کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1176894 961899 2011 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Immunodiagnostic reagents using llama single domain antibody–alkaline phosphatase fusion proteins
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Immunodiagnostic reagents using llama single domain antibody–alkaline phosphatase fusion proteins
چکیده انگلیسی

Naive libraries of single domain antibodies (sdAbs) enable rapid isolation of binders to nearly any target. These binders, however, lack the benefits bestowed by in vivo affinity maturation and typically have low affinity toward their targets. We expressed five low-affinity toxin binding sdAbs, previously selected from a naive library derived from variable regions of llama heavy chain-only antibodies, as fusions with a hyperactive mutant Escherichia coli alkaline phosphatase (AP) and examined the impact on apparent affinity and utility. AP spontaneously dimerizes in solution, effectively dimerizing the fused sdAbs, imparting avidity in place of the lower affinity monomeric interactions. The sdAb–AP fusion also combines the target recognition domain with a signal transduction domain, commonly used in enzyme-linked immunosorbent assays (ELISAs). The functional affinity of the sdAb–AP fusions, often increased by a factor of 10 over unfused sdAbs, and their utility as tracer reagents in ELISAs was dramatically improved, giving limits of detection of 300 ng/ml or less, whereas parental sdAbs gave no discernible signal at the toxin concentrations tested. The fusion of sdAbs to AP presents a valuable route to facilitate the implementation of sdAb-based immunoreagents rapidly selected from existing naive libraries toward new or emerging threats.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 417, Issue 2, 15 October 2011, Pages 188–194
نویسندگان
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