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Enhancement of scFv fragment reactivity with target antigens in binding assays following mixing with anti-tag monoclonal antibodies.
J Immunol Methods. 2004 Nov; 294(1-2):23-35.JI

Abstract

The phage display Ab library technology has been found to be a useful method to isolate antigen-specific Ab fragments, since the repertoire of antibody specificities is broad and since it bypasses the need of immunization. However, when screening clones isolated from a phage display Ab library, the yield of isolating antigen-specific Ab fragments is low and the rate of false negative results is high. This limitation reflects the low affinity/avidity of Ab fragments and/or the low density of the target antigen. To facilitate the isolation of Ab fragments with a broad range of affinities to antigens of interest from phage display Ab libraries, we have developed a simple method to increase the sensitivity of binding assays to detect the reactivity of single-chain fragments of antibody variable regions (scFv) with target antigens. This method involves the mixing of scFv fragments, expressing a c-myc epitope tag, with anti-tag mAb 9E10 prior to their use in binding assays in order to form stable dimeric Ab fragment-anti-tag mAb complexes. The increase in the reactivity of scFv fragments with the corresponding antigen is observed over a broad range of scFv fragment (6-800 microg/ml) and mAb 9E10 (0.5-30 microg/ml) concentrations, thereby facilitating the testing of scFv fragment preparations with unknown scFv fragment concentrations. Use of this method in binding assays resulted in a twofold increase in the reactivity of low-affinity purified scFv fragments with the corresponding antigen. Moreover, application of this method to screen clones isolated from phage display scFv libraries resulted in a reproducible increase in both the yield of antigen-specific scFv clones and the titer of scFv fragment preparations by a factor of 5 and 2- to 32-fold, respectively. Lastly, this method can be applied in both ELISA and flow cytometry and is independent of the characteristics of the antigen (i.e. whole cells, carbohydrates and purified protein) and/or of the library (synthetic scFv Library (#1), a large semi-synthetic phage display scFv library and the human synthetic VH+VL scFv library (Griffin.1 library)) used. Therefore, the method we have described represents a sensitive, simple and reproducible technique that will facilitate the isolation and use of scFv fragments.

Authors+Show Affiliations

Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15604013

Citation

Wang, Xinhui, et al. "Enhancement of scFv Fragment Reactivity With Target Antigens in Binding Assays Following Mixing With Anti-tag Monoclonal Antibodies." Journal of Immunological Methods, vol. 294, no. 1-2, 2004, pp. 23-35.
Wang X, Campoli M, Ko E, et al. Enhancement of scFv fragment reactivity with target antigens in binding assays following mixing with anti-tag monoclonal antibodies. J Immunol Methods. 2004;294(1-2):23-35.
Wang, X., Campoli, M., Ko, E., Luo, W., & Ferrone, S. (2004). Enhancement of scFv fragment reactivity with target antigens in binding assays following mixing with anti-tag monoclonal antibodies. Journal of Immunological Methods, 294(1-2), 23-35.
Wang X, et al. Enhancement of scFv Fragment Reactivity With Target Antigens in Binding Assays Following Mixing With Anti-tag Monoclonal Antibodies. J Immunol Methods. 2004;294(1-2):23-35. PubMed PMID: 15604013.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Enhancement of scFv fragment reactivity with target antigens in binding assays following mixing with anti-tag monoclonal antibodies. AU - Wang,Xinhui, AU - Campoli,Michael, AU - Ko,Eric, AU - Luo,Wei, AU - Ferrone,Soldano, PY - 2004/05/04/received PY - 2004/08/04/revised PY - 2004/08/10/accepted PY - 2004/12/18/pubmed PY - 2005/2/4/medline PY - 2004/12/18/entrez SP - 23 EP - 35 JF - Journal of immunological methods JO - J. Immunol. Methods VL - 294 IS - 1-2 N2 - The phage display Ab library technology has been found to be a useful method to isolate antigen-specific Ab fragments, since the repertoire of antibody specificities is broad and since it bypasses the need of immunization. However, when screening clones isolated from a phage display Ab library, the yield of isolating antigen-specific Ab fragments is low and the rate of false negative results is high. This limitation reflects the low affinity/avidity of Ab fragments and/or the low density of the target antigen. To facilitate the isolation of Ab fragments with a broad range of affinities to antigens of interest from phage display Ab libraries, we have developed a simple method to increase the sensitivity of binding assays to detect the reactivity of single-chain fragments of antibody variable regions (scFv) with target antigens. This method involves the mixing of scFv fragments, expressing a c-myc epitope tag, with anti-tag mAb 9E10 prior to their use in binding assays in order to form stable dimeric Ab fragment-anti-tag mAb complexes. The increase in the reactivity of scFv fragments with the corresponding antigen is observed over a broad range of scFv fragment (6-800 microg/ml) and mAb 9E10 (0.5-30 microg/ml) concentrations, thereby facilitating the testing of scFv fragment preparations with unknown scFv fragment concentrations. Use of this method in binding assays resulted in a twofold increase in the reactivity of low-affinity purified scFv fragments with the corresponding antigen. Moreover, application of this method to screen clones isolated from phage display scFv libraries resulted in a reproducible increase in both the yield of antigen-specific scFv clones and the titer of scFv fragment preparations by a factor of 5 and 2- to 32-fold, respectively. Lastly, this method can be applied in both ELISA and flow cytometry and is independent of the characteristics of the antigen (i.e. whole cells, carbohydrates and purified protein) and/or of the library (synthetic scFv Library (#1), a large semi-synthetic phage display scFv library and the human synthetic VH+VL scFv library (Griffin.1 library)) used. Therefore, the method we have described represents a sensitive, simple and reproducible technique that will facilitate the isolation and use of scFv fragments. SN - 0022-1759 UR - https://www.unboundmedicine.com/medline/citation/15604013/Enhancement_of_scFv_fragment_reactivity_with_target_antigens_in_binding_assays_following_mixing_with_anti_tag_monoclonal_antibodies_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-1759(04)00283-2 DB - PRIME DP - Unbound Medicine ER -