Mimtags: The use of phage display technology to produce novel protein-specific probes

Ahmed, Nayyar; Dhanapala, Pathum; Sadli, Nadia; Barrow, Colin J.; Suphioglu, Cenk

doi:10.1016/j.jim.2014.02.001

Cited by 3 publications

(6 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This was performed by using direct ELISA, in which the peptide was allowed to interact with the IL-4Rα in the absence of the cytokines to determine its avidity to the target antigen. Evidently, the differences in absorbance readings obtained showed that N1 biopep had specifically bound IL-4Rα, when compared to the control, which we have recently reported in a previous study [28]. A trend was seen with increasing concentration of the peptide, where higher absorbance readings were recorded on the spectrophotometer [28].…”

Section: Direct Elisasupporting

confidence: 67%

“…Evidently, the differences in absorbance readings obtained showed that N1 biopep had specifically bound IL-4Rα, when compared to the control, which we have recently reported in a previous study [28]. A trend was seen with increasing concentration of the peptide, where higher absorbance readings were recorded on the spectrophotometer [28]. This indicated that the streptavidin-HRP had indeed recognized the biotin on peptide N1, which in turn interacted with the IL-4Rα target antigen [28].…”

Section: Direct Elisasupporting

confidence: 66%

“…A trend was seen with increasing concentration of the peptide, where higher absorbance readings were recorded on the spectrophotometer [28]. This indicated that the streptavidin-HRP had indeed recognized the biotin on peptide N1, which in turn interacted with the IL-4Rα target antigen [28].…”

Section: Direct Elisamentioning

confidence: 87%

“…Amplification of 10 individual plaques from the fifth round of panning was subjected to amplification and sequencing by following the manufacturer's instructions and our established methods [28]. Sequencing was performed by the Micromon DNA Sequencing Facility at Monash University (Melbourne, Australia).…”

Section: Amplification and Sequencing Of Phage Clonesmentioning

confidence: 99%

“…Four rows of a 96-well microtiter plate were coated with 90 μl of the target IL-4Rα (10nM) protein and the M13 ELISA was performed according to manufacturer's instructions and our established methods [28]. The colour development on the plates was read by using Thermo Labsystems Multiscan plate reader, set at 492 nm of wavelength.…”

Section: M13 Binding Elisamentioning

confidence: 99%

See 4 more Smart Citations

Identification and Characterization of a Novel IL-4 Receptor α Chain (IL-4Rα) Antagonist to Inhibit IL-4 Signalling

Ahmed

Dhanapala

Suphioglu

2015

Cell Physiol Biochem

Self Cite

View full text Add to dashboard Cite

Background/Aims: In recent times, allergy has become a financial, physical and psychological burden to the society as a whole. In allergic cascades, cytokine IL-4 binds to IL-4 receptor (IL-4R), consequently producing allergen-specific IgE antibodies by B cells. In addition, among other functions, IL-4 is also responsible for B and T cell proliferation and differentiation. Hence, characterization of novel antagonists that inhibit IL-4 signalling forms the overall aim of this study. Methods: Phage display was used to screen a random 12-mer synthetic peptide library with a human IL-4Rα to identify peptide candidates. Once identified, the peptides were commercially synthesized and used for in vitro immunoassays. Results: We have successfully used phage display to identify M13 phage clones that demonstrated specific binding to IL-4Rα. The peptide N1 was synthesized for use in ELISA, demonstrating significant binding to IL-4Rα and inhibiting interaction with cytokine IL-4. Furthermore, the peptide was tested in a transfected HEK-Blue IL-4 reporter cell line model, which produces alkaline phosphatase (AP). QUANTI-Blue, a substrate, breaks down in the presence of AP producing a blue coloration. Using this colorimetric analysis, >50% inhibition of IL-4 signalling was achieved. Conclusion: We have successfully identified and characterised a synthetic peptide antagonist against IL-4Rα, which effectively inhibits IL-4 interaction with the IL-4Rα in vitro. Since IL-4 interaction with IL-4Rα is a common pathway for many allergies, a prophylactic treatment can be devised by inhibiting this interaction for future treatment of allergies.

show abstract

Section: Direct Elisasupporting

confidence: 67%

Section: Direct Elisasupporting

confidence: 66%

Section: Direct Elisamentioning

confidence: 87%

Section: Amplification and Sequencing Of Phage Clonesmentioning

confidence: 99%

Section: M13 Binding Elisamentioning

confidence: 99%

See 3 more Smart Citations

Identification and Characterization of a Novel IL-4 Receptor α Chain (IL-4Rα) Antagonist to Inhibit IL-4 Signalling

Ahmed

Dhanapala

Suphioglu

2015

Cell Physiol Biochem

Self Cite

View full text Add to dashboard Cite

show abstract

Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5

Hashimoto

Zhou

Hamauchi

et al. 2018

Sci Rep

View full text Add to dashboard Cite

The production of antibodies against the extracellular regions (ECR) of multispanning membrane proteins is notoriously difficult because of the low productivity and immunogenicity of membrane proteins due to their complex structure and highly conserved sequences among species. Here, we introduce a new method to generate ECR-binding antibodies utilizing engineered liposomal immunogen prepared using a wheat cell-free protein synthesis system. We used claudin-5 (CLDN-5) as the target antigen, which is a notoriously difficult to produce and poorly immunogenic membrane protein with two highly conserved extracellular loops. We drastically improved the productivity of CLDN-5 in the cell-free system after suppressing and normalizing mRNA GC content. To overcome its low immunogenicity, two engineered antigens were designed and synthesized as proteoliposomes: a human/mouse chimeric CLDN-5, and a CLDN-5-based artificial membrane protein consisting of symmetrically arranged ECRs. Intraperitoneal immunization of both engineered CLDN-5 ECR antigens induced ECR-binding antibodies in mice with a high success rate. We isolated five monoclonal antibodies that specifically recognized CLDN-5 ECR. Antibody clone 2B12 showed high affinity (<10 nM) and inhibited CLDN-5-containing tight junctions. These results demonstrate the effectiveness of the methods for monoclonal antibody development targeting difficult-to-produce membrane proteins such as CLDNs.

show abstract

Nothing to be Sneezed at: Th2 Cytokines as Novel Therapeutic Targets for the Production of Anti- Allergy Agents

2016

Frontiers in Clinical Drug Research - Anti Allergy Agents

View full text Add to dashboard Cite

Mimtags: The use of phage display technology to produce novel protein-specific probes

Cited by 3 publications

References 46 publications

Identification and Characterization of a Novel IL-4 Receptor α Chain (IL-4Rα) Antagonist to Inhibit IL-4 Signalling

Identification and Characterization of a Novel IL-4 Receptor α Chain (IL-4Rα) Antagonist to Inhibit IL-4 Signalling

Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5

Nothing to be Sneezed at: Th2 Cytokines as Novel Therapeutic Targets for the Production of Anti- Allergy Agents

Contact Info

Product

Resources

About