HIGH SENSITIVE DETECTION FOR gp41 AND p24 of HUMAN IMMUNODEFICIENCY VIRUS BY THE USE OF CHEMICAL SENSOR

AIDS传感器在实际应用中，应克服以下几个问题：1.可以进行针对HIV蛋白的gp 14和p24的两种抗体的检测。特别是，在本研究中，感染的确认可以在测量后立即进行; 2.分析时间应在几分钟的顺序; 3.当血清样品应用于该系统时，可以进行高灵敏度检测。本研究利用表面等离子体共振（SPR）的省时、定量分析的特点，在检测抗HIV抗体之前，先将抗原化学固定在传感器针尖表面的金膜上，实现了对抗体的检测。金膜在室温下暴露于10 μ M的4，4-二硫代丁酸中1小时。清洗后，将传感器芯片浸入含有95%二氧六环的1-乙基-3-（3-二甲氨基丙基）-碳二亚胺盐酸盐（EDPC）和N-羟基琥珀酰亚胺（NHS）中， ...更多信息 冲洗3小时，然后用纯水洗涤。经此处理后，制备了一种稳定的SPR免疫传感器，在该传感器中，抗gp 41抗体的检测范围为1 ~ 20 μ g/ml。真实的监测免疫反应，通过处理计算机中输入的数据，从动力学容易地确定亲和常数Ka为2.7 × 107/M。为了便于实际应用，研究了血清对反射率的影响。在反应中观察到巨大的影响，这可能是由血清中成分的非特异性反应引起的。在SPR检测中，人血清影响抗体的灵敏度和检测范围。在检测p24抗体的情况下，首先可以检测溶于PBS中的抗p24抗体，范围为0 - 40 μ g/ml。其次，研究了血清对SPR反射信号的影响。人血清对抗体测定的灵敏度和检测范围产生巨大影响。然而，当用人血清进行封闭并进一步将血清在56 ℃的条件下加热30分钟时，血清中成分的非特异性反应被显著消除。在20 μ g/ml以下可定量检测人血清中的抗体，灵敏度基本恢复到初始值，而在20 μ g/ml以上仍有效果。少

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 … More 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