![]() Since the seminal work of Arnon and colleagues showing that protein-reactive antibodies could be elicited by a synthetic peptide 11, the versatility of peptide polyclonal and monoclonal antibodies has led to increasing interest in using protein fragments (100–150 residues) and peptides (10–20 residues) as surrogates of whole proteins 12, 13, 14. Whole proteins have traditionally been used as immunogens for antibody production. The robust workflow lowers the barrier to quality mAb production by dramatically reducing the experimental burden associated with hybridoma screening, antibody characterization and validation. To meet the expanding need for fit-for-purpose antibodies, we developed a peptide-mediated method for producing high affinity monoclonal antibodies (mAbs) against multiple sites on a target protein. Corroboration with at least two validated antibodies to non-overlapping epitopes on the same protein is required for data reliability 10. ![]() High quality antibodies are also urgently needed for large-scale initiatives like the Human Atlas Project to map organ, tissue and cellular distribution of proteins in the human proteome. The antibody used in early high-profile reports was later found to cross-react with a closely-related family member, myostatin (GDF8), raising concerns over the validity of the original findings 8, 9. For example, inadequate antibody characterization have led to controversies surrounding the role of growth differentiation factor 11 (GDF11) in age-related cardiac, muscle and cognitive decline 7. However, performance inconsistencies and poor validation are often encountered with commercial antibodies, contributing to irreproducible and misleading data 1, 2, 3, 4, 5, 6. This robust method motivates its ready adoption for other protein targets.Īntibodies are protein workhorses with broad utility in research, diagnostic and therapeutic applications. The use of short antigenic peptides of known sequence facilitated direct epitope mapping crucial for antibody characterization. Antibodies against spatially distant sites on hANKRD1 facilitated validation schemes applicable to two-site ELISA, western blotting and immunocytochemistry. ELISA assay miniaturization afforded by novel DEXT microplates allowed rapid hybridoma screening with concomitant epitope identification. Antigenic peptides (13–24 residues long) presented as three-copy inserts on the surface exposed loop of a thioredoxin carrier produced high affinity mAbs that are reactive to native and denatured hANKRD1. The workflow is illustrated by generating mAbs against multiple in silico-predicted epitopes on human ankyrin repeat domain 1 (hANKRD1) in a single hybridoma production cycle. We describe an epitope-directed monoclonal antibody (mAb) production method that addresses issues of antibody quality, validation and utility. We believe that changes in scaffold pore size and related physical properties can have a profound impact on cell behavior, such as adhesion, proliferation and infiltration, and the significance of their influence on applications of electrospun tissue engineering scaffolds is worthy of further investigation in the future.High quality, well-validated antibodies are needed to mitigate irreproducibility and clarify conflicting data in science. In the present review, we have summarized the preparation of macroporous electrospun scaffold techniques for the skin, blood vessels, bone, cartilage and nerve tissue engineering for different applications, and further discuss the influence of changing pore-enlarging process parameters on the properties of the scaffolds, such as mechanical properties, and hydrophilicity and hydrophobicity, etc. Many methods for enlarging the pore size of electrospun scaffolds have been described in the literature. In order to achieve satisfactory results for use in tissue engineering, the pore size of the scaffold should be increased to a size dependent on the specific cells being cultured. Despite electrospinning having multiple advantages over other methods such as creating materials with a superfine fiber diameter, high specific surface area, and good mechanical properties, the pore diameter of scaffolds prepared directly using conventional electrospinning is often smaller than a few tens of microns, which may not be suitable for three-dimensional (3-D) cell culture and tissue growth.
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