Document Detail


The use of phage display to generate conformation-sensor recombinant antibodies.
MedLine Citation:
PMID:  23154784     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We describe a phage display approach that we have previously used to generate conformation-sensor antibodies that specifically recognize and stabilize the oxidized, inactive conformation of protein tyrosine phosphatase 1B (PTP1B). We use a solution-based panning and screening strategy conducted in the presence of reduced active PTP1B, which enriches antibodies to epitopes unique to the oxidized form while excluding antibodies that recognize epitopes common to oxidized and reduced forms of PTP1B. This strategy avoids conventional solid-phase immobilization owing to its inherent potential for denaturation of the antigen. In addition, a functional screening strategy selects single-chain variable fragments (scFvs) directly for their capacity for both specific binding and stabilization of the target enzyme in its inactive conformation. These conformation-specific scFvs illustrate that stabilization of oxidized PTP1B is an effective strategy to inhibit PTP1B function; it is possible that this approach may be applicable to the protein tyrosine phosphatase (PTP) family as a whole. With this protocol, isolation and characterization of specific scFvs from immune responsive animals should take ~6 weeks.
Authors:
Aftabul Haque; Nicholas K Tonks
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2012-11-15
Journal Detail:
Title:  Nature protocols     Volume:  7     ISSN:  1750-2799     ISO Abbreviation:  Nat Protoc     Publication Date:  2012 Dec 
Date Detail:
Created Date:  2012-11-16     Completed Date:  2013-04-16     Revised Date:  2013-07-18    
Medline Journal Info:
Nlm Unique ID:  101284307     Medline TA:  Nat Protoc     Country:  England    
Other Details:
Languages:  eng     Pagination:  2127-43     Citation Subset:  IM    
Affiliation:
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA.
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MeSH Terms
Descriptor/Qualifier:
Cell Surface Display Techniques / methods*
Epitopes / metabolism
Immunologic Techniques / methods*
Oxidation-Reduction
Protein Tyrosine Phosphatase, Non-Receptor Type 1 / metabolism*
Recombinant Proteins / metabolism*
Single-Chain Antibodies / metabolism*
Grant Support
ID/Acronym/Agency:
CA53840/CA/NCI NIH HHS; GM55989/GM/NIGMS NIH HHS; R01 CA053840/CA/NCI NIH HHS; R01 GM055989/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Epitopes; 0/Recombinant Proteins; 0/Single-Chain Antibodies; EC 3.1.3.48/Protein Tyrosine Phosphatase, Non-Receptor Type 1
Comments/Corrections

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