کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10533012 | 961836 | 2005 | 10 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Dissociation-independent selection of high-affinity anti-hapten phage antibodies using cleavable biotin-conjugated haptens
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
The engineering of hapten-specific antibodies with affinity constant higher (Ka values > 1010 Mâ1) than those of conventional antibodies promises hapten immunoassays exhibiting sub-femtomole range sensitivity, based on the conventional competitive assay principle. Here we report a simple method to select phage particles displaying anti-hapten antibody fragments with exceptionally high affinity. 11-Deoxycortisol (11-DC), selected as a model target hapten, was covalently conjugated to biotin via a spacer that included a reductively cleavable disulfide bond. Phage particles displaying high-affinity, single-chain Fv fragments (scFvs) specific for 11-DC (Ka1.3 Ã 1010 Mâ1) were incubated with the “cleavable biotin”-conjugated 11-DC, and the resulting complexes was captured on immobilized NeutrAvidin. Mild reductive conditions that did not decrease phage infectivity easily cleaved the disulfide bond, allowing the recovery of target phage particles; this process is fully independent of the dissociation of the antigen-antibody interaction. Five serial rounds of selection enabled the isolation and enrichment of the anti-11-DC phage (specific phage ratio > 90%) from among a 100,000-fold excess of nonspecific phage particles. This method will be applicable for selection of extra-high-affinity phage antibodies against a wide variety of haptens.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 347, Issue 2, 15 December 2005, Pages 287-296
Journal: Analytical Biochemistry - Volume 347, Issue 2, 15 December 2005, Pages 287-296
نویسندگان
Norihiro Kobayashi, Tsuyoshi Karibe, Junichi Goto,