Development of Immunochromatographic Tests with Commercial Dyes and Their Applications

• Main Authors: Shwu-Jinng Wang, 王淑璟
• Other Authors: Yung-Chuan Liu
• Format: Others
• Language: en_US
• Online Access: http://ndltd.ncl.edu.tw/handle/61047073034999339652

博士 === 國立中興大學 === 化學工程學系所 === 98 === This study was divided into three parts involving the feasibility of using reactive dyes as an immunoassay marker in immunochromatographic test (ICT) , an improved dye immunochromatographic test (DICT) using dextran as conjugate spacer, and the feasibility of using disperse dyes as a chromogenic marker for the detection of antibodies against infectious bursal disease virus. For investigating the feasibility of using reactive dyes as an immunoassay marker, a dichlorine triazine dye, Procion Blue MX-7RX (PBM), was employed to stain the antibody against human serum albumin (anti-HSA). With the color intensity revealed in the ICT strip as the objective variables, the optimal dyeing conditions were found as follows: pH 11.4, temperature 35.7 ℃, molar ratio 188 (mol dye/mol antibody), and reaction time 45.6 min. The dyed-anti-HSA revealed a maximal color intensity of 8738 without apparent loss of antigen binding affinity. An improved dye immunochromatographic test (DICT) using dextran as conjugate spacer loading dye molecules to enhance chromophor color intensity with the potential of simultaneous multi-colored assay was developed. To construct this new effective chromophor, a dyeing process was carried out by coupling a reactive dye PBM to modified dextran (MD). The optimal conjugate condition between PBM and MD was studied. It showed that under the optimized dyeing conditions, a molar ratio (glucose units/chloroacetic acid) of 1:3, dextran MW of 10 kDa, and a dyeing time of 10 h were obtained. The resulting dyed-dextran chromophor, used as both spacer and color intensifier, was further labeled to a model antibody, anti-human serum albumin (antiHSA), to build a PBM-dextran-antiHSA (PDA) conjugate. The PDA thus obtained generated the highest color intensity of 29,898 assayed by DICT strips and densitometer scanning. As compared to protein direct dyeing, this indirect dyeing protocol yielded a chromophor 3.42 times increase in color intensity calculated on equal molar HSA basis. A competitive DICT for determination of HSA using a handheld rapid-test scanner was carried out. A linear range between 0 to 50 μg/ml with a detection limit of 0.12 μg/ml was observed. For investigating the feasibility of using disperse dyes as a chromogenic marker, a disperse dye, DADISPERSE NAVY BLUE SP, was selected in analyzing antibody against infectious bursal disease virus (anti-IBDV). With the color intensity revealed in the disperse dye ICT strip as the objective function, the optimal dyeing conditions were found as follows: dye concentration absorbance (at λmax = 587 nm) = 3, pH 7, 50oC, for 10 min. Under these conditions, the resultant dyed-antibody (rabbit anti-chicken) can produce an optimal color intensity reading of 55,054 on the strip. For performing qualitative immunoassay, chicken sera samples taken from different farms were used for the anti-IBDV titre assessment. The results of DICT strips showed very high sensitivity and specificity as compared to that analyzed by FlockChek enzyme linked immunosorbent assay (F-ELISA) kits. For quantitative immunoassay, it was found that the color intensity measured with DICT was linearly correlated to that of F-ELISA titre (r2 = 0.9687). Therefore, DICT was further applied to the detection of chicken anti-IBDV sera under vaccination in the farms. The average titres of the sampling groups exhibited a strong agreement to that of F-ELISA. Accordingly, the DICT method developed in this study, shown to be reliable, cheap and simple in both qualitative and quantitative immunoassays, is particularly suitable for point-of-need testing (PONT) in agricultural applications.