トリインフルエンザにおける抗核蛋白抗体の迅速検出法の確立

禽流感抗核蛋白抗体快速检测方法的建立

• 批准号: 62760263
• 负责人: 井土 俊郎
• 金额: $ 0.58万
• 依托单位: Nippon Institute for Biological Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas
• 财政年份: 1987
• 资助国家: 日本
• 起止时间: 1987 至 1988
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-62760263/
• 关键词: 鶏インフルエンザ; 抗S抗体; 免疫; 寒天ゲル内沈降反応; EIISA; 診断

鶏インフルエンザウイルス(AIV)感染に伴って産生される抗S抗体の簡便な検出法の確立を目的として研究を行い, 本年度は以下の成果が得られた.1.各種のAIVに対する抗血清の作成A/C/Jpn24及びA/WS/島根/42/80株についてはSPF鶏を用いて感染及び不活化IV免疫血清を作成した. また, A/PR/8/34. A/D/Ukraine/1/63, A/D/Czech/56及びA/C/Germany/N/49株については不活化IV免疫血清を作成した.2.IVのS抗原(核蛋白)の精製法の検討超遠心法で精製したIV浮遊液, IV感染発育鶏卵漿尿膜の20%乳剤及び初代培養鶏腎, 継代犬腎(MDCK)又は豚胎児腎(ESK)細胞の各々5%乳剤の3方法で調整した5材料から, 超遠心法及びゲル濾過法でS抗原の精製を試みた. その結果, 精製IV粒子を破壊して得たS抗原は夾雑蛋白は少なく, 寒天ゲル内沈降(AGP)反応性がよく良質であったが収量が少なかった. 漿尿膜S抗原は夾雑蛋白が多く, 抗原価も低かった. 培養細胞由来S抗原は夾雑物が比較的少なく, 抗原価が高く反応も明瞭で, 3方法の中で最良であった. この性状はPAGEによって確認された.3.S抗原に対する抗体(抗S抗体)検出法の検討培養細胞由来のS抗原を供試して, 抗血清中の抗S抗体を免疫方法との関連においてAGP及びELISA法によって検索した. その結果, 抗SAGP抗体はAIV感染鶏ではHI抗体価64倍以上の個体は全例陽性であったが, 非精製不活化IV接種鶏ではHI抗体価512倍の個体でも陰性であった. しかし, IV粒子が破壊されるような条件で精製した不活化IV接種鶏では抗SAGP抗体が産生された. ELISA法による抗S抗体検出法は特異性に問題があった. 次年度では抗S抗体産生と免疫法及びELISA法について詳細に検討し, 本症診断上の意義づけを行う.

The following results were obtained this year: 1. Antisera against various AIV strains were prepared: A/C/Jpn24 and A/WS/Shimane/42/80 strains.また, A/PR/8/34. A/D/Ukraine/1/63, A/D/Czech/56 and A/C/Germany/N/49 strains were used to prepare inactivated IV immune serum. 2. Study on purification method of S antigen (nuclear protein) of IV suspension, IV infection development, 20% milk of oocyte plasma urine membrane and 5% milk of primary cultured kidney, MDCK and ESK cells. 3 methods were used to adjust 5 materials. Ultra-remote method and filtration method are used to purify S antigen. As a result, purified IV particles were obtained and S antigen was added to the protein. In cold weather, AGP was found to be of good quality. S antigen in plasma urine membrane is high in protein and low in antigen. Cultured cells from S antigen to the inclusion of substances compared with the few, antigen to the high anti-anti-clear, 3 methods of the best. These characteristics were confirmed by PAGE. 3. Antibodies against S antigen (anti-S antibodies) were detected by ELISA and AGP. The results showed that anti-SAGP antibody against AIV infection was more than 64 times higher than HI antibody in all positive cases, and 512 times higher than HI antibody in non-purified inactivated IV vaccination. IV particles were isolated under conditions that did not activate IV inoculation and anti-SAGP antibodies were produced. ELISA method for anti-S antibody detection is not specific. In the following year, the production of anti-S antibody, immune method and ELISA method were discussed in detail, and the diagnostic significance of this disease was discussed.

项目成果

Toshiro IZUCHI: The Japanese Journal of Veterinary Science.

Toshiro IZUCHI：日本兽医科学杂志。