Immunoassay-based diagnostic tools for persolnalized therapy

Abstract

An immunoassay is a method that utilizes an antigen-antibody reaction to detect a specific target substance. It is used for disease diagnosis, drug monitoring, and research to track various biochemical substances. In particular, it is widely used to detect biomarkers in disease diagnosis. This immunoassay has gradually been applied in various methods. It has been applied to developing point-of-care diagnostic tools because they are fast, simple, and cost-effective. In this study, an immunoassay was applied to develop a diagnostic tool for personalized therapy. In Chapter 2, the microarray-based immunoassay was applied to single-cell analysis to evaluate the drug response to anti-cancer drugs. The proposed device comprised a function to capture a single lung cancer cell (single-cell capture efficiency, 89.5%), an antibody microarray for detecting biomolecules released from individual cancer cells (selectivity for each target, > 89.5%), a micro-valve for mixing solution, and pumps (mixing efficiency, > 95%). The device obtained target protein information according to drug combination from each cancer cell and analyzed it. Consequently, heterogeneity within the lung cancer cell line could be identified and provide heterogeneity information for single-cell proteomics, often concealed in bulk analysis. In Chapter 3, a competitive lateral flow immunoassay (LFIA) was developed to monitor vancomycin (VAN) in the peritoneal dialysis effluent (PDE) of patients with peritonitis. An LFIA sensor was also developed to monitor vancomycin in the whole blood. VAN, a glycopeptide antibiotic effective against gram-positive bacteria, is commonly used as a first-line treatment for severe infections and methicillin-resistant Staphylococcus aureus (MRSA). However, VAN has side effects that cause renal dysfunction and ototoxicity at high doses. Thus, therapeutic drug monitoring(TDM) is recommended. VAN is administered intraperitoneally (Intraperitoneal, IP) and intravenously (IV). The peritoneal injection is mainly used for peritoneal dialysis patients with peritonitis. Therefore, an LFIA sensor was developed to detect vancomycin in PDE. The sensor's detection range was from 1 ng/mL to 10 µg/mL. When comparing the concentration detected by ELISA and the developed LFIA in PDE samples of patients who were prescribed VAN, the VAN concentration in sample 1, sample 2, and sample 3 showed slightly lower results (8.5%, 5.7%, and 8.3%, respectively) in LFIA. However, both methods showed consistent trends, ensuring the reliability and consistency of the LFIA results in clinical samples. In addition, LFIA, which can directly detect VAN in blood without pre-treatment, was developed to monitor the concentration of VAN in blood. The detection range in blood was from 1ng/mL to 10 µg/mL, with a detection limit of 5 ng/mL. A strong correlation (rs=0.89) was observed when comparing the results with ELISA, a commonly used method. This finding indicates the effectiveness of VAN detection through LFIA. Overall, this study proposed the immunoassay-based microarray capable of analyzing single-cell analysis and competitive LFIA for monitoring VAN. In the first study, we developed a device capable of single-cell analysis and analyzed multiple proteins to confirm the heterogeneity of cancer cells and drug response in single cells. Second, we developed and verified the performance of LFIA for monitoring VAN in human derivatives (peritoneal dialysis effluent, whole blood). The developed tools can be applied clinically and are expected to provide valuable information for personalized therapy.