|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1996 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1995 : ¥1,400,000 (Direct Cost : ¥1,400,000)
In the actual application of AIDS sensor, following items should be overcome ; 1. Detection of both antibodies against gp14 and p24 of the HIV proteins can be performed. Especially, in this study, confirmation of infection can be done just after the measurement ; 2. Analytical time should be in the order of few minutes ; 3. High sensitive detection can be performed when serum samples are applied to this system. This study has clarified the obove items by the use of surface plasmon resonance (SPR) which has charasteristics of time saving measurement and quantitative analysis.
Prior sensing of the anti HIV antibodies, the method of chemical fixation of antigen on gold film of the surface of sensor tip was accomplished. Gold filmwas exposed to 10uM 4,4-dithiobutyric acid for one hour at room temperature. After washing, the sensor chip was immersed in 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDPC) and N-hydrpxy-succinimide (NHS) containing 95% dioxane with thorough st
irring for 3 hr, followed by washing with pure water. By this treatment, we had a stable immunosensor tip for SPR.In the sensor, anti gp41 antibody could be detected ranging from 1 to 20 ug/ml. The immunoreaction was monitored in real time and the affinity constant Ka was easily determined from the kinetics as being 2.7 x 107/M by processing the data inputted in a computer. The influence of serum on the reflectance was examined for the purpose of practical application. Huge effects were observed in the response, which was presumably caused by non-specific reactions of ingredients in the serum. Human serum influenced both the sensitivity to and the detection range for the antibody in SPR measurement. In the case of detection of p24 antibody, anti p24 antibody dissolved in PBS could be detected, at first, ranging from 0 to 40 ug/ml. Secondly, the influence of serum on the reflectance signal of SPR was examined. The human serum generated huge effects on both sensitivity to and detection range in the measurement of the antibody. However, when blocking was carried out by human serum and further the serum was heated under the condition of 56ﾟC for 30 min, non-specific reaction by the ingredients in the serum was considerably eliminated. The antibody in the human sera samples could be quantitatively detected under 20 ug/ml and the sensitivity was almost recovered to the initial value while the effects still remained over 20 ug/ml of the antibody. Less