DETECTION OF PROTEINS OF HUMAN IMMUNODEFICIENCY VIRUS BY USING CHEMICAL SENSOR

针对HIV感染的筛查，已报道了酶联免疫吸附试验(EL ISA)、明胶凝集等技术。此外，对阳性标本的确认采用了另一种方法--免疫印迹法。由于艾滋病病毒及其抗体难以处理，有关艾滋病的化学传感器的报道到目前为止还没有见过。作者建立了几种针对HIV每种蛋白和相应的单抗的重组体。本研究利用gp41和单抗构建了针对HIV的免疫传感器GD41基因保守区的氨基酸序列已确定为H-RGPDREGIEEEGGERDRDC-OH(19mer)。用Gp41…多肽制备抗GP41单抗(41S-2)从氨基酸的表位图谱来看，gd41多肽的中心区主要被单抗识别，位于19肽中心区的GIEEE氨基酸序列在合成的5聚肽中显示出最强的亲和力，而序列EGIEE，亲和力第三强，最适合构建免疫亲和力传感器。前者亲和力太强，不能用作免疫亲和力传感器。根据流动系统仪器检测gp41(19聚体)。在传感实验之前，用过氧化氢酶标记的EGIEE与固定在免疫膜上的单抗反应，膜固定在流线上。其次，gp41多肽被漂浮。由于EGIEE与gp41多肽的亲和力比gp41多肽小，因此gp41多肽应该被gp41的19聚体取代。免疫反应后，从膜亲和力释放的过氧化氢酶标记的EGIEE与底物相遇，过氧化氢酶能分解H_2O_2·H_2O_2，然后在流水线中插入氧电极进行检测，用记录仪监测酶反应，电流响应与抗原gp41(19聚体)浓度的关系良好。在该体系中，gp41的检测限为1-2 mg/ml，抗体的检测限达到0.1 mg/ml。考虑到抗原的多肽合成和表位定位技术的巨大发展，推测免疫亲和技术可以构建艾滋病传感器。较少

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