| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1991: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1990: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
Many cases of intestinal anisakiasis as well as gastric anisakiasis have been reported in Japan. As compared to gastric anisakiasis which is more easily diagnosed, intestinal ansakiasis can be misdiagnosed as ileus caused by other diseases. We have reported that serial measurements of serum anti-anisakis IgE antibodies are very useful In the diagnosis of intestinal anisakiasis as well as acute gastric anisakiasis.
In this research project, We attempted to prepare the 76k, 64k and 6OkDa proteins of Anisakis larva which are defined by serum antibodies from patients, in order to establish a specific ELISA assay system for the diagnosis of intestinal and gastric anisakiasis
1. Monoclonal antibody production : Mice(n-8)were immunized by implantation of live Anisakis larvae into the abdominal cavity every one week. After 4-6 weeks anti-anisakis antibody were detected in all of 8 mice, assayed by ELISA using larval homogenates as antigen.
2. Screening of hybridoma : Spleen cells of each immunized mouse were fused with mouse myeloma cells(P3U-1)by using 45% polyethylene glycol-4000. Screening and selection of hybridoma cells was performed. Each of 20 hybridomas produced plenty amount, of an tibody, which reacted with Anisakis larva by ELISA system.
3. Western immunoblotting : Hybridoma cells were screened finally by using Western ; immunoblotting in order to identify their specific correspondence to 76, 64 or 60 kDa proteins, now in progress.
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