Construction of naïve camelids VHH repertoire in phage display-based library

Sabir, Jamal S.M.; Atef, Ahmed; El-Domyati, Fotouh M.; Edris, Sherif; Hajrah, Nahid H.; Alzohairy, Ahmed M.; Bahieldin, Ahmed

doi:10.1016/j.crvi.2014.02.004

Cited by 39 publications

(35 citation statements)

References 28 publications

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“…Synthetic VHH libraries do not require any experimental animal handling. Such libraries are built based on a well characterized VHH of which the conserved FRs are retained and amino acids in the CDRs are altered by saturating site specific mutagenesis [11,12,13,14]. Immune libraries are often the first choice to isolate high affinity VHHs because natural somatic antibody maturation can create an enormous diversity.…”

Section: Introductionmentioning

confidence: 99%

Single-Domain Antibodies and Their Formatting to Combat Viral Infections

et al. 2018

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Since their discovery in the 1990s, single-domain antibodies (VHHs), also known as Nanobodies®, have changed the landscape of affinity reagents. The outstanding solubility, stability, and specificity of VHHs, as well as their small size, ease of production and formatting flexibility favor VHHs over conventional antibody formats for many applications. The exceptional ease by which it is possible to fuse VHHs with different molecular modules has been particularly explored in the context of viral infections. In this review, we focus on VHH formats that have been developed to combat viruses including influenza viruses, human immunodeficiency virus-1 (HIV-1), and human respiratory syncytial virus (RSV). Such formats may significantly increase the affinity, half-life, breadth of protection of an antiviral VHH and reduce the risk of viral escape. In addition, VHHs can be equipped with effector functions, for example to guide components of the immune system with high precision to sites of viral infection.

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Section: Introductionmentioning

confidence: 99%

Single-Domain Antibodies and Their Formatting to Combat Viral Infections

et al. 2018

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“…It was then modified for expression of functional single-chain Fv antibody—pIII fusion proteins on the surface of M13 bacteriophages [44]. pSex81 was also used successfully for construction of naïve sdAb library [45] production of complete human IgG [46], and for directed protein evolution [47]. Transformation efficiency of pSex81 is declared ~1 × 10 8 CFU/μg DNA with chemically competent E. coli (XL1-Blue).…”

Section: Discussionmentioning

confidence: 99%

Joining the in vitro immunization of alpaca lymphocytes and phage display: rapid and cost effective pipeline for sdAb synthesis

et al. 2017

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BackgroundCamelids possess unique functional heavy chain antibodies, which can be produced and modified in vitro as a single domain antibody (sdAb or nanobody) with full antigen binding ability. Production of sdAb in conventional manner requires active immunization of Camelidae animal, which is laborious, time consuming, costly and in many cases not feasible (e.g. in case of highly toxic or infectious antigens).ResultsIn this study, we describe an alternative pipeline that includes in vitro stimulation of naïve alpaca B-lymphocytes by antigen of interest (in this case endothelial cell binding domain of OspA of Borrelia) in the presence of recombinant alpaca interleukins 2 and 4, construction of sdAb phage library, selection of antigen specific sdAb expressed on phages (biopanning) and confirmation of binding ability of sdAb to the antigen. By joining the in vitro immunization and the phage display ten unique phage clones carrying sdAb were selected. Out of ten, seven sdAb showed strong antigen binding ability in phage ELISA. Furthermore, two soluble forms of sdAb were produced and their differential antigen binding affinity was measured with bio-layer interferometry.ConclusionA proposed pipeline has potential to reduce the cost substantially required for maintenance of camelid herd for active immunization. Furthermore, in vitro immunization can be achieved within a week to enrich mRNA copies encoding antigen-specific sdAbs in B cell. This rapid and cost effective pipeline can help researchers to develop efficiently sdAb for diagnostic and therapeutic purposes.

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“…The actual size of the VHH library was 5 × 10 8 . Considering that we started from 2 × 10 8 lymphocytes, the proportion of B cell-producing antibodies was 20%-30% of the total of lymphocytes, and VHHs represented 50% of the total camel antibodies (Monegal et al, 2009;Sabir et al, 2014). The theoretical maximal library diversity would be 3 × 10 7 , so the size of the naive camel VHH library covered the whole genetic diversity based on the initial lymphocytes.…”

Section: Construction Of a Naive Camel Phage Displayed Vhh Librarymentioning

confidence: 99%

Anti-idiotypic VHH mediated environmentally friendly immunoassay for citrinin without mycotoxin

Xiong

Zhang

et al. 2020

Food and Agricultural Immunology

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Competitive enzyme-linked immunosorbent assay (ELISA) is the most widely used method for rapidly screening of mycotoxins in contaminated food products; however, it involves the use of toxin-protein conjugates as a competitive antigen and toxin standards as calibrators. In this work, a single domain heavy chain antibody (VHH)-based environmentally friendly ELISA (VHH-ELISA) was developed to measure citrinin concentration in Monascusfermented rice. With this novel indirect competitive VHH-ELISA, the use of a synthetic antigen and pure toxins for operation and environmental safety simultaneously avoided. The assay exhibited a limit of detection of 2.25 ng/mL, with a linear range of 5.0 −500.0 ng/mL. VHH-ELISA was then utilized for sample testing, and excellent agreement was observed between VHH-ELISA and high-performance liquid chromatography as a reference. This study provided a novel platform to develop environmentally friendly immunoassays for other toxic small molecules in the application of anti-idiotypic VHHs.

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Construction of naïve camelids VHH repertoire in phage display-based library

Cited by 39 publications

References 28 publications

Single-Domain Antibodies and Their Formatting to Combat Viral Infections

Single-Domain Antibodies and Their Formatting to Combat Viral Infections

Joining the in vitro immunization of alpaca lymphocytes and phage display: rapid and cost effective pipeline for sdAb synthesis

Anti-idiotypic VHH mediated environmentally friendly immunoassay for citrinin without mycotoxin

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