to detect human H-FABP, and to compare different immobilization methods applied to immobilize the anti-human H-FABP monoclonal antibodies ( MAbs ) on a gold surface including adsorption, Protein A, cystamine, N-succinimidyl-3-(2-pyridyldithio)propionate ( SPDP ) - Protein A, cystamine - glutaraldehyde - Protein A ( CGP ) method. The recent results indicate that the reusability of the sensor chip adopting the cystamine method was found to be better than the other immobilization methods. Ten cycles of measurements could be performed on the same chip regenerated with a 0.1 M glycine-HCl buffer, a 10 M NaOH solution was used for clearing nonspecific binding in mouse serum. A linear relationship between SPR angle shift and the log values of H-FABP concentrations in the range from 0.2 to 100 ng/mL in phosphate buffered saline ( PBS ) and in mouse serum. When used for 10 days, the angle shifts were all > 95% of those on the response at the first day. Correlation coefficient was 0.9986 between this SPR immunosensor system and ELISA for determination of H-FABP in mouse serum samples.
This self-assembled SPR optical immunosensor system offers advantages of simplicity of immobilization, low sample requirement, label-free, no pretreatment, high sensitivity, high specificity and high reusability, it will be used for home care and point-of-care in early diagnosis of AMI, and for preventive medicine research. |
