TRIM21 chimeric protein as a new molecular tool for multispecies IgG detection

Abstract

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.

Keywords: Antibody; ELISA; Immunoglobulin; Protein A; Protein G; Protein engineering.

Fig. 1

Schematic diagram showing chimeric protein. A A 3D-structure of Mouse IgG (blue) bound to the PRYSPRY domain of wild-type mouse TRIM21 (red). B The primary and quaternary structure of SA-TRIM21. In red is represented the PRYSPRY domain of TRIM21. Purple represented the rigid EAAAK linker, orange shows the streptavidin domain and yellow the Poli-histidine tag. C The graphical summary of SA-TRIM21 recognizing the antigen/antibody complex and performing signaling by biotin-HRP capture

Fig. 2

SA-TRIM21 functional assays. A The performance of streptavidin domain: SA-TRIM21 (0–500 ng) was coated into polystyrene plates followed by detection using Biotin-HRP (1:2500). B Detection of immobilized mouse serum. Mouse serum (50 ng) was coated onto polystyrene plates and detected using HRP-coupled anti-mouse IgG (Sigma, 1:10.000) or 500 ng SA-TRIM21 followed by incubation with biotinylated HRP (dil:1:2500). Control experiments were performed using mouse IgG and biotinylated HRP in the absence of SA-TRIM21. Experiments were performed in triplicates, and error bars represent standard deviation. A, B Wells were washed with PBST, 50 μL chromogenic substrate TMB was added to the wells, incubated for 10 min, followed by the addition of 50 μL H₂SO₄ to quench the reaction followed by OD₄₅₀ nm reading using an ELISA plate reader

Fig. 3

TNF-α/Infliximab detection on indirect ELISA using SA-TRIM21. SA-TRIM21 (0.25 μg/well) and biotinylated HRP (1:5000) were added to the plates for TNF α/Infliximab complex detection

Fig. 4

The graph represents the recognition of multispecies sera by the SA-TRIM21-HRP protein. Animal sera (50 ng) were coated onto polystyrene plates, blocked with 1%BSA, incubated with SA-TRIM21 (0.5 μg/well), and biotinylated HRP (1:2500). Control experiments were performed using immobilized BSA into wells. After washing with PBST (low salt), 50 μL chromogenic substrate TMB was added to the wells, incubated for 10 min, followed by the addition of 50 μL H2SO4 to quench the reaction followed by OD₄₅₀ nm reading using an ELISA plate reader. The data shows the ratio of serum/control OD ₄₅₀ reading. Experiments were performed in triplicates, and error bars represent standard deviation

Fig. 5

Performance of SA-TRIM21 for IgG detection on veterinary ELISA kits: Anti-Leishmania IgG detection in dog serum. Wells were coated with specific L. infantum antigens, followed by incubation with dog serum samples. Wells were washed followed by incubation with HRP coupled anti-IgG or SA-TRIM21. After washing, the chromogen TMB was added to the wells followed by incubation during 10 min. Reactions were quenched using Stop solution and absorbance was read at 450 nm

Fig. 6

Detection of anti-equine infectious anemia IgG on horse serum. Wells were coated with viral antigens, followed by incubation with horse serum samples. Wells were washed followed by incubation with HRP coupled anti-IgG or SA-TRIM21. After washing, the chromogen TMB was added to the wells followed by incubation during 10 min. Reactions were quenched using Stop solution and absorbance was read at 450 nm

Fig. 7

The figure depicts the alignment of IgG heavy chain Fc regions from different mammalian species. The residues in gray are conserved among all sequences. The residues marked in bold represent the Mouse IgG Fc hot spots (H433, N434, and H435) necessary for binding to TRIM21 identified in previous studies [6]

Fig. 8

Impact of imidazole elution on IgG binding. Direct ELISA assays using 50 ng immobilized human serum or 50 ng BSA, followed by SA-TRIM21-HRP (0.5 μg) or HRP-coupled Protein A/G incubation and washing with imidazole-containing buffers. Chromogenic substrate TMB was added to the wells, incubated for 10 min, followed by the addition of 50 μL H 2SO4 and OD450 reading. The data shows the ratio of serum/control OD450 reading. Experiments were performed in triplicates, and error bars represent standard deviation

Fig. 9

Western blotting detection of CYP1A from protein extracts of mullet fish microsomes. Lane 1, 250 ng of protein extract; lane 2, 125 ng; lane 3, 62.5 ng; lane 4 3.12 ng; lane 5 1.56 ng; lane 6 0.78 ng. Rabbit IgG anti CYP1A was used as the primary antibody at a dilution ratio of 1:7500 in TBS/1% casein. SA-TRIM21 at 0.25 μg/mL was added to the nitrocellulose membrane in TBS/1% casein for rabbit IgG recognition. Biotin HRP (1:2500) was added followed by detection using Luminol based reagent (ECL Scienco Biotech) and chemioluminescent detector