ABSTRACT
mWasabi is a bright monomeric green fluorescent protein. It can be used as a fusion tag to monitor various biological events, e.g. protein localization. Here we report the selection of camelid-derived single-domain antibody fragments (nanobodies) against mWasabi. In this work, phage-display approach was employed to select the high affinity mWasabi-specific Nb (nanobodies). These nanobodies were able to recognize mWasabi or in a fused fashion with PD1. The interesting binding characteristics of these two mWasabi-specific nanobodies could be valuable for design new tools for cellular tracing or targeting based on the mWasabi-fusing protein in many different biological research fields.
Subject(s)
Cell Surface Display Techniques/methods , Luminescent Proteins/chemistry , Single-Domain Antibodies/immunology , Single-Domain Antibodies/isolation & purification , Amino Acid Sequence , Animals , Camelidae/blood , Camelidae/immunology , HEK293 Cells , Humans , Immunoglobulin G/blood , Luminescent Proteins/immunology , Luminescent Proteins/isolation & purification , Programmed Cell Death 1 Receptor/analysis , Programmed Cell Death 1 Receptor/metabolism , Protein Binding , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence AlignmentABSTRACT
Nanobodies are antigen binding variable domains of heavy-chain antibodies without light-chains, and these biomolecules occur naturally in the serum of Camelidae species. Nanobodies have a compact structure and low molecular weight when compared with antibodies, and are the smallest active antigen-binding fragments. Because of their remarkable stability and manipulable characteristics, nanobodies have been incorporated into biomaterials and used as molecular recognition and tracing agents, drug delivery systems, molecular imaging tools and disease therapeutics. This review summarizes recent progress in this field focusing on nanobodies as versatile biomolecules for biomedical applications.
Subject(s)
Biocompatible Materials/chemistry , Biomedical Research , Camelidae/immunology , Nanostructures/chemistry , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/immunology , Animals , Antigens/chemistry , Antigens/immunology , Camelidae/bloodABSTRACT
Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) is a critical negative immunomodulatory receptor that is normally expressed in activated T cells and noticeably, in many cancerous cells. Indeed, molecular detection of CTLA-4 protein is crucial in basic research. In this work, the extracellular domain of the human CTLA-4 (hCTLA-4) protein was cloned, expressed, and purified. Subsequently, this protein was used as an antigen for camel (Camelus dromedarius) immunization to obtain polyclonal camelid sera against this protein. Furthermore, we evaluated the benefits of applying camelid hyperimmune sera containing heavy-chain antibodies in different antibody-based techniques. Our results indicated that hCTLA-4 protein was successfully expressed in the prokaryotic system. The polyclonal antibody (pAb) that raised against recombinant hCTLA-4 protein was able to detect the CTLA-4 protein in antibody-based techniques, such as enzyme-linked immunosorbent assay, western blotting, flow cytometry and immunohistochemistry (IHC) staining. This study shows that, due to the advantages such as multi-epitope-binding ability, camelid pAbs are potent to efficiently apply for molecular detection of CTLA-4 receptors in fundamental antibody-based researches such as IHC.
