Enhancement of scFv fragment reactivity with target antigens in binding assays following mixing with anti-tag monoclonal antibodies.
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.
Wang
Xinhui
X
Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.
Campoli
Michael
M
Ko
Eric
E
Luo
Wei
W
Ferrone
Soldano
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000915:Antibody Affinity / Q000235:genetics* / Q000276:immunology; D001666:Binding Sites, Antibody / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D015723:Gene Library; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007135:Immunoglobulin Variable Region; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins