DETECTION OF PROTEINS OF HUMAN IMMUNODEFICIENCY VIRUS BY USING CHEMICAL SENSOR

一些技术，如酶联免疫吸附试验（ELISA），明胶聚集等已被报道用于筛选HIV（人类免疫缺陷病毒）感染。此外，使用血清的另一种方法western blotting来确认阳性样品。由于难以处理艾滋病病毒和抗体，迄今为止有关艾滋病化学传感器的报告仅见。作者已经建立了几种HIV蛋白的重组体和相应的单克隆抗体。本研究利用gp41和单克隆抗体构建艾滋病免疫传感器。gp41是HIV包膜的糖蛋白之一，是判断HIV感染的重要蛋白。gd41保守区氨基酸序列已确定为H-RGPDREGIEEEGGERDRDC-OH （19 mer）。将上述gp41多肽与钥匙孔帽贝偶联制备抗gp41单克隆抗体（41S-2）。从氨基酸表位定位来看，gd41多肽的中心区域主要被单克隆抗体识别。在合成的5个聚肽中，位于19个聚肽中心的GIEEE氨基酸序列亲和性最强。另一方面，该序列EGIEE具有第三强亲和力，最适合构建免疫亲和传感器。前者强度太大，不能用作免疫亲和传感器。Gp41 （19mer）用流动系统仪检测。在传感实验前，用过氧化氢酶标记的EGIEE与固定在免疫膜上的单克隆抗体反应。膜设置在流水线上。其次，流动gp41多肽。由于EGIEE与gp41多肽相比对单克隆抗体的亲和力较低，因此应将该多肽替换为gp41的19 mer。免疫反应后，从亲和膜上释放的过氧化氢酶标记的EGIEE与底物接触，过氧化氢酶分解h_2o_2，然后在流水线上插入氧电极检测h_2o_2。用记录仪监测酶的反应。目前的反应与抗原gp41 （19mer）的浓度关系良好。在本系统中，gp41的检出限为1 ~ 2mg/ml。抗体达到0.1mg/ml。考虑到肽合成和抗原表位定位技术的巨大发展，推测可以利用免疫亲和技术构建艾滋病传感器。少

Some techniques such as the enzyme linked immunosorbent assay (ELISA), gelatin aggregation etc.have been reported for the screening of HIV (Human Immunodeficiency Virus) infection.Furthermore, the confirmation of the positive sample is performed using the another method, western blotting, of the serum.The reports of the chemical sensor with regard to AIDS have merely seen so far for the reason of the difficulty of handling AIDS virus and the antibodies.Authors have established some kinds of the recombinants for each protein of HIV and the corresponding monoclonal antibodies.In this study, gp41 and the monoclonal antibody were used to construct the immunosensor for AIDS.gp41 is one of the glycoproteins of the envelope of HIV,which is the important protein for judgment of HIV infection.Amino acid sequence of the conservative region of gd41 has already determined to be H-RGPDREGIEEEGGERDRDC-OH (19 mer).Anti gp41 monoclonal antibody (41S-2) was produced using the above polypeptide of gp41 … More conjugated with key hole limpet.From the epitope mapping of the amino acid, the central region of gd41 polypeptide was mainly recognized by the monoclonal antibody.Amino acid sequence of GIEEE which locates at the central region of 19 mer polypeptide displayd the strongest affinity in the synthesized 5 mer peptides.On the other hand, the sequence, EGIEE,showed the third strongest affinity which had the most preferable feature for the construction of immunoaffinity sensor.The former was too strong to use as the immunoaffinity sensor.gp41 (19mer) was detected in accordance with the flow system apparatus.EGIEE labeled with catalase was reacted with the monoclonal antibody fixed on the immuno-membrane prior to the sensing experiment.The membrane was set in the flow line.Secondly, gp41 polypeptide was flowed.As EGIEE has less affinity compared with the gp41 polypeptide against the monoclonal antibody, the peptide should be replaced by the 19 mer of gp41.After the immunoreaction, the EGIEE labeled with catalase which was released from the affinity membrane meets to the substrate, H_2O_2.H_2O_2 was decomposed by the catalase and then detected with the oxygen electrode inserted in the flow line.The enzyme reaction was monitored with the recorder.The relation of the current response and the concentration of antigen, gp41 (19mer) showed good relation.In this system, the detection limit of gp41 was 1-2mg/ml.And that of antibody reached to 0.1mg/ml.Considering the huge development of peptide synthesis and epitope mapping techniques of the antigen, it is inferred that AIDS sensor can be constructed by the immuno affinity technique. Less