- 期刊
Immunochemical Characterization of Cobrotoxin Modified with Glutaraldehyde
以雙作用性試藥Glutaraldehyde修飾Cobrotoxin之免疫化學特性
摘要
從眼鏡蛇（Taiwan cobra;Naja naja atra）毒分離出來之Cobrotoxin，是一種腱後神經毒素（postsynaPtic neurotoxin; a-neur-otoxin )，分子含有62個腔基酸殘基及4個雙硫鍵，是屬於短鏈神經毒素（short neurotoxin )。近十年來，我們曾經以化學修飾方法探究其構造與生物活性之關係以及修飾毒素之免疫化學特性。 雙作用性試藥（bifunctional reagents）常被利用於蛋白質之化學構造，尤其多元體（olig-omers）之立體構造及子單元（subuaits）之結合之研討以及在溶液中蛋白質分子內腔基酸殘基間之距離的測定等。最近有人初步利用glutaral-dehyde修飾蛇毒或竭毒（scorplon venom）分離出來之神經毒素，結果修飾毒素之毒性顯著減低。利用這些修飾毒素免疫家兔結果，則得到能中和原毒甚高之抗血清。 本實驗，以9摩爾比之glutaraldehyde修飾cobrotoxin後形成之交叉結合（cross-linked ）修飾毒素，首先以Sephadex G-50可得到五個分劃，其中四個主要分劃再分別以Sephadex G-75或G-50純化兩次，結果得到單元體、取元體、三元體及多元體等交叉結合之修飾毒素。測定這些修飾毒素致死毒性之結果，多元體則完全沒有毒性，而其餘的毒性亦幾乎消失。利用定量沈澱反應測定結果，這些修飾毒素對於抗cobro-toxin抗體仍具有很強之抗原性。利用修飾毒素（未經純化之混合體、單元體、眾元體以及多元體）免疫於家兔所得到之抗血清，除了抗多元體血清外，其他抗血清與抗cobrotoxin一樣，具有很強之中和力價，其中抗單元體血清甚至比抗cobrotoxin血清之中和能力強1.4倍。雖然它們對 cobrotoxin 反應形成沈澱之沈澱性抗體含量低，但利用親和層析法（affiulty chromat-ography）測定抗血清中之非沈搬性抗體（non-precipitating antibody）結果，除了抗多元體血清外，其餘的含有相當高之非沈澱性抗體。一方面，這些抗修飾毒素血清與對應的（homolog-ous）抗原反應，仍形成較多量之沈澱。 從以上實驗結果得知，cobrotoxin經glu-taraldehyde修飾後毒性幾乎消失，因此可大量免疫動物，在較短期間內可產生中和力價很高之抗血清，而且其非沈澱性抗體含量較高，因此這種修飾毒素可為一個優良之免疫原以產生抗血清，來提高蛇毒咬傷患者之治療效果。
關鍵字
無資料並列摘要
Modification of cobrotoxin with glutaraldehyde, a bifunctional reagent, resulted in the formation of cross-inked derivatives.The intra-molecular cross-linked monomeric derlvatives as well as dimers, trilllers, and higher molecular weigllt derivatives arising from intermolecular reactions were separated by gel filtration. Polymeric dsrivatives were completely detoxified and the others lost almost completely their lethality. However, all the modified derlvatives retained relatively high antigenic activities towards the antibody against native toxin . Rabbits immunized with these modified derivatives (except for trimeric derivatives) produced considsrable amounts of non-precipitating antibody but much less precipitating antibody to cobrotoxin. The neutralizing capacity of anti-onomeric derivatives sera had 1.4 times more potent than that of anti-cobrotoxin sera. Antisera prepared by immunization with the unfractionated reaction products of cobrotoxin or with dimeric derivatives had the same or almost the same capacity as that of anti-cobrotoxin sera for neutralizing the lethality of cobrotoxin. These antisera were obtained in only one-half period required for anti-cobrotoxin sera. However, polymeric cobrotoxin derivatives were poor linmunogens for the production of neutralizing antibody against native toxin.