| Summary: | 碩士 === 國立中興大學 === 生命科學院碩士在職專班 === 104 === According to The Taiwan Centers for Disease Control’s (Taiwan CDC) surveillance data Acinetobacter baumannii is gaining importance as a cause of nosocomial infections. The issue of healthcare-associated infections caused by multidrug resistant A. baumannii is becoming a major concern globally in healthcare sector. Rapid pathogen identification methods are necessary to ensure timely accurate diagnosis of disease in patients with infections requiring immediate treatment. In the clinical microbiology laboratory where many traditional culture-based methods take days to yield results, providing diagnostic information in a matter of hours using enzyme-linked immunosorbent assay (ELISA) holds the potential for vast improvements in patient care. In the present study, recombinant proteins rTonB-R, rNcsP, rPase1, and rPase2 were used to develop a polyclonal antibody based indirect competitive ELISA (icELISA) method for detecting A. baumannii antigens. Recombinant Escherichia coli BL21(DE3) expressing rTonB-R, rNcsP, rPase1, and rPase2 antigens were prepared and were purified with Ni affinity chromatography. Box titration was carried out with different dilutions of coating antigens and antibodies. Optimal dilutions of antigens and antibodies in the ELISA were determined by box titrations. The calibration curve was fitted based on the average of 3 separate assays in triplicate and showed well correlation. Sensitivity was evaluated according to the binding rate, and the data were calculated using the B/B0% values, which B0 represents the binding with no analyte present and B represents the binding with analyte present. ELISA showed that the serum level of antibody against recombinant protein in A. baumannii patients was stronger than in normal human. However, A. baumannii patients and normal human were indistinguishable by icELISA. In summary, the developed icELISA was a feasible method for detecting A. baumannii antigens. The potential serodiagnosis of the four antigens awaits further efforts.
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