王玉麒Wang, Yu-Chie林哲雄Lin, Che-Hsiung2019-09-052018-08-252019-09-052018http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22G0897430105%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/103991本研究主要是探討將分子量小且不具免疫原性的半抗原直接聚合成為免疫原的方法。三聚氰胺是一種半抗原，它無法有效激活動物的體液免疫反應；為了使動物產生對應的抗體，並免除載體蛋白的使用，我們嘗試以戊二醛引發三聚氰胺的交聯聚合反應，並以此複合物對動物進行免疫注射。研究結果顯示，在所設定的實驗條件下，已有75〜80％的三聚氰胺被聚合，這些聚合的三聚氰胺顆粒分析其重量平均分子量（Mw）為17842 Da；我們證明聚合的三聚氰胺可以成功地激活小鼠和兔子的免疫反應產生對應抗體，所生產的單株抗體顯示對三聚氰胺具高度專一性，而對類緣物資三聚氰胺二醯胺、三聚氰胺一醯胺與三聚氰酸等，皆僅有相當低的交叉反應性（0.92％，1.31％，0.89％）。藉此開發的競爭性酵素連結免疫吸附分析方法，其適用於檢測三聚氰胺水溶液濃度範圍為10-1至104 ng / mL；另外，我們使用自由態的三聚氰胺作為包被抗原使其直接吸附於親水性的免疫分析盤上，而不需要另將其結合於蛋白質上。 我們的研究透露出具有特定官能基（如胺基或羧基）的半抗原可以藉由加入特定的交聯劑而聚合形成大的聚合物，本方法不僅簡化了免疫原製備的程序，而且還可消除使用載體時所附隨的相關問題，因此，它可以說是免疫原製備的全新發展。In this study, we provide methods for directly polymerizing small and non-immunogenic haptens into immunogens. Melamine is a hapten and ineffective in inducing humoral immune response in animals. In order to make melamine hapten immunogen successfully generate specific antibodies, we tried to cross-link melamine via glutaraldehyde mediated polymerization. Our results showed that 75~80% of melamine had been polymerized under the adopted experimental condition. The polymerized melamine analysed that the weight-average molecular weight (Mw) of particle is 17842 dalton. We proved that the polymerized melamine is an effevtive immunogen to elicit antibodies in both mice and rabbits. The elicited monoclonal antibodies exhibited a high specificity with low cross-reactivity (0.92%, 1.31%, 0.89%) toward ammeline, ammelide and cyanurate. The developed competitive enzyme-linked immunosorbent assay (cELISA) method is applicable in the range of 10-1 to 104 ng/mL melamine in buffer solution. In addition, we found the free form of melamine coulded be used as coating antigen directly attaching to hydrophilic ELISA plate and with no conjugated protein. Our study discloses the fact that a hapten with specific functional group (such as amine or carboxyl group) may be polymerized into a big complex by adding a particular cross-linking agent. The method not only simplifies the procedures of immunogen preparation but also eliminate the involvement of carriers and their concomitant problems. Therefore, it can be considered as a new development of immunogen preparation.半抗原免疫原三聚氰胺戊二醛競爭性酵素連結免疫吸附分析haptenimmunogenmelamineglutaraldehydecompetitive enzyme-linked immunosorbent assay (cELISA)