Generation of Recombinant Antibodies Against Toxins and Viruses by Phage Display for Diagnostics and Therapy

Unkauf, Tobias; Miethe, Sebastian; Fühner, Viola; Schirrmann, Thomas; Frenzel, André; Hust, Michael

doi:10.1007/978-3-319-32805-8_4

Cited by 19 publications

(11 citation statements)

References 134 publications

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“…Phage antibody display is an in vitro technology for the production of recombinant antibodies in scFv format (Unkauf et al, ). The panning process is an important step in phage display, involving binding of phage‐scFv to a specific antigen, washing away of nonspecific binders, and elution of the target‐binding phages (Frenzel et al, ).…”

Section: Discussionmentioning

confidence: 99%

A comparison of three strategies for biopanning of phage‐scFv library against diphtheria toxin

Lakzaei

Rasaee

Fazaeli

et al. 2018

Journal Cellular Physiology

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The biopanning process is a critical step in phage display for isolating peptides or proteins with specific binding properties. Conventional panning methods are sometimes not so effective and may result in nonspecific or low‐yield positive results. In this study, three different strategies including soluble antibody‐capturing, pH‐stepwise elution, and conventional panning were used for enrichment of specific clones against diphtheria toxoid. The reactivity of the selected clones was evaluated using an indirect enzyme‐linked immunosorbent assay. The positive clones were screened using Vero cell viability assay. The neutralizing clones were expressed in HB2151 strain of Escherichia coli and soluble single‐chain fragment variable (scFv) fragments were purified by nickel‐nitrilotriacetic acid affinity chromatography. Finally, the ability of scFv fragments for neutralizing diphtheria toxin (DT) were evaluated again using Vero cell viability assay. After four rounds of panning, the soluble antibody‐capturing method yielded 15 positive phage‐scFv clones against diphtheria toxoid. Conventional panning and pH‐stepwise elution model resulted from nine and five positive phage‐scFv clones, respectively. Among all positive clones, three clones were able to neutralize DT in Vero cell viability assay. Two of these clones belonged to a soluble antibody‐capturing method and one of them came from conventional panning. Three neutralizing clones were used for soluble expression and purification of scFvs fragments. It was found that these soluble scFv fragments possessed neutralizing activity ranging from 0.15 to 0.6 µg against two‐fold cytotoxic dose 99% of DT. In conclusion, the results of our study indicate that soluble antibody‐capturing method is an efficient method for isolation of specific scFv fragments.

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Section: Discussionmentioning

confidence: 99%

A comparison of three strategies for biopanning of phage‐scFv library against diphtheria toxin

Lakzaei

Rasaee

Fazaeli

et al. 2018

Journal Cellular Physiology

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“…Various sources have been used for the therapeutic antibody production against viruses, including total B cells [ 75 ], single memory B cells [ 21 ], and single plasma B cells [ 21 ]. Several reports have also shown the efficacy of isolating the antibodies from native phage libraries which possess potent neutralization activities against viruses [ 76 ]. The downside of this technique is that it cannot be used to identify new neutralizing epitopes of virus [ 21 ].…”

Section: Challenges In Antibody-based Therapiesmentioning

confidence: 99%

Antibody-Mediated Therapy against HIV/AIDS: Where Are We Standing Now?

Awi

Teow

2018

Journal of Pathogens

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Acquired immunodeficiency syndrome (AIDS) cases are on the rise globally. To date, there is still no effective measure to eradicate the causative agent, human immunodeficiency virus (HIV). Highly active antiretroviral therapy (HAART) is being used in HIV/AIDS management, but it results in long-term medication and has major drawbacks such as multiple side effects, high cost, and increasing the generation rate of escape mutants. In addition, HAART does not control HIV-related complications, and hence more medications and further management are required. With this, other alternatives are urgently needed. In the past, small-molecule inhibitors have shown potent antiviral effects, and some of them are now being evaluated in clinical trials. The challenges in developing these small molecules for clinical use include the off-target effect, poor stability, and low bioavailability. On the other hand, antibody-mediated therapy has emerged as an important therapeutic modality for anti-HIV therapeutics development. Many antiviral antibodies, namely, broad neutralizing antibodies (bnAbs) against multiple strains of HIV, have shown promising effects in vitro and in animal studies; further studies are ongoing in clinical trials to evaluate their uses in clinical applications. This short review aims to discuss the current development of therapeutic antibodies against HIV and the challenges in adopting them for clinical use.

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“…Single-chain fragment variable (scFv) was first described and constructed more than three decades ago by Bird et al [ 17 ] and Huston et al [ 18 ]. Since then, selections of genetically engineered scFvs have been seen as effective therapeutics in many diseases such as cancer, influenza, and enteric colibacillosis [ 19 , 20 , 21 , 22 ]. ScFv antibody consists of a variable region of heavy chain (V H ) and light chain (V L ) joined by an adjustable peptide linker.…”

Section: Introductionmentioning

confidence: 99%

In Vitro Characterization of Neutralizing Hen Antibodies to Coxsackievirus A16

Mwale

Lee

Huang

et al. 2021

IJMS

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Coxsackievirus A16 (CA16) is one of the major causative agents of hand, foot, and mouth disease (HFMD). Children aged <5 years are the most affected by CA16 HFMD globally. Although clinical symptoms of CA16 infections are usually mild, severe complications, such as aseptic meningitis or even death, have been recorded. Currently, no vaccine or antiviral therapy for CA16 infection exists. Single-chain variable fragment (scFv) antibodies significantly inhibit viral infection and could be a potential treatment for controlling the infection. In this study, scFv phage display libraries were constructed from splenocytes of a laying hen immunized with CA16-infected lysate. The pComb3X vector containing the scFv genes was introduced into ER2738 Escherichia coli and rescued by helper phages to express scFv molecules. After screening with five cycles of bio-panning, an effective scFv antibody showing favorable binding activity to proteins in CA16-infected lysate on ELISA plates was selected. Importantly, the selected scFv clone showed a neutralizing capability against the CA16 virus and cross-reacted with viral proteins in EV71-infected lysate. Intriguingly, polyclonal IgY antibody not only showed binding specificity against proteins in CA16-infected lysate but also showed significant neutralization activities. Nevertheless, IgY-binding protein did not cross-react with proteins in EV71-infected lysate. These results suggest that the IgY- and scFv-binding protein antibodies provide protection against CA16 viral infection in in vitro assays and may be potential candidates for treating CA16 infection in vulnerable young children.

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Generation of Recombinant Antibodies Against Toxins and Viruses by Phage Display for Diagnostics and Therapy

Cited by 19 publications

References 134 publications

A comparison of three strategies for biopanning of phage‐scFv library against diphtheria toxin

A comparison of three strategies for biopanning of phage‐scFv library against diphtheria toxin

Antibody-Mediated Therapy against HIV/AIDS: Where Are We Standing Now?

In Vitro Characterization of Neutralizing Hen Antibodies to Coxsackievirus A16

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