Background The production of monoclonal antibodies for immunoglobulin detection is not cost-effective, while polyclonal antibody production depends on laboratory animals, raising concerns on animal welfare. The widespread use of immunoglobulins in the pharmaceutical industry and the increasing number and variety of new antibodies entering the market require new detection and purification strategies. The Tripartite motif-containing protein 21 is a soluble intracellular immunoglobulin G receptor that binds to the constant region of immunoglobulin G from various species with high affinity. We hypothesized that using this protein as an antibody-binding module to create immunoglobulin detection probes will improve the portfolio of antibody affinity ligands for diagnostic or therapeutic purposes. Results We created a chimeric protein containing a mutated form of the C-terminal domain of mouse Tripartite motif-containing protein 21 linked to streptavidin to detect immunoglobulin G from various species of mammals. The protein is produced by heterologous expression and consists of an improved molecular tool, expanding the portfolio of antibody-affinity ligands for immunoassays. We also demonstrate that this affinity ligand may be used for purification purposes since imidazole elution of antibodies can be achieved instead of acidic elution conditions of current antibody purification methods. Conclusion Data reported here provides an additional and superior alternative to the use of secondary antibodies, expanding the portfolio of antibodies affinity ligands for detection and purification purposes.

The binding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein to the angiotensin-converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein.Therefore, developing high-affinity and cost-effective ACE2 mimetic ligands that disrupt this protein-protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2-5 kDa) and highly stable proteins containing solvent-exposed alphahelix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha-helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS-CoV-2 spike protein. The engineered proteins (h-deface2, p-deface2, and p-deface2-MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7 C), high-affinity binding to the spike protein with apparent K d values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h-deface2, p-deface2, and p-deface2-MUT, respectively, and were used in a diagnostic assay that detected SARS-CoV-2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha-helices in a constrained form for designing of high-affinity ligands.

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The binding of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike protein to the Angiotensin-Converting Enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high-affinity and cost-effective ACE2 peptidomimetic ligands that disrupt this protein-protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small and highly stable proteins, and engineered the amino acid residues on its conformationally constrained alpha-helices to mimic the critical residues on the ACE2 helix 1 that interacts with the Spike-protein. The engineered proteins were soluble and purified to homogeneity with high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability, high-affinity binding to the Spike protein with dissociation constants in the low nanomolar range, and were used in a diagnostic assay that detected SARS-CoV-2 neutralizing antibodies. This work addresses the challenge of developing helical peptidomimetics by demonstrating that defensins provide promising scaffolds to engineer alpha-helices in a constrained form for designing high-affinity ligands.

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Introduction: Primary antibody detection and purification are crucial procedures in research, diagnosis and therapeutics. Molecular tools currently available consist of secondary antibodies as well as bacterial proteins such as Proteins A and G. Disadvantages of the current methods include complex and expensive production of secondary antibodies. In the case of Proteins A and G, they either do not recognize all IgG isotypes or require acidic elution conditions for antibody purification, which may lead to antibody denaturation. Recently, an IgG binding protein was discovered and its use to produce a chimeric protein fused to streptavidin, named YT1902, is of great impact to detect and purify IgGs, being an attractive strategy to overcome current limitations. Objective: To develop a new molecular tool for primary antibody detection using chimeric bacterial protein (YT1902) comprising of an IgG binding module linked to enzymatic probes. To develop a chromatographic column for antibody purification using novel chimeric bacterial protein (YT1902). Methodology: Coding gene of YT1902 protein (Trim21-Streptavidin) was purchased from Genescript and cloned into pET15b(+) vector. The resultant recombinant vectors were expressed into E. coli BL21 (DE3) PlysS and induced at 17 oC during 24 hours in the presence of 0.01 mM IPTG. YT1902 was present in inclusion bodies and refolding was performed by washing steps followed by aggregates dissolution with 6M Guanidine Hydrochloride. Refolding was performed by dialysis followed by affinity purification using Ni-NTA resin. Immunoassays. ELISA was performed by coating on 96 wells microtiters with serum from different animal species or purified IgGs. After blocking with 1% skim milk, HRP labeled YT1902 was used to detect immobilized IgGs. Western blotting analysis was performed using HRP of Alkaline fosfatase labeled YT1902. Results: We have produced chimeric proteins, using an IgG binding protein recently discovered (TRIM21) linked to streptavidin. TRIM21 is a cytosolic protein that binds all IgG isotypes from many species. Bacterial expression, purification and refolding were successful. YT1902 was incubated with biotinylated HRP or alkaline phosphatase for immunoassays. ELISA, western blotting and immunohistochemistry experiments demonstrated that the chimeric protein efficiently detected monoclonal and polyclonal antibodies from a wide range of species, including human, mouse, rat, dog, horse and bovine.Purification experiments are underway using biotin agarose and immobilized YT1902 for antibody purification. Conclusion: YT1902 is able to detect efficiently IgGs from a wide range of mammalian species. The production costs of such proteins is considerable lower than current secondary antibody production. IgG purification using this strategy, will likely be superior than Protein A or G, since elution is not dependent on pH change, which often cause antibody denaturation.The patent of the current invention has been deposited and will provide a great tool for antibody detection and pu...

G-type immunoglobulins (IgGs) are extensively used in the pharmaceutical industry against various diseases, being also crucial in multiple immunoassays. The production of secondary monoclonal antibodies (Abs) for IgG detection is not cost-effective, while polyclonal antibody production still depends on laboratory animals, which raises concerns regarding animal welfare. As alternatives, bacterial proteins (A and G) have been widely exploited; however, several difficulties are encountered regarding their use for IgG detection and purification. The widespread use of IgGs in the pharmaceutical industry and the increasing number and variety of new Abs entering the market impose the need to develop new detection and purification strategies. The TRIM21 protein is a soluble intracellular IgG receptor that binds to the Fc region of many species with high affinity. We created a chimeric protein containing a mutated form of the C-terminal domain of mouse TRIM21 linked to a streptavidin moiety to detect IgGs from a wide range of species. The protein is promptly produced by heterologous expression and consists of an improved molecular tool, expanding the portfolio of Ab-affinity ligands for immunoassays.

Primary antibody detection is crucial for biomarker detection in research and diagnosis. Molecular tools for antibody detection and purification currently available consist of secondary antibodies as well as bacterial proteins such as Proteins A and G. Disadvantages of the current methods include the fact that secondary antibody production is expensive and may require the use of animals. In the case of Proteins A and G, they either do not recognize all IgG isotypes or require acidic elution conditions for antibody purification, which may lead to antibody denaturation. We have produced chimeric proteins, using an IgG binding protein recently discovered (TRIM21) linked to detection proteins such as streptavidin, and alkaline phosphatase. TRIM21 is a cytosolic protein that binds all IgG isotypes from many species. Bacterial expression, purification and refolding of TRIM21‐streptavidin as well as TRIM21‐alkaline phosphatase were successful. TRIM21‐streptavidin chimeric protein was incubated with biotinylated HRP or alkaline phosphatase for immunoassays. ELISA and western blotting experiments demonstrated that the chimeric proteins efficiently detect monoclonal and polyclonal antibodies from a wide range of species, including human, mouse, rat, dog, horse and bovine. Binding experiments are currently underway to determine the binding constants for TRIM21 chimeric proteins and IgG from different species. Future experiments will be performed using imobilized TRIM21 for antibody purification.Support or Funding InformationThis work was supported by the “ State University of Santa Catarina” (UDESC), “Fundação de Amparo a Pesquisa e Inovação do Estado de Santa Catarina” (FAPESC) and CNPQ.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.