Adult-onset immunodeficiency syndrome (AOID) patients with autoantibodies (autoAbs) against interferon-gamma (IFN-γ) generally suffer from recurrent and recalcitrant disseminated non-tuberculous mycobacterial diseases. Since the early stages of AOID do not present specific symptoms, diagnosis and treatment of the condition are not practical. A simplified diagnostic method for differentiating AOID from other immunodeficiencies, such as HIV infection, was created. Anti-IFN-γ is generally identified using enzyme-linked immunosorbent assay (ELISA), which involves an instrument and a cumbersome process. Recombinant IFN-γ indirectly conjugated to colloidal gold was used in the modified immunochromatographic (IC) strips. The biotinylated-IFN-γ was incorporated with colloidal-gold-labeled 6HIS-maltose binding protein-monomeric streptavidin (6HISMBP-mSA) and absorbed at the conjugate pad. The efficacy of the IC strip upon applying an anti-IFN-γ autoAb cut-off ELISA titer of 2500, the sensitivity and specificity were 84% and 90.24%, respectively. When a cut-off ELISA titer of 500 was applied, the sensitivity and specificity were 73.52% and 100%, respectively.
Several anti-HIV scaffolds have been proposed as complementary treatments to highly active antiretroviral therapy. AnkGAG1D4, a designed ankyrin repeat protein, formerly demonstrated anti-HIV-1 replication by interfering with HIV-1 Gag polymerization. However, the improvement of the effectiveness was considered. Recently, the dimeric molecules of AnkGAG1D4 were accomplished in enhancing the binding activity against HIV-1 capsid (CAp24). In this study, the interaction of CAp24 against the dimer conformations was elucidated to elaborate the bifunctional property. The accessibility of the ankyrin binding domains was inspected by bio-layer interferometry. By inverting the second module of dimeric ankyrin (AnkGAG1D4NC-CN), the CAp24 interaction KD was significantly reduced. This reflects the capability of AnkGAG1D4NC-CN in simultaneously capturing CAp24. On the contrary, the binding activity of dimeric AnkGAG1D4NC-NC was indistinguishable from the monomeric AnkGAG1D4. The bifunctional property of AnkGAG1D4NC-CN was subsequently confirmed in the secondary reaction with additional p17p24. This data correlates with the MD simulation, which suggested the flexibility of the AnkGAG1D4NC-CN structure. The CAp24 capturing capacity was influenced by the distance of the AnkGAG1D4 binding domains to introduce the avidity mode of AnkGAG1D4NC-CN. Consequently, AnkGAG1D4NC-CN showed superior potency in interfering with HIV-1 NL4-3 WT and HIV-1 NL4-3 MIRCAI201V replication than AnkGAG1D4NC-NC and an affinity improved AnkGAG1D4-S45Y.
Protein and DNA interactions are crucial for many cellular processes. Biolayer Interferometry (BLI) is a label-free technology for determining kinetic biomolecular interactions with high accuracy results. In the present study, we determined the kinetic binding of a zinc finger scaffold, 2LTRZFP, which formerly constructed the interfering effect on HIV-1 integration process using BLI. The competitive Enzyme-linked immunosorbent assay (ELISA) was used to initially show the specific binding of 2LTRZFP. The percentages of inhibition were 62% and 22% in double-stranded 2LTR (ds2LTR) and irrelevant DNA (dsNeg), respectively. Consequently, the binding affinity of 2LTRZFP against ds2LTR target analyzed by BLI was 40 nM, which is stronger than the interaction of HIV-1 integrase (IN) enzyme to the 2LTR circle junction. Additionally, the 2LTRZFP did not interact with the genomic DNA extracted from SupT1 cell line. This result indicates that 2LTRZFP did not exhibit off-target effects against human genome. The knowledge obtained from this study supports the prospect of using 2LTRZFP in HIV-1 gene therapy.
Assembly and budding in the late-stage of human immunodeficiency virus type 1 (HIV-1) production relies on the polymerization of Gag protein at the inner leaflet of the plasma membrane. We previously generated an ankyrin repeat protein (Ank1D4) that specifically interacts with the CAp24 protein. This study aimed to improve the binding activity of Ank1D4 by generating two platforms for the Ank1D4 dimer. The design of these constructs featured a distinct orientation of monomeric Ank1D4 connected by a linker peptide (G S) . The binding surfaces in either dimer generated from the C-terminus of the Ank1D4 monomer linked with the N-terminus of another monomer (Ank1D4 ) or its inverted form (Ank1D4 ), similar to monomeric Ank1D4. The interaction of Ank1D4 with CAp24 from capture ELISA was significantly greater than that of Ank1D4 and the parental Ank1D4. The bifunctional characteristic of Ank1D4 was further demonstrated using sandwich ELISA. The binding kinetics of these ankyrins were evaluated using bio-layer interferometry analysis. The K of Ank1D4 , Ank1D4 and monomeric Ank1D4 was 3.5 nM, 53.7 nM, and 126.2 nM, respectively. The dynamics of the interdomain linker and the behavior of ankyrin dimers were investigated in silico. Upon the binding distance calculation from the candidate structures, the achievement in obtaining double active sites is more possible in Ank1D4 . The CD spectroscopic data indicated that secondary structure of dimer forms resemble Ank1D4 monomer α-helical content. This finding confers the strategy to generate dimer from rigid scaffold for acquiring the binding avidity.
Background: Ankyrin (Ank) is a protein family with crucial roles in retaining normal cellular physiology. In addition, ankyrin offers the potential as a non-antibody binder against various biomolecules. The designed ankyrin repeat protein (DARPin) selected from phage display libraries is useful for molecular detection and therapy. Monoclonal antibodies (mAbs) specific to the common epitope of DARPin are required to detect protein-protein interaction. Objectives: This study aimed to establish mAbs against common antigenic determinant of ankyrins for further application in immunological techniques. Materials and methods: Ank1D4 monomer and dimer were generated in the Escherichia coli expression system for immunogen preparation and validation of established mAbs. The binding activity of anti-Ank mAb obtained from different hybridoma clones was characterized using Ank1D4 by indirect ELISA. Candidate anti-Ank mAbs were validated for their cross-reactivity against irrelevant ankyrin (Ank2D3). The binding kinetic of mAbs from three candidate hybridoma clones (Ank-54, Ank-59, and Ank-94) was evaluated using bio-layer interferometry (BLI). The highest affinity clone (Ank-94 mAb ) was further validated for its specificity against Ank1D4 and dimeric Ank1D4 using indirect ELISA. The interaction of three anti-Ank mAbs and ankyrins was compared by western immunoblotting analysis. The specificity of Ank-94 mAb was determined using a closely related scaffold, i.e., alpha-helicoidal HEAT-like repeat protein scaffold (αRep) by indirect ELISA. Ankyrins were detected by sandwich ELISA using Ank-94 mAb. Results: The culture supernatant from hybridoma clones were characterized for their anti-ankyrin binding properties. Using indirect ELISA, three clones exhibited positive reactivity against the immunized ankyrin antigen (Ank1D4). The interactive epitope was found to rely on common antigenic determinants found in Ank1D4, dimeric Ank1D4, and an irrelevant ankyrin, Ank2D3. The immunoblotting results suggest that all mAbs interact with the sequential epitope of ankyrins. The cross-reactivity of Ank-94 mAb was not observed with αRep. Ank-94 mAb was selected for further purification and evaluation of binding properties due to its highest degree of binding affinity against Ank1D4. Conclusion: The establishment of a novel Ank-94 mAb could be a valuable research tool in tracing the target of DARPins or developing immunoassays. Ank-94 mAb is superior over formerly produced Ank mAbs since it recognizes a common epitope on DARPins and relies on sequential epitope. Ank-94 mAb has no cross-reactivity with another scaffold, αRep.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.