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Fulltext scFv antibody: principles and clinical application

Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M

Clin. Dev. Immunol., 2012;2012:980250.
PMID: 22474489 DOI: 10.1155/2012/980250

To date, generation of single-chain fragment variable (scFv) has become an established technique used to produce a completely functional antigen-binding fragment in bacterial systems. The advances in antibody engineering have now facilitated a more efficient and generally applicable method to produce Fv fragments. Basically, scFv antibodies produced from phage display can be genetically fused to the marker proteins, such as fluorescent proteins or alkaline phosphatase. These bifunctional proteins having both antigen-binding capacity and marker activity can be obtained from transformed bacteria and used for one-step immunodetection of biological agents. Alternatively, antibody fragments could also be applied in the construction of immunotoxins, therapeutic gene delivery, and anticancer intrabodies for therapeutic purposes. This paper provides an overview of the current studies on the principle, generation, and application of scFv. The potential of scFv in breast cancer research is also discussed in this paper.

Matched MeSH terms: Peptide Library
Use of superparamagnetic beads for the isolation of a peptide with specificity to cymbidium mosaic virus

Ooi DJ, Dzulkurnain A, Othman RY, Lim SH, Harikrishna JA

J Virol Methods, 2006 Sep;136(1-2):160-5.
PMID: 16781785

A modified method for the rapid isolation of specific ligands to whole virus particles is described. Biopanning against cymbidium mosaic virus was carried out with a commercial 12-mer random peptide display library. A solution phase panning method was devised using streptavidin-coated superparamagnetic beads. The solution based panning method was more efficient than conventional immobilized target panning when using whole viral particles of cymbidium mosaic virus as a target. Enzyme-linked immunosorbent assay of cymbidium mosaic virus-binding peptides isolated from the library identified seven peptides with affinity for cymbidium mosaic virus and one peptide which was specific to cymbidium mosaic virus and had no significant binding to odontoglossum ringspot virus. This method should have broad application for the screening of whole viral particles towards the rapid development of diagnostic reagents without the requirement for cloning and expression of single antigens.

Matched MeSH terms: Peptide Library
Fulltext Targeted Delivery of Cell Penetrating Peptide Virus-like Nanoparticles to Skin Cancer Cells

Gan BK, Yong CY, Ho KL, Omar AR, Alitheen NB, Tan WS

Sci Rep, 2018 05 31;8(1):8499.
PMID: 29855618 DOI: 10.1038/s41598-018-26749-y

Skin cancer or cutaneous carcinoma, is a pre-eminent global public health problem with no signs of plateauing in its incidence. As the most common treatments for skin cancer, surgical resection inevitably damages a patient's appearance, and chemotherapy has many side effects. Thus, the main aim of this study was to screen for a cell penetrating peptide (CPP) for the development of a targeting vector for skin cancer. In this study, we identified a CPP with the sequence NRPDSAQFWLHH from a phage displayed peptide library. This CPP targeted the human squamous carcinoma A431 cells through an interaction with the epidermal growth factor receptor (EGFr). Methyl-β-cyclodextrin (MβCD) and chlorpromazine hydrochloride (CPZ) inhibited the internalisation of the CPP into the A431 cells, suggesting the peptide entered the cells via clathrin-dependent endocytosis. The CPP displayed on hepatitis B virus-like nanoparticles (VLNPs) via the nanoglue successfully delivered the nanoparticles into A431 cells. The present study demonstrated that the novel CPP can serve as a ligand to target and deliver VLNPs into skin cancer cells.

Matched MeSH terms: Peptide Library
Solution structure and in silico binding of a cyclic peptide with hepatitis B surface antigen

Muhamad A, Ho KL, Rahman MB, Uhrín D, Tan WS

Chem Biol Drug Des, 2013 Jun;81(6):784-94.
PMID: 23405984 DOI: 10.1111/cbdd.12120

A specific ligand targeting the immunodominant region of hepatitis B virus is desired in neutralizing the infectivity of the virus. In a previous study, a disulfide constrained cyclic peptide cyclo S(1) ,S(9) Cys-Glu-Thr-Gly-Ala-Lys-Pro-His-Cys (S(1) , S(9) -cyclo-CETGAKPHC) was isolated from a phage displayed cyclic peptide library using an affinity selection method against hepatitis B surface antigen. The cyclic peptide binds tightly to hepatitis B surface antigen with a relative dissociation constant (KD (rel) ) of 2.9 nm. The binding site of the peptide was located at the immunodominant region on hepatitis B surface antigen. Consequently, this study was aimed to elucidate the structure of the cyclic peptide and its interaction with hepatitis B surface antigen in silico. The solution structure of this cyclic peptide was solved using (1) H, (13) C, and (15) N NMR spectroscopy and molecular dynamics simulations with NMR-derived distance and torsion angle restraints. The cyclic peptide adopted two distinct conformations due to the isomerization of the Pro residue with one structured region in the ETGA sequence. Docking studies of the peptide ensemble with a model structure of hepatitis B surface antigen revealed that the cyclic peptide can potentially be developed as a therapeutic drug that inhibits the virus-host interactions.

Matched MeSH terms: Peptide Library
Selection of high affinity ligands to hepatitis B core antigen from a phage-displayed cyclic peptide library

Ho KL, Yusoff K, Seow HF, Tan WS

J Med Virol, 2003 Jan;69(1):27-32.
PMID: 12436474

M13 phages that display random disulfide constrained heptapeptides on their gpIII proteins were used to select for high affinity ligands to hepatitis B core antigen (HBcAg). Phages bearing the amino acid sequences C-WSFFSNI-C and C-WPFWGPW-C were isolated, and a binding assay in solution showed that these phages bind tightly to full-length and truncated HBcAg with K D rel values less than 25 nM, which is at least 10 orders of magnitude higher than phage carrying the peptide sequence LLGRMK selected from a linear peptide library. Both the phages that display the constrained peptides were inhibited from binding to HBcAg particles by a monoclonal antibody that binds specifically to the immunodominant region of the particles. A synthetic heptapeptide with the amino acid sequence WSFFSNI derived from one of the fusion peptides inhibits the binding of large surface antigen (L-HBsAg) to core particles with an IC50 value of 12 +/- 2 microM. This study has identified a smaller peptide with a greater inhibitory effect on L-HBsAg-HBcAg association.

Matched MeSH terms: Peptide Library*
Fulltext Selection and characterisation of Affimers specific for CEA recognition

Shamsuddin SH, Jayne DG, Tomlinson DC, McPherson MJ, Millner PA

Sci Rep, 2021 01 12;11(1):744.
PMID: 33436840 DOI: 10.1038/s41598-020-80354-6

Carcinoembryonic antigen (CEA) is the only blood based protein biomarker at present, used for preoperative screening of advanced colorectal cancer (CRC) patients to determine the appropriate curative treatments and post-surveillance screening for tumour recurrence. Current diagnostics for CRC detection have several limitations and development of a highly sensitive, specific and rapid diagnostic device is required. The majority of such devices developed to date are antibody-based and suffer from shortcomings including multimeric binding, cost and difficulties in mass production. To circumvent antibody-derived limitations, the present study focused on the development of Affimer proteins as a novel alternative binding reagent for CEA detection. Here, we describe the selection, from a phage display library, of Affimers specific to CEA protein. Characterization of three anti-CEA Affimers reveal that these bind specifically and selectively to protein epitopes of CEA from cell culture lysate and on fixed cells. Kinetic binding analysis by SPR show that the Affimers bind to CEA with high affinity and within the nM range. Therefore, they have substantial potential for used as novel affinity reagents in diagnostic imaging, targeted CRC therapy, affinity purification and biosensor applications.

Matched MeSH terms: Peptide Library*
Review of phage display: A jack-of-all-trades and master of most biomolecule display

Song BPC, Ch'ng ACW, Lim TS

Int J Biol Macromol, 2024 Jan;256(Pt 2):128455.
PMID: 38013083 DOI: 10.1016/j.ijbiomac.2023.128455

Phage display was first described by George P. Smith when it was shown that virus particles were capable of presenting foreign proteins on their surface. The technology has paved the way for the evolution of various biomolecules presentation and diverse selection strategies. This unique feature has been applied as a versatile platform for numerous applications in drug discovery, protein engineering, diagnostics, and vaccine development. Over the decades, the limits of biomolecules displayed on phage particles have expanded from peptides to proteomes and even alternative scaffolds. This has allowed phage display to be viewed as a versatile display platform to accommodate various biomolecules ranging from small peptides to larger proteomes which has significantly impacted advancements in the biomedical industry. This review will explore the vast array of biomolecules that have been successfully employed in phage display technology in biomedical research.

Matched MeSH terms: Peptide Library*
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.

Matched MeSH terms: Peptide Library*
Phage display of recombinant antibodies toward Burkholderia pseudomallei exotoxin

Nathan S, Li H, Mohamed R, Embi N

J. Biochem. Mol. Biol. Biophys., 2002 Feb;6(1):45-53.
PMID: 12186782

We have used the phagemid pComb3H to construct recombinant phages displaying the single chain variable fragment (ScFv) towards exotoxin of Burkholderia pseudomallei. Variable heavy and light chain fragments were amplified from the hybridoma 6E6A8F3B line, with a wide spectrum of primers specific to mouse antibody genes. Through overlapping extension polymerase chain reaction, the heavy and light chain fragments were linked to form the ScFv which was subsequently cloned into the phage display vector and transformed into ER2537 cells to yield a complexity of 10(8) clones. The transformants were screened by four rounds of biopanning against the exotoxin and resulted in selective enrichment of exotoxin-binding antibodies by 301 fold. The phage pool from the final round of selection displayed antibodies of high-affinity to the exotoxin as demonstrated by ELISA. Several clones were selected randomly from this pool and analysed by restriction enzyme digestion, fingerprinting and sequencing. Restriction analysis confirmed that all clones carried a 700-800 bp insert whose sequences, in general, corresponded to that of mouse IgG. Fingerprinting profiles delineated the antibodies into two families with different CDR sequences.

Matched MeSH terms: Peptide Library
Peptide mimotopes of complex carbohydrates in Salmonella enterica serovar typhi which react with both carbohydrate-specific monoclonal antibody and polyclonal sera from typhoid patients

Thong KL, Tang SS, Tan WS, Devi S

Microbiol. Immunol., 2007;51(11):1045-52.
PMID: 18037781

Polyclonal sera from typhoid patients and a monoclonal antibody, mAb ATVi, which recognizes the capsular polysaccharide Vi antigen (ViCPS), were used to select for peptides that mimic the ViCPS by using a phage-displayed random 12-mer peptide library. Two major common mimotopes selected from the library carried the amino acid sequences TSHHDSHGLHRV and ENHSPVNIAHKL. Enzyme-linked immunosorbent assays (ELISAs) showed that these peptides carry mimotopes to ViCPS. Phage clones that contained the 12-mer peptides were also tested against pooled/individual typhoid patients' sera and found to have 3 to 5 times higher binding compared to normal sera. By using Phage-ELISA assays, the derived synthetic peptides, TSHHDSHGLHRV and ENHSPVNIAHKL, were tested against a monoclonal antibody mAb ATVi and over 2-fold difference in binding was found between these peptides and a control unrelated peptide, CTLTTKLYC. Inhibition of the mAb's binding to ViCPS indicated that the synthetic peptides successfully competed with the capsular polysaccharide for antibody binding.

Matched MeSH terms: Peptide Library
Peptide inhibitors against influenza virus

Rajik M, Yusoff K

Antivir Chem Chemother, 2011;21(4):151-4.
PMID: 21602612 DOI: 10.3851/IMP1728

Influenza A virus is a particularly problematic virus because of its ability to cause high levels of morbidity on a global scale within a remarkably short period of time. It also has the potential to kill very large numbers of people as occurred in the Spanish influenza pandemic in 1918. Options for antiviral therapy are limited because of the paucity of available drugs and the rapid mutation rate of the virus leading to the emergence of drug-resistant strains. The current H1N1 pandemic and potential threats posed by other strains highlight the need to develop novel therapeutic and prophylactic strategies. Here, we summarize the current state and recent developments of peptide-based inhibitors of influenza A virus.

Matched MeSH terms: Peptide Library
Pathotyping of Newcastle disease virus with a filamentous bacteriophage

Ramanujam P, Tan WS, Nathan S, Yusoff K

Biotechniques, 2004 Feb;36(2):296-300, 302.
PMID: 14989094

A filamentous phage bearing the peptide sequence TLTTKLY was isolated from a heptapeptide phage display library against a velogenic Newcastle disease virus (NDV). In order to investigate the potential of this specific phage as an immunological reagent in virus pathotyping, an enzyme-linked immunosorbent assay (ELISA)-based method was developed. This method can differentiate the velogenic strains from the mesogenic and lentogenic strains. An equilibrium-binding assay in solution showed that the interactions between the phage and all the NDV strains gave rise to two widely differing dissociation constants (Kdrel). Based upon the first Kdrel values, NDV strains can be classified into two groups; the first comprises the velogenic strains, and the second consists of the mesogenic and lentogenic strains. These results indicate a high degree of correlation between the binding affinities and pathotyping of NDV strains using the TLTTKLY phage.

Matched MeSH terms: Peptide Library
Novel peptides that inhibit the propagation of Newcastle disease virus

Ramanujam P, Tan WS, Nathan S, Yusoff K

Arch Virol, 2002 May;147(5):981-93.
PMID: 12021868

A disulfide constrained random heptapeptide library displayed on filamentous bacteriophage M13 was applied to select specific ligands that interact with Newcastle disease virus (NDV). A fusion phage carrying the amino acid sequence TLTTKLY was selected from the panning procedure. An antibody competition assay showed that the selected phage was capable of competing with the polyclonal antibodies raised against NDV for binding sites on the virus. Determination of the binding affinity of this phage with NDV by an equilibrium binding assay in solution revealed two different dissociation constants, suggesting that there could be two distinct binding sites for the phage on NDV. Synthetic peptides with the sequence CTLTTKLYC, either in linear or cyclic conformations inhibited the binding of phage bearing the same sequence to NDV. These peptides also inhibited the hemolytic activity of the virus as well as its propagation in embryonated chicken eggs.

Matched MeSH terms: Peptide Library
Neutralization of Burkholderia pseudomallei protease by Fabs generated through phage display

Nathan S, Rader C, Barbas CF

Biosci Biotechnol Biochem, 2005 Dec;69(12):2302-11.
PMID: 16377887

The isolation of therapeutic and functional protease inhibitors in vitro via combinatorial chemistry and phage display technology has been described previously. Here we report the construction of a combinatorial mouse-human chimeric antibody fragment (Fab) antibody library targeted against the protease of the tropical pathogen, Burkholderia pseudomallei. The resulting library was biopanned against the protease, and selected clones were analyzed for their ability to function as protease inhibitors. Three families of Fabs were identified by restriction fingerprinting, all of which demonstrated high specificity towards the protease of B. pseudomallei. Purified Fabs also demonstrated the capacity to inhibit B. pseudomallei protease activity in vitro, and this inhibitory property was exclusive to the pathogenic protease. Thus these recombinant antibodies are candidates for immunotherapy and tools to aid in further elucidation of the mechanism of action of the B. pseudomallei protease.

Matched MeSH terms: Peptide Library*
Fulltext Naïve Human Antibody Libraries for Infectious Diseases

Chan SK, Rahumatullah A, Lai JY, Lim TS

Adv Exp Med Biol, 2017;1053:35-59.
PMID: 29549634 DOI: 10.1007/978-3-319-72077-7_3

Many countries are facing an uphill battle in combating the spread of infectious diseases. The constant evolution of microorganisms magnifies the problem as it facilitates the re-emergence of old infectious diseases as well as promote the introduction of new and more deadly variants. Evidently, infectious diseases have contributed to an alarming rate of mortality worldwide making it a growing concern. Historically, antibodies have been used successfully to prevent and treat infectious diseases since the nineteenth century using antisera collected from immunized animals. The inherent ability of antibodies to trigger effector mechanisms aids the immune system to fight off pathogens that invades the host. Immune libraries have always been an important source of antibodies for infectious diseases due to the skewed repertoire generated post infection. Even so, the role and ability of naïve antibody libraries should not be underestimated. The naïve repertoire has its own unique advantages in generating antibodies against target antigens. This chapter will highlight the concept, advantages and application of human naïve libraries as a source to isolate antibodies against infectious disease target antigens.

Matched MeSH terms: Peptide Library*
Mimotopes of the Vi antigen of Salmonella enterica serovar typhi identified from phage display peptide library

Tang SS, Tan WS, Devi S, Wang LF, Pang T, Thong KL

Clin Diagn Lab Immunol, 2003 Nov;10(6):1078-84.
PMID: 14607870

The capsular polysaccharide Vi antigen (ViCPS) is an essential virulence factor and also a protective antigen of Salmonella enterica serovar Typhi. A random 12-mer phage-displayed peptide library was used to identify mimotopes (epitope analogues) of this antigen by panning against a ViCPS-specific monoclonal antibody (MAb) ATVi. Approximately 75% of the phage clones selected in the fourth round carried the peptide sequence TSHHDSHGLHRV, and the rest of the clones harbored ENHSPVNIAHKL and other related sequences. These two sequences were also obtained in a similar panning process by using pooled sera from patients with a confirmed diagnosis of typhoid fever, suggesting they mimic immunodominant epitopes of ViCPS antigens. Binding of MAb ATVi to the mimotopes was specifically blocked by ViCPS, indicating that they interact with the same binding site (paratope) of the MAb. Data and reagents generated in this study have important implications for the development of peptide-base diagnostic tests and peptide vaccines and may also provide a better understanding of the pathogenesis of typhoid fever.

Matched MeSH terms: Peptide Library*
Mimotopes of heat shock proteins of Salmonella enterica serovar Typhi identified from phage-displayed peptide library

Yuen HL, Shamala D, Thong KL

J Infect Dev Ctries, 2008 Aug 30;2(4):313-23.
PMID: 19741295

BACKGROUND: Heat shock proteins (HSPs) are known to be involved in the pathogenesis of Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever. The objective of this study was to apply a phage display library to identify mimotopes of two HSPs, HSP90 and DnaK in S. Typhi.
METHODOLOGY: A 12-mer random peptide library expressed on the surface of the filamentous phage, M13, was used to select the mimotopes of two S. Typhi heat shock proteins by biopanning with monoclonal antibodies (mAbs), DnaK and HSP90. The immunogenicity of the selected peptides was determined through binding affinity with polyclonal antibodies from pooled typhoid-confirmed patients' sera and purified HSPs mAb using Western blotting and ELISA.
RESULTS: Five rounds of biopanning resulted in enrichment of phage clones expressing the binding motifs TDxSTRP and FPSHYWLYPPPT, respectively. The selected peptides showed strong immunoreactivity with patients' sera. Thus, monoclonal antibodies against HSP and patient sera can select common mimotopes from the random peptide library.
CONCLUSION: These findings may provide fundamental information for further studies on diagnostic application or vaccine design against this aetiologic agent of typhoid fever.

Matched MeSH terms: Peptide Library
Mass Spectrometry Immuno Assay (MSIA™) Streptavidin Disposable Automation Research Tips (D.A.R.T's®) Antibody Phage Display Biopanning

Chin CF, Choong YS, Lim TS

Methods Mol Biol, 2018;1701:285-299.
PMID: 29116511 DOI: 10.1007/978-1-4939-7447-4_15

Antibody phage display has been widely established as the method of choice to generate monoclonal antibodies with various efficacies post hybridoma technology. This technique is a popular method which takes precedence over ease of methodology, time- and cost-savings with comparable outcomes to conventional methods. Phage display technology manipulates the genome of M13 bacteriophage to display large diverse collection of antibodies that is capable of binding to various targets (nucleic acids, peptides, proteins, and carbohydrates). This subsequently leads to the discovery of target-related antibody binders. There have been several different approaches adapted for antibody phage display over the years. This chapter focuses on the semi-automated phage display antibody biopanning method utilizing the MSIA™ streptavidin D.A.R.T's® system. The system employs the use of electronic multichannel pipettes with predefined programs to carry out the panning process. The method should also be adaptable to larger liquid handling instrumentations for higher throughput.

Matched MeSH terms: Peptide Library*
Magnetic bead-based semi-automated phage display panning strategy for the directed evolution of antibodies

Ch'ng ACW, Konthur Z, Lim TS

Methods Enzymol, 2020;630:159-178.
PMID: 31931984 DOI: 10.1016/bs.mie.2019.10.023

Directed evolution is a proven approach to fine tune or modify biomolecules for various applications ranging from research to industry. The process of evolution requires methods that are capable of not only generating genetic diversity but also to distinguish the variants of desired characteristics. One method that is synonymous with directed evolution of proteins is phage display. Here, we present a protocol describing the application of magnetic nanoparticles coupled with a processor to carry out the identification of monoclonal antibodies (mAbs) from a diverse antibody library via phage display. Target antigens are coupled to magnetic nanoparticles as the solid phase for the isolation of the binding mAbs via affinity. A gradual enrichment in clones would result in increasing ELISA readouts with increasing rounds of panning. During monoclonal level analysis, positivity can be deduced with comparison to background and controls. The biopanning process can also be adopted for the directed evolution of enzymes, scaffold proteins or even peptides.

Matched MeSH terms: Peptide Library*
Magnetic Nanoparticle-Based Semi-Automated Panning for High-Throughput Antibody Selection

Ch'ng ACW, Hamidon NHB, Konthur Z, Lim TS

Methods Mol Biol, 2018;1701:301-319.
PMID: 29116512 DOI: 10.1007/978-1-4939-7447-4_16

The application of recombinant human antibodies is growing rapidly mainly in the field of diagnostics and therapeutics. To identify antibodies against a specific antigen, panning selection is carried out using different display technologies. Phage display technology remains the preferred platform due to its robustness and efficiency in biopanning experiments. There are both manual and semi-automated panning selections using polystyrene plastic, magnetic beads, and nitrocellulose as the immobilizing solid surface. Magnetic nanoparticles allow for improved antigen binding due to their large surface area. The Kingfisher Flex magnetic particle processing system was originally designed to aid in RNA, DNA, and protein extraction using magnetic beads. However, the system can be programmed for antibody phage display panning. The automation allows for a reduction in human error and improves reproducibility in between selections with the preprogrammed movements. The system requires minimum human intervention to operate; however, human intervention is needed for post-panning steps like phage rescue. In addition, polyclonal and monoclonal ELISA can be performed using the semi-automated platform to evaluate the selected antibody clones. This chapter will summarize the suggested protocol from the panning stage till the monoclonal ELISA evaluation. Other than this, important notes on the possible optimization and troubleshooting are also included at the end of this chapter.

Matched MeSH terms: Peptide Library*

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