| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1994: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1993: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
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
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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
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