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

在艾滋病传感器的实际应用中，需要克服以下几点：1.可以同时检测抗HIV蛋白gp14和p24的抗体。特别是，在本研究中，可以在测量后立即确认感染；2.分析时间应在几分钟左右；3.将血清样本应用于该系统时，可以进行高灵敏度的检测。本研究利用表面等离子体共振(SPR)技术，在检测抗HIV抗体前，完成了将抗原化学固定在传感器尖端表面金膜上的方法，并利用表面等离子体共振技术(SPR)进行了检测。金膜在10uM的4，4-二硫代丁酸中室温暴露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-羟基丁二酰亚胺(NHS)中，进行彻底的st…反复搅拌3小时，然后用清水洗净。通过这种处理，我们得到了稳定的SPR免疫传感器尖端。在该传感器中，抗gp41抗体的检测范围为1~20ug/ml，实时监测免疫反应，通过计算机处理数据，很容易从动力学中确定亲和常数Ka为2.7×107/M。为了实际应用，考察了血清对反射率的影响。在反应中观察到了巨大的影响，这可能是由血清中成分的非特异性反应引起的。在SPR测定中，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，首先可以检测到溶解在PBS中的抗p24抗体，范围为0~40ug/ml。其次，考察了血清对SPR反射信号的影响。在抗体检测中，人血清对抗体的灵敏度和检测范围都有很大影响。而用人血清封闭后，在56ﾟC条件下加热30min，可显著消除血清成分的非特异性反应。人血清中的抗体在20微克/毫升以下即可定量检测，灵敏度基本恢复到初始值，而效价仍维持在20微克/毫升以上。较少

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