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.
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.
Introduction:Antibodies are molecules extensively used in in vitro diagnostic and therapeutic applications. However, identifying new antibodies requires cost-effective and large-scale screening of binding molecules. Therefore, the development of improved microscale purification techniques may assist the rapid screening of molecules allowing the discovery of new and improved antibodies for diagnostic and therapeutic purposes TRIM21 is a soluble IgG-binding protein that has been shown to detect IgG from several mammalian species. In addition, two histidine residues mediate the molecular interaction between TRIM21 and IgGs. These findings suggest that the use of imidazole-containing buffers may disrupt the interaction between these molecules, generating a neutral pH-based elution method for IgG purification. Therefore, we investigated using TRIM21 immobilized into polystyrene plates to capture and purify IgGs on a microscale.Objective: This project aimed to investigate the ability of TRIM21 to capture and purify IgGs from serum based on neutral pH conditions.Methodology: TRIM21 was coated into polystyrene plates, followed by incubation with serum, removing nonbinding species, washing with PBS-based buffers, and elution with increasing imidazole concentrations. Eluted fractions were evaluated by ELISA and dot blots using anti-human, anti-rabbit, anti-horse, anti-mouse, anti-sheep, anti-cattle secondary antibodies. Results:The use of 0.5M imidazole buffer allowed purification of IgGs from different species validating this method as a promising tool for antibody purification. Conclusion:The promising results prove the principle of plate-based chromatographic applications-a good alternative for developing purification processes.
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