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Interleukin 23 and autoimmune diseases: current and possible future therapies

Abdo AIK, Tye GJ

Inflamm Res, 2020 May;69(5):463-480.
PMID: 32215665 DOI: 10.1007/s00011-020-01339-9

PURPOSE: IL-23 is a central proinflammatory cytokine with a wide range of influence over immune response. It is implicated in several autoimmune diseases due to the infinite inflammatory loops it can create through the positive feedbacks of both IL-17 and IL-22 arms. This made IL-23 a key target of autoimmune disorders therapy, which indeed was proven to inhibit inflammation and ameliorate diseases. Current autoimmune treatments targeting IL-23 are either by preventing IL-23 ligation to its receptor (IL-23R) via antibodies or inhibiting IL-23 signaling by signaling downstream mediators' inhibitors, with each approach having its own pros and cons.
METHODS: Literature review was done to further understand the biology of IL-23 and current therapies.
RESULTS: In this review, we discuss the biological features of IL-23 and its role in the pathogenesis of autoimmune diseases including psoriasis, rheumatoid arthritis and inflammatory bowel diseases. Advantages, limitations and side effects of each concept will be reviewed, suggesting several advanced IL-23-based bio-techniques to generate new and possible future therapies to overcome current treatments problems.
Minireview: applied structural bioinformatics in proteomics

Choong YS, Tye GJ, Lim TS

Protein J, 2013 Oct;32(7):505-11.
PMID: 24096348 DOI: 10.1007/s10930-013-9514-1

The limited sequence similarity of protein sequences with known structures has led to an indispensable need for computational technology to predict their structures. Structural bioinformatics (SB) has become integral in elucidating the sequence-structure-function relationship of a protein. This report focuses on the applications of SB within the context of protein engineering including its limitation and future challenges.
Enhancement of immune response against Mycobacterium tuberculosis HspX antigen by incorporation of combined molecular adjuvant (CASAC)

Lew MH, Norazmi MN, Tye GJ

Mol Immunol, 2020 Jan;117:54-64.
PMID: 31739193 DOI: 10.1016/j.molimm.2019.10.023

Tuberculosis (TB) is one of the deadliest human diseases worldwide caused by mycobacterial infection in the lung. Bacillus Calmette-Guerin (BCG) vaccine protects against disseminated TB in children, but its effectiveness is still questionable due to highly variable protections in adolescence and elderly individuals. Targeting the latency M.tb antigen is a recent therapeutic approach to eradicate dormant pathogen that could possibly lead to disease activation. In this study, we aimed to potentiate immune responses elicited against 16 kDa α-crystalline (HspX) tuberculosis latency antigen by incorporation of Combined Adjuvant for Synergistic Activation of Cellular immunity (CASAC). Histidine-tagged recombinant HspX protein was initially produced in Escherichia coli and purified using Ni-NTA chromatography. To evaluate its adjuvanticity, C57BL/6 mice (n = 5) were initially primed and intradermally immunised in 2-weeks interval for 4 rounds with recombinant HspX, formulated with and without CASAC. Humoral and cell-mediated immune responses elicited against HspX antigen were evaluated using ELISA and Flow Cytometry. Our findings showed that CASAC improved humoral immunity with increased antigen-specific IgG1 and IgG2a antibody response. Stronger CD8+ and Th1-driven immunity was induced by CASAC formulation as supported by elevated level of IFN-γ, TNF-α, IL-12 and IL-17A; and with low IL-10 secretion. Interestingly, adjuvanted HspX vaccine triggered a higher percentage of effector memory T-cell population than those immunised with unadjuvanted vaccine. In conclusion, CASAC adjuvant has great potential to enhance immunogenicity elicited against HspX antigen, which could be an alternative regimen to improve the efficacy of future therapeutic vaccine against Mycobacterium tuberculosis.
CRISPR-Cas9 Genome Editing Tool for the Production of Industrial Biopharmaceuticals

Khan AH, Tye GJ, Noordin R

Mol Biotechnol, 2020 Sep;62(9):401-411.
PMID: 32749657 DOI: 10.1007/s12033-020-00265-9

A broad range of cell lines with characteristic features are used as bio-factories to produce recombinant proteins for basic research and therapeutic purposes. Genetic engineering strategies have been used to manipulate the genome of mammalian cells, insects, and yeasts for heterologous expression. One reason is that the glycosylation pattern of the expression hosts differs somehow from mammalian cells, which may cause immunogenic reactions upon administration in humans. CRISPR-Cas9 is a simple, efficient, and versatile genome engineering tool that can be programmed to precisely make double-stranded breaks at the desired loci. Compared to the classical genome editing methods, a CRISPR-Cas9 system is an ideal tool, providing the opportunity to integrate or delete genes from the target organisms. Besides broadened applications, limited studies have used CRISPR-Cas9 for editing the endogenous pathways in expression systems for biopharmaceutical applications. In the present review, we discuss the use of CRISPR-Cas9 in expression systems to improve host cell lines, increase product yield, and humanize glycosylation pathways by targeting intrinsic genes.
DNA-templated silver nanocluster for live-intracellular FOXP3 detection

Lee SY, Fazlina N, Tye GJ

Anal Biochem, 2019 09 15;581:113352.
PMID: 31260647 DOI: 10.1016/j.ab.2019.113352

DNA-templated silver nanocluster (AgNC), a new promising fluorescence probe has gained importance in biosensing and bioimaging in recent years. We employed a label-free AgNC to detect an intracellular transcription factor known as forkhead box p3 (FOXP3), which is the master regulator of regulatory T cells (Tregs) suppressive function. We developed an optimized method for the detection of messenger ribonucleic acid (mRNA) of FOXP3 by hybridizing AgNC and G-rich to the target FOXP3 mRNA of a MCF-7 cells. MCF-7 cells are chosen as a model as it readily expresses FOXP3. The hybridized samples were examined with UV illuminator and further verified with fluorescence spectroscopy, fluorescence microscope and flow cytometry. The successful hybridization of a three-way junction with AgNC, G-rich and mRNA FOXP3 target generated an improved fluorescence intensity with a spectral shift. We have successfully delivered the green fluorescing AgNC and G-rich into MCF-7 cells, producing a shift to red fluorescing cells corroborated by flow cytometry results. In summary, our approach enables the detection of intracellular FOXP3 nucleic acid and holds considerable potential in establishing a non-lethal intracellular detection system which would be crucial for the isolation of regulatory T-cells (Tregs) when combined with other cell surface markers.
Selection and evaluation of single domain antibody against p19 subunit of IL-23 by phage display for potential use as an autoinflammatory therapeutic

Abdo AIK, Nordin F, Tye GJ

Int Immunopharmacol, 2024 Aug 20;137:112371.
PMID: 38852516 DOI: 10.1016/j.intimp.2024.112371

IL-23 is a double-subunit cytokine that plays an important role in shaping the immune response. IL-23 was found to be associated with several autoinflammatory diseases by generating sustained inflammatory loops that lead to tissue damage. Antibody neutralization of IL-23 was proven to be effective in ameliorating associated diseases. However, antibodies as large proteins have limited tissue penetration and tend to elicit anti-drug antibodies. Additionally, anti-IL-23 antibodies target only one subunit of IL-23 leaving the other one unneutralized. Here, we attempted to isolate a recycling single domain antibody by phage display. One of IL-23 subunits, p19, was expressed in E. coli fused to Gamillus protein to stabilize the α-helix-only p19. To remove Gamillus binders, two biopanning methods were investigated, first, preselection with Gamillus and second, challenge with IL-23 then on the subsequent round challenge with p19-Gam. The isolation of calcium-dependent and pH-dependent recycling binders was performed with EDTA and citrate buffers respectively. Both methods of panning failed to isolate high-affinity and specific p19 recycling binders, while from the second panning method, a high affinity and specific p19 standard binder, namely H11, was successfully isolated. H11 significantly inhibited the gene expression of IL-17 and IL-22 in IL-23-challenged PBMCs indicating H11 specificity and neutralizing ability for IL-23. The new binder due to its small size can overcome antibodies limitations, also, it can be further engineered in the future for antigen clearance such as fusing it to cell penetrating peptides, granting H11 the ability to clear excess IL-23 and enhancing its potential therapeutic effect.
Fulltext Applications of Recombinant Monoclonal Antibodies against Filarial Antigen Proteins

Rahumatullah A, Yunus MH, Tye GJ, Noordin R

Am J Trop Med Hyg, 2020 03;102(3):578-581.
PMID: 31933469 DOI: 10.4269/ajtmh.19-0777

This study investigated the applications of recombinant monoclonal antibodies (rmAbs) produced against two recombinant filarial proteins of diagnostic value. Ab5B and Ab3A were produced against recombinant BmSXP, and Ab4 and Ab4-fragment crystallizable (Fc) against recombinant BmR1. Ab5B and Ab4-Fc were found to be useful as quality control (QC) reagents for two commercial rapid test kits, such as Brugia RapidTM and BLF Rapid® (Reszon Diagnostics International Sdn. Bhd., 47600 Subang Jaya, Selangor, Malaysia), respectively. The two rmAbs reacted positively with the corresponding recombinant proteins lined on the nitrocellulose strips of the cassette tests, thus may replace or reduce the need for patient serum samples as positive controls for QC of the commercial kits. They were also successfully conjugated to gold nanoparticles and reacted positively with the test lines containing the corresponding recombinant proteins when directly applied to the cassette tests. The gold-conjugated reagents can be used to confirm the antigenicity of test lines after the storage of the rapid tests for a prolonged period or under unfavorable conditions. Furthermore, Ab5B and Ab3A were shown to be able to capture the target recombinant proteins through immunoaffinity purification, enabling their use for applications that need very highly purified proteins. In conclusion, this study demonstrated several potential uses of rmAb proteins produced against recombinant filarial proteins.
Immune Stimulation of RAP domain binding protein (rTgRA15) from Toxoplasma gondii

Lew MH, Noordin R, Monsur Alam Khan M, Tye GJ

Pathog Glob Health, 2018 10;112(7):387-394.
PMID: 30332344 DOI: 10.1080/20477724.2018.1536854

Toxoplasmosis, a parasitic disease in human and animals, is caused by Toxoplasma gondii. Our previous study has led to the discovery of a novel RAP domain binding protein antigen (TgRA15), an apparent in-vivo induced antigen recognised by antibodies in acutely infected individuals. This study is aimed to evaluate the humoral response and cytokine release elicited by recombinant TgRA15 protein in C57BL/6 mice, demonstrating its potential as a candidate vaccine for Toxoplasma gondii infection. In this study, the recombinant TgRA15 protein was expressed in Escherichia coli, purified and refolded into soluble form. C57BL/6 mice were immunised intradermally with the antigen and CASAC (Combined Adjuvant for Synergistic Activation of Cellular immunity). Antigen-specific humoral and cell-mediated responses were evaluated using Western blot and ELISA. The total IgG, IgG1 and IgG2a antibodies specific to the antigen were significantly increased in treatment group compare to control group. A higher level of interferon gamma (IFN-γ) secretion was demonstrated in the mice group receiving booster doses of rTgRA15 protein, suggesting a potential Th1-mediated response. In conclusion, the rTgRA15 protein has the potential to generate specific antibody response and elicit cellular response, thus potentially serve as a vaccine candidate against T. gondii infection.
The potential applications of T cell receptor (TCR)-like antibody in cervical cancer immunotherapy

Dass SA, Selva Rajan R, Tye GJ, Balakrishnan V

Hum Vaccin Immunother, 2021 09 02;17(9):2981-2994.
PMID: 33989511 DOI: 10.1080/21645515.2021.1913960

Cervical cancer is ranked as the fourth most common cancer in women worldwide. Monoclonal antibody has created a new dimension in the immunotherapy of many diseases, including cervical cancer. The antibody's ability to target various aspects of cervical cancer (oncoviruses, oncoproteins, and signaling pathways) delivers a promising future for efficient immunotherapy. Besides, technologies such as hybridoma and phage display provide a fundamental platform for monoclonal antibody generation and create the opportunity to generate novel antibody classes including, T cell receptor (TCR)-like antibody. In this review, the current immunotherapy strategies for cervical cancer are presented. We have also proposed a novel concept of T cell receptor (TCR)-like antibody and its potential applications for enhancing cervical cancer therapeutics. Finally, the possible challenges in TCR-like antibody application for cervical cancer therapeutics have been addressed, and strategies to overcome the challenges have been highlighted to maximize the therapeutic benefits.
Soil-transmitted helminthic vaccines: Where are we now?

Wong MTJ, Anuar NS, Noordin R, Tye GJ

Acta Trop, 2023 Mar;239:106796.
PMID: 36586174 DOI: 10.1016/j.actatropica.2022.106796

It has been tested and proven that vaccination is still the best strategy to combat infectious diseases. However, to date, there are still no vaccines against human soil-transmitted helminthic diseases, despite their high prevalence globally, particularly in developing countries and rural areas with tropical climates and poor sanitation. The development of vaccines against helminths is riddled with obstacles. Helminths have a complex life cycle, multiple stages within the same host with stage-specific antigen expression, and the ability to regulate host immune reactions to evade the immune response. These elements contribute to the main challenge of helminthic vaccines: the identification of effective vaccine candidates. Therefore, this article reviews the current progress and potential future direction of soil-transmitted helminthic vaccines, particularly against Trichuris trichiura, Ascaris lumbricoides, Strongyloides stercoralis, Necator americanus and Ancylostoma duodenale. The study design employed was a systematic review, using qualitative meta-summary synthesis. Preclinical studies and clinical trials on the development of protein subunit vaccines against the five soil-transmitted helminths were searched on PubMed and Scopus. Effectiveness was indicated by a reduction in worm burden or larval output, an increase in specific IgG levels, or an increase in cytokine production. Our findings show that only the hookworm vaccine against N. americanus is in the clinical trial phase, while the rest is still in exploratory research and pre-clinical development phase.
Fulltext Expression of mammalian proteins for diagnostics and therapeutics: a review

Mark JKK, Lim CSY, Nordin F, Tye GJ

Mol Biol Rep, 2022 Nov;49(11):10593-10608.
PMID: 35674877 DOI: 10.1007/s11033-022-07651-3

BACKGROUND: Antibodies have proven to be remarkably successful for biomedical applications. They play important roles in epidemiology and medicine from diagnostics of diseases to therapeutics, treating diseases from incessant chronic diseases such as rheumatology to pandemic outbreaks. With no end in sight for the demand for antibody products, optimizations and new techniques must be expanded to accommodate this.
METHODS AND RESULTS: This review discusses optimizations and techniques for antibody production through choice of discovery platforms, expression systems, cell culture mediums, and other strategies to increase expression yield. Each system has its own merits and demerits, and the strategy chosen is critical in addressing various biological aspects.
CONCLUSIONS: There is still insufficient evidence to validate the efficacy of some of these techniques, and further research is needed to consolidate these industrial production systems. There is no doubt that more strategies, systems, and pipelines will contribute to enhance biopharmaceutical production.
Generation of IgG antibodies against Strongyloides stercoralis in mice via immunization with recombinant antigens A133 and Ss-IR

Wong MTJ, Anuar NS, Noordin R, Tye GJ

Acta Trop, 2024 Mar;251:107122.
PMID: 38246399 DOI: 10.1016/j.actatropica.2024.107122

Strongyloidiasis, caused by the nematode Strongyloides stercoralis, remains a threat to global public health, and a vaccine would be useful to control the disease, especially in developing countries. This study aimed to evaluate the efficacy of recombinant proteins, A133 and Ss-IR, as potential vaccine candidates against strongyloidiasis by investigating the humoral and cellular immune responses in immunized mice. Respective antigens were adjuvanted with Complete Freund's Adjuvant (prime) and Incomplete Freund's Adjuvant (boost) and administered intraperitoneally (prime) and subcutaneously (boost) to female BALB/c mice. For antigen-only doses, only antigens were injected without adjuvants. Altogether, 1 prime dose, 4 booster doses, and 2 antigen-only doses were administered successively. ELISAs were conducted to assess the antibody responses, along with flow cytometry and cytokine ELISA to elucidate the cellular immune responses. Results showed that A133 and Ss-IR induced the production of IgG1 and IgG2a, with A133 generating more robust IgG2a responses than Ss-IR. Flow cytometry findings indicated that effector CD8+T-cells and memory B-cells activity were upregulated significantly for A133 only, whereas cytokine ELISA demonstrated that a Th1/Th2/Th17 mixed cell responses were triggered upon vaccination with either antigen. This preliminary study illustrated the good potential of recombinant A133 and Ss-IR as vaccine candidates against S. stercoralis. It provided information on the probable immune mechanism involved in host defence and the elicitation of protection against S. stercoralis.
Fulltext General overview on structure prediction of twilight-zone proteins

Khor BY, Tye GJ, Lim TS, Choong YS

Theor Biol Med Model, 2015;12:15.
PMID: 26338054 DOI: 10.1186/s12976-015-0014-1

Protein structure prediction from amino acid sequence has been one of the most challenging aspects in computational structural biology despite significant progress in recent years showed by critical assessment of protein structure prediction (CASP) experiments. When experimentally determined structures are unavailable, the predictive structures may serve as starting points to study a protein. If the target protein consists of homologous region, high-resolution (typically <1.5 Å) model can be built via comparative modelling. However, when confronted with low sequence similarity of the target protein (also known as twilight-zone protein, sequence identity with available templates is less than 30%), the protein structure prediction has to be initiated from scratch. Traditionally, twilight-zone proteins can be predicted via threading or ab initio method. Based on the current trend, combination of different methods brings an improved success in the prediction of twilight-zone proteins. In this mini review, the methods, progresses and challenges for the prediction of twilight-zone proteins were discussed.
Identification of significant hub genes and pathways associated with metastatic breast cancer and tolerogenic dendritic cell via bioinformatics analysis

Ching KWC, Mokhtar NF, Tye GJ

Comput Biol Med, 2024 Nov 15;184:109396.
PMID: 39549529 DOI: 10.1016/j.compbiomed.2024.109396

Metastatic breast cancer (MBC) is an advanced-stage breast cancer associated with more than 90 % of cancer-related deaths. Immunosuppressive properties of tolerogenic dendritic cells (tolDCs) in tumour immune microenvironment (TIME) may be a risk factor for the rapid progression to MBC. However, the exact connections between the two are unknown. The aim of the current study is to uncover gene signatures and key pathways associated with MBC and tolDCs via an integrated bioinformatics approach. Gene expression profiles of MBC and tolDCs were retrieved from Gene Expression Omnibus (GEO) to identify common differentially expressed genes (DEGs). From DGE analysis, 529 upregulated common DEGs and 367 downregulated common DEGs had been identified. In enrichment analysis, common DEGs enriched in GO terms of defense response to virus and KEGG pathway of transcriptional misregulation in cancer were reported to be significantly associated with MBC and tolDCs. From the constructed PPI networks, 23 hub genes were identified, although only 5 genes were significant; 3 upregulated (ISG15, OAS2 and RSAD2) and 2 downregulated (eEF2 and PPARG) as they were found to be significantly correlated and had the same expression trend as predicted in validation analysis of overall survival (OS) analysis, expression levels, immune infiltration analysis and immunohistochemistry (IHC) analysis. These 5 hub genes can now be exploited in developing novel therapeutic interventions and as diagnostic biomarkers for enhancing the clinical outcomes of MBC patients.
Principles and application of antibody libraries for infectious diseases

Lim BN, Tye GJ, Choong YS, Ong EB, Ismail A, Lim TS

Biotechnol Lett, 2014 Dec;36(12):2381-92.
PMID: 25214212 DOI: 10.1007/s10529-014-1635-x

Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombinant antibody technology allows the generation of fully human antibodies. Phage display is the gold standard for the production of human antibodies in vitro. To generate monoclonal antibodies by phage display, the generation of antibody libraries is crucial. Antibody libraries are classified according to the source where the antibody gene sequences were obtained. The most useful library for infectious diseases is the immunized library. Immunized libraries would allow better and selective enrichment of antibodies against disease antigens. The antibodies generated from these libraries can be translated for both diagnostic and therapeutic applications. This review focuses on the generation of immunized antibody libraries and the potential applications of the antibodies derived from these libraries.
Fulltext Assembly and stability of Salmonella enterica ser. Typhi TolC protein in POPE and DMPE

Leong SW, Lim TS, Tye GJ, Ismail A, Aziah I, Choong YS

J Biol Phys, 2014 Sep;40(4):387-400.
PMID: 25011632 DOI: 10.1007/s10867-014-9357-9

In this work we assessed the suitability of two different lipid membranes for the simulation of a TolC protein from Salmonella enterica serovar Typhi. The TolC protein family is found in many pathogenic Gram-negative bacteria including Vibrio cholera and Pseudomonas aeruginosa and acts as an outer membrane channel for expulsion of drug and toxin from the cell. In S. typhi, the causative agent for typhoid fever, the TolC outer membrane protein is an antigen for the pathogen. The lipid environment is an important modulator of membrane protein structure and function. We evaluated the conformation of the TolC protein in the presence of DMPE and POPE bilayers using molecular dynamics simulation. The S. typhi TolC protein exhibited similar conformational dynamics to TolC and its homologues. Conformational flexibility of the protein is seen in the C-terminal, extracellular loops, and α-helical region. Despite differences in the two lipids, significant similarities in the motion of the protein in POPE and DMPE were observed, including the rotational motion of the C-terminal residues and the partially open extracellular loops. However, analysis of the trajectories demonstrated effects of hydrophobic matching of the TolC protein in the membrane, particularly in the lengthening of the lipids and subtle movements of the protein's β-barrel towards the lower leaflet in DMPE. The study exhibited the use of molecular dynamics simulation in revealing the differential effect of membrane proteins and lipids on each other. In this study, POPE is potentially a more suitable model for future simulation of the S. typhi TolC protein.
DNA fluorescence shift sensor: a rapid method for the detection of DNA hybridization using silver nanoclusters

Lee SY, Hairul Bahara NH, Choong YS, Lim TS, Tye GJ

J Colloid Interface Sci, 2014 Nov 01;433:183-188.
PMID: 25129336 DOI: 10.1016/j.jcis.2014.07.033

DNA-templated silver nanoclusters (AgNC) are a class of subnanometer sized fluorophores with good photostability and brightness. It has been applied as a diagnostic tool mainly for deoxyribonucleic acid (DNA) detection. Integration of DNA oligomers to generate AgNCs is interesting as varying DNA sequences can result in different fluorescence spectra. This allows a simple fluorescence shifting effect to occur upon DNA hybridization with the hybridization efficiency being a pronominal factor for successful shifting. The ability to shift the fluorescence spectra as a result of hybridization overcomes the issue of background intensities in most fluorescent based assays. Here we describe an optimized method for the detection of single-stranded and double-stranded synthetic forkhead box P3 (FOXP3) target by hybridization with the DNA fluorescence shift sensor. The system forms a three-way junction by successful hybridization of AgNC, G-rich strand (G-rich) to the target DNA, which generated a shift in fluorescence spectra with a marked increase in fluorescence intensity. The DNA fluorescence shift sensor presents a rapid and specific alternative to conventional DNA detection.
Fulltext Development of an antigen-DNAzyme based probe for a direct antibody-antigen assay using the intrinsic DNAzyme activity of a daunomycin aptamer

Omar N, Loh Q, Tye GJ, Choong YS, Noordin R, Glökler J, et al.

Sensors (Basel), 2013;14(1):346-55.
PMID: 24379042 DOI: 10.3390/s140100346

G-Quadruplex (G-4) structures are formed when G-rich DNA sequences fold into intra- or intermolecular four-stranded structures in the presence of metal ions. G-4-hemin complexes are often effective peroxidase-mimicking DNAzymes that are applied in many detection systems. This work reports the application of a G-rich daunomycin-specific aptamer for the development of an antibody-antigen detection assay. We investigated the ability of the daunomycin aptamer to efficiently catalyze the hemin-dependent peroxidase activity independent of daunomycin. A reporter probe consisting of biotinylated antigen and daunomycin aptamer coupled to streptavidin gold nanoparticles was successfully used to generate a colorimetric readout. In conclusion, the daunomycin aptamer can function as a robust alternative DNAzyme for the development of colorimetric assays.
Phage display antibodies for diagnostic applications

Hairul Bahara NH, Tye GJ, Choong YS, Ong EB, Ismail A, Lim TS

Biologicals, 2013 Jul;41(4):209-16.
PMID: 23647952 DOI: 10.1016/j.biologicals.2013.04.001

With major developments in molecular biology, numerous display technologies have been successfully introduced for recombinant antibody production. Even so, phage display still remains the gold standard for recombinant antibody production. Its success is mainly attributed to the robust nature of phage particles allowing for automation and adaptation to modifications. The generation of monospecific binders provides a vital tool for diagnostics at a lower cost and higher efficiency. The flexibility to modify recombinant antibodies allows great applicability to various platforms for use. This review presents phage display technology, application and modifications of recombinant antibodies for diagnostics.
Application of streptavidin mass spectrometric immunoassay tips for immunoaffinity based antibody phage display panning

Chin CF, Ler LW, Choong YS, Ong EB, Ismail A, Tye GJ, et al.

J Microbiol Methods, 2016 Jan;120:6-14.
PMID: 26581498 DOI: 10.1016/j.mimet.2015.11.007

Antibody phage display panning involves the enrichment of antibodies against specific targets by affinity. In recent years, several new methods for panning have been introduced to accommodate the growing application of antibody phage display. The present work is concerned with the application of streptavidin mass spectrometry immunoassay (MSIA™) Disposable Automation Research Tips (D.A.R.T's®) for antibody phage display. The system was initially designed to isolate antigens by affinity selection for mass spectrometry analysis. The streptavidin MSIA™ D.A.R.T's® system allows for easy attachment of biotinylated target antigens on the solid surface for presentation to the phage library. As proof-of-concept, a domain antibody library was passed through the tips attached with the Hemolysin E antigen. After binding and washing, the bound phages were eluted via standard acid dissociation and the phages were rescued for subsequent panning rounds. Polyclonal enrichment was observed for three rounds of panning with five monoclonal domain antibodies identified. The proposed method allows for a convenient, rapid and semi-automated alternative to conventional antibody panning strategies.

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