醫院廢水中含有許多抗藥性病原體，尤其是困難梭狀芽孢桿菌 (Clostridioides difficile)，這是一種革蘭氏陽性、專性厭氧、會形成孢子的細菌，可造成嚴重的抗生 素相關性腹瀉。C. difficile 孢子對於酒精和漂白劑，以及萬古黴素等用於治療 C. difficile infection (CDI) 的抗生素有耐受性，是造成 CDI 復發和醫院或社區環境中擴散的主要 原因。因此，檢測醫院廢水中和病患糞便中的 C. difficile 孢子對病患健康和環境消毒 至關重要。我們在每週監測中注意到醫院廢水中孢子的數量具有波動性且與醫院內的 抗生素使用量和溫度有相關性。我們也發現另一種院內感染病源體，耐萬古黴素腸球 菌 (VRE) 經常出現在 CDI 患者的糞便中。另一方面，現行的孢子檢測方法至少需要 七天的培養才具有足夠被監測到的菌量，而在免疫檢測方面，目前市面上無法買到成 對用於檢測孢子的抗體。為了生產檢測孢子單株抗體 (mAbs)，我們用純化的孢子免 疫小鼠。另一方面，我們建構重組桿狀病毒 SRC 和 SBA 來分別表現孢子外壁蛋白質 CdeC 和 CotA。我們發現在 ELISA 中，免疫孢子的小鼠對於孢子和表現孢子外壁蛋 白質的細胞裂解物比未免疫組產生更高的抗體反應，我們因此犧牲免疫孢子的小鼠取 出脾臟細胞。將脾臟細胞與骨髓瘤細胞融合後，產出了能夠表達抗體的 384 株融合瘤 細胞。使用我們製作的重組桿狀病毒 SRC 和 SBA 感染 Sf21 細胞的裂解物和孢子來 檢驗融合瘤的抗原性，發現 20 株融合瘤中有兩個選殖株有較高且穩定的讀值。這些 mAbs 將被進一步用來開發一套能更快速、準確地檢測病人糞便和醫院廢水中的孢子 檢測系統。

C. difficile infection (CDI) results in symptoms ranging from mild diarrhea to severe pseudomembranous colitis and toxic megacolon. The spores can persist in the environment for several months and cannot be eliminated by alcohol, bleach, or antibiotics. Therefore, detecting spores is crucial for patient health and public health. Fluctuations in spore amounts in hospital wastewater were noted during our weekly surveillance. Changes in spore amounts in hospital wastewater are correlated with the antibiotics consumption and room temperature. The current method for detecting spores takes seven days, and for detection via immunoassay, there is no specific antibody pair against spores available on the market. Thus, we aim to produce monoclonal antibodies (mAbs) that can recognize the spores for developing a spore detection platform. We immunized mice with purified spores and constructed recombinant baculoviruses SRC and SBA to express the spore exosporium proteins CdeC and CotA, respectively. We found that in ELISA, mice immunized with spores developed higher antibody responses to spores and cell lysates expressing exosporium proteins than unimmunized mice. We then sacrificed the immunized mice to generate hybridoma. After fusing mouse splenocytes with myeloma cells, we successfully produced 384 hybridoma clones and used spores or cells expressing exosporium proteins as antigens to initially screen out 20 hybridoma clones. Two of the selected clones showed higher and stable ELISA OD450 values. These mAbs will be further used to develop a system for more rapid and accurate detection of spores in patient feces and hospital wastewater.

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