Heavy chain single-domain antibodies to detect native human soluble epoxide hydrolase

Cui, Yongliang; Li, Dongyang; Morisseau, Christophe; Dong, Jie; Yang, Jun; Wan, Debin; Rossotti, Martín A.; Gee, Shirley J.; González‐Sapienza, Gualberto; Hammock, Bruce D.

doi:10.1007/s00216-015-8889-6

Cited by 14 publications

(25 citation statements)

References 46 publications

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“…The VHH phage display library was built as previously described 4,25 . A three-year-old llama ( Lama glama ) from the Montevideo municipal zoo was immunized every two weeks with 6 doses of 500 µg of recombinant, affinity purified human sEH in incomplete Freund adjuvant by sub-cutaneous injection.…”

Section: Methodsmentioning

confidence: 99%

Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays

Rossotti¹,

Pírez²,

González-Techera³

et al. 2015

Anal. Chem.

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Single domain heavy-chain binders (nanobodies) obtained from camelid antibody libraries hold a great promise for immunoassay development. However, there is no simple method to select the most valuable nanobodies from the crowd of positive clones obtained after the initial screening. In this paper, we describe a novel nanobody-based platform that allows comparison of the reactivity of hundreds of clones with the labeled antigen, and identifies the best nanobody pairs for two-site immunoassay development. The output clones are biotinylated in vivo in 96-well culture blocks and then used to saturate the biotin binding capacity of avidin coated wells. This standardizes the amount of captured antibody allowing their sorting by ranking their reactivity with the labeled antigen. Using human soluble epoxide hydrolase (sEH) as a model antigen, we were able to classify 96 clones in four families and confirm this classification by sequencing. This provided a criterion to select a restricted panel of five capturing antibodies and to test each of them against the rest of the 96 clones. The method constitutes a powerful tool for epitope binning, and in our case allowed development of a sandwich ELISA for sEH with a detection limit of 63 pg/mL and four log dynamic range, which performed with excellent recovery in different tissue extracts. This strategy provides a systematic way to test nanobody pairwise combinations and would have a broad utility for the development of highly sensitive sandwich immunoassays.

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Section: Methodsmentioning

confidence: 99%

Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays

Rossotti¹,

Pírez²,

González-Techera³

et al. 2015

Anal. Chem.

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“…Furthermore, because VHH has only one variable region, specific antibodies can be screened at 1x10 6 . However, in ordinary antibody Fab or ScFv libraries, the library needs to be large enough to more easily screen for antibodies (93,96,97,102,103).…”

Section: Construction Of Library and Panning Of Nanobodiesmentioning

confidence: 99%

“…The phage display technology is used to screen binders for various targets from diverse and large libraries and is widely employed by various research teams. Since the difference between VHH and VH is mainly due to the absence of the CH1 region, the work of building an immune and natural library focuses on isolating antibodies lacking CH1 from IgG, which can be achieved by one-step ( 96 ) or two-step nested PCR amplification ( 97 ). The key of PCR is to design PCR primers using the conserved nucleotide sequence of the antibody back-bone region.…”

Section: Construction Of Library and Panning Of Nanobodiesmentioning

confidence: 99%

“…The VHH fragment is amplified by the second PCR ( 98 ). The amplified antibody sequence is ligated to the phagemid vector, such as pMECS ( 95 , 97 ), pHEN ( 99 ), pAX50 ( 100 ), and pComb3 ( 96 , 101 ). The remaining steps are the same as the ordinary process of constructing an antibody library ( 98 ).…”

Section: Construction Of Library and Panning Of Nanobodiesmentioning

confidence: 99%

“…After several rounds of panning and enrichment, VHH with high affinity is obtained, and a positive VHH is identified by indirect ELISA ( 98 ). Or, as an alternative, it can also be panned in the liquid phase ( 96 ). After blocking, binding, elution and amplification, specific binding between streptavidin-coated magnetic beads and biotin-labeled antigen can also be used to obtain antigen-specific antibodies.…”

Section: Construction Of Library and Panning Of Nanobodiesmentioning

confidence: 99%

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Nanobodies targeting immune checkpoint molecules for tumor immunotherapy and immunoimaging (Review)

et al. 2020

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The immune checkpoint blockade is an effective strategy to enhance the anti-tumor T cell effector activity, thus becoming one of the most promising immunotherapeutic strategies in the history of cancer treatment. Several immune checkpoint inhibitor have been approved by the FDA, such as anti-CTLA-4, anti-PD-1, anti-PD-L1 monoclonal antibodies. Most tumor patients benefitted from these antibodies, but some of the patients did not respond to them. To increase the effectiveness of immunotherapy, including immune checkpoint blockade therapies, miniaturization of antibodies has been introduced. A single-domain antibody, also known as nanobody, is an attractive reagent for immunotherapy and immunoimaging thanks to its unique structural characteristic consisting of a variable region of a single heavy chain antibody. This structure confers to the nanobody a light molecular weight, making it smaller than conventional antibodies, although remaining able to bind to a specific antigen. Therefore, this review summarizes the production of nanobodies targeting immune checkpoint molecules and the application of nanobodies targeting immune checkpoint molecules in immunotherapy and immunoimaging.

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Novel application of anti‐human Fc nanobody for screening high‐producing CHO cells for monoclonal antibody

Zhu

Wang²,

Yong³

et al. 2022

Engineering in Life Sciences

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Animal-derived anti-IgG secondary antibodies are currently employed to stain and screen of human monoclonal antibody(mAb)-producing cells, but using animal-derived antibodies may raise the concerns of high cost, complicated operations and biological safety issues in biopharmaceutical manufacturing. Nanobodies(VHHs) are attractive forms of antibodies for their straightforward engineering and expression in both eukaryotic and prokaryotic systems. Using phage-displayed immune llama VHH library, we identified new anti-Fc VHHs that could bind to human Fc with high affinity. In GFP fusion format, the anti-Fc VHH-GFP generated dramatically stronger FACS signals than AF488 conjugated anti-IgG antibodies when used for staining mAb-producing CHO cells. Furthermore, preparative sorting of CHO cells based on anti-Fc VHH-GFP staining resulted in the enrichment of cell lines capable of synthesizing mAb at high productivity. This safe and cost-efficient anti-Fc VHH-GFP may optimize the process of generating highly productive cell lines for therapeutic mAb production compared to conventional animal-derived fluorescent antibodies.

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Heavy chain single-domain antibodies to detect native human soluble epoxide hydrolase

Cited by 14 publications

References 46 publications

Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays

Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays

Nanobodies targeting immune checkpoint molecules for tumor immunotherapy and immunoimaging (Review)

Novel application of anti‐human Fc nanobody for screening high‐producing CHO cells for monoclonal antibody

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