| Project/Area Number |
61870024
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
Virology
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| Research Institution | Yamaguchi Univ. Sch. Med. |
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Principal Investigator |
YAMAMOTO Naoki Dept. Viro. Parasitol., Yamaguchi unive. Sch. Med., Professor, 医学部, 教授 (00094053)
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| Co-Investigator(Kenkyū-buntansha) |
KOBAYASHI Susumu Dept. Virol. Parasitol., Yamaguchi Univ. Sch. Med., Lecturer, 医学部, 講師 (70034931)
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| Project Period (FY) |
1986 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥10,000,000 (Direct Cost: ¥10,000,000)
Fiscal Year 1987: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1986: ¥8,800,000 (Direct Cost: ¥8,800,000)
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| Keywords | AIDS virus / Neutralizing antibody / 測定法 / AIDSウイルス |
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| Research Abstract |
The aim of this study is to develop accurate and sentisive system using MT-4 cells, Hich is very susceptibel to hyman immunodeficiency verus (HIV ), to measure neutralizign antibody ( NA ) against HIV. NA is bery improtant in respect of evaluation of host defense activity ans clinical course of AIDS in the host. However, higherto known assay systems presented several problems such as low sensitivity. Low specificity. the use of radioisotope and complicatedness. Thus, we first attempted to establish in the first year of this project a new assay system for HIV NA by the use of 3H-thymidine. This procedure is based on the rapid inhibition of the DNA synthesis in MT-4 cells after HIV infection. which makes if possible to know the presence and antibody titer bvy the neutralization reaction. Due to its high sensitivity some sera represented 1:9000 or more in NA titer. Also. it seemed that NA titers decresed with the progress of disease. In the secound year of this project we developed an enz
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yme-linked immunosorbent assay (ELISA) which was comparable to of better than 3H-thymidine incorporation assay in the sensitivity. For this we also used the MT-4 cells after adsorbtion onto the surface of microp lates ans the synthesis ovf viral antigen was measured after HIV infection by ELISA. This procedure detected HIV antigen specifically and was relatively sensitive. Moreover, procedure was rather simple. One of the disadvantages encountered with this assay, however, was that nonspecific reaction was seen at the low dilution of the serum. For this reason antibody-regative sera were only recorded-as less than 1:120 To overcome this problen. follwing experiment is planned : the MT-4 cells are infected with virus and cultured in suspension as done in 3H-tynmidine assay. Then, the antigen synthesis is measured by ELISA on the nitreocellulose filter after fixation. On the other hand, clarification of antigen epitope, which is essential for HIV binding. to CD4 molocule, on the viral bylcoprotein is not reached to the level as we initially scheduled. For this we are persuing the experiment in which synthetic peptides deduced from nucleotide sequence of the env gene are use. Patent applications are sial planned as to the 3H-thymidine assay and ELISA assay for the measurement of the amount of HIV and NA. Less
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