Immunochromatographic assay for biomarkers under challenging physiological condition

Abstract

Immunochromatographic assay (ICA) is high potential point of care testing (POCT) platform by the rapid, easy-to-use, cost-effective properties. However, conventional ICA structure has limitation to cover various physiological condition biomarkers which have various physiological condition such as concentration, matrix effect, and glycan chain structure. In order to overcome limitation, this study of dissertation deals with several physiological condition of biomarker, with developed detection strategy for the biomarkers, and advanced ICA structure. Chapter 2 describes the elimination of Hook-effect on ICA structure. The Hook effect is false negative interference which occurred when excess amount of target biomarkers are introduced in ICA. The excess amount of target biomarker simultaneously bind to pair antibody of immunoassay, resulting in preventing immunoassay. Therefore, despite of high concentration of target biomarker in the sample solution, weak signal intensity is obtained in ICA. This phenomenon is usually confirmed in C-reactive protein (CRP) and human chorionic gonadotropin (hCG). In order to overcome the Hook-effect on ICA, the migration of detection probe (gold nanoparticle; AuNP) and protein on the several ICA structure was analyzed, and the migration of AuNP and protein on the ICA was separated. As a results, increasing signal intensity of titration curve on developed ICA structure was obtained without decreasing of signal intensity. The practicality of developed ICA structure was proved by applying 33 clinical sample. Chapter 3 describes the determination of cerebrospinal fluid (CSF) leakage through selective deletion of glycol-isoform of transferrin. CSF has critical role in human fluid for protection of brain and cerebrospinal. The leakage of CSF can induce sever disease such as meningitis, brain abscess, and pneumocephalus, rapid detection of CSF leakage is important. The conventional method for determination of CSF leakage is imaging methods (magnetic resonance imaging; MRI, computerized tomography; CT), as the biomedical assay, transferrin assay have mainly performed. The glycan chain of transferrin has different structure in serum and CSF. In the CSF, transferrin has absent sialic acid terminal glycan chain structure. Based on the different glycan chain structure of transferrin between serum and CSF, ICA structure was developed. Several deletion lines, which immobilized with sialic acid specific lectin, deletes serum specific transferrin in forward on ICA structure. By detecting residue of transferrin on the sample in rare test line, the CSF leakage is determined. In order to reduce the interference by other sialo-protein in the human serum, sample pre-treatment process was introduced. 13 positive, 34 negative and 13 artificial mixture sample was applied to the method, through receiver operating characteristic analysis, significant difference between positive, mixture, and negative sample was verified. Lastly, in the Chapter 4, determination of sialylation of PSA for improving specificity of total PSA assay was described. PSA is critical biomarker for determination of prostate cancer. However, the concentration of PSA also raised in benign prostatic hyperplasia (BPH), and prostatitis. This physiological property decreases the specificity of conventional total PSA assay. Aberrant sialylation of glycan chain, which is typical modification of cancer biomarker, indicated in PSA, therefore, demonstration of sialylation proportion of PSA determine prostate cancer from BPH and prostatitis. By determine total and sialylated PSA on the ICA, and calculating the relative sialylation proportion through calculation of the two signal intensity, determination of prostate cancer from BPH and prostatitis was obtained. The verification of the developed methods was evaluated with receiver operating characteristic analysis In this dissertation, various ICA structure was described and the ICA structure and detection strategy was evaluated. Through the advance ICA structure, several physiological interference of biomarker was overcome. The validation of the methods was proved through application of clinical samples, the determination of diseases was significantly performed.