Generation and validation of murine monoclonal and camelid recombinant single domain antibodies specific for human pancreatic glycoprotein 2

Schlör, Anja; Holzlöhner, Pamela; Listek, Martin; Grieß, Cindy; Butze, Monique; Micheel, Burkhard; Hentschel, C; Sowa, Mandy; Roggenbuck, Dirk; Schierack, Peter; Füner, Jonas; Schliebs, Erik; Goihl, Alexander; Reinhold, Dirk; Hanack, Katja

doi:10.1016/j.nbt.2018.03.006

Cited by 5 publications

(7 citation statements)

References 25 publications

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“…Peripheral blood mononuclear cells (PBMCs) were isolated from fresh whole blood from three different naïve camelids (llama, alpaca, and huarizo) according to [ 44 , 45 ]. Finally, pellets were homogenized in 5 mL RNAPure TM peqGOLD (VWR, Dresden, Germany) and stored at −80 °C for further treatments.…”

Section: Methodsmentioning

confidence: 99%

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Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA

Czarnecka

Weichelt

Rödiger

et al. 2022

IJMS

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The discovery that certain diseases have specific miRNA signatures which correspond to disease progression opens a new biomarker category. The detection of these small non-coding RNAs is performed routinely using body fluids or tissues with real-time PCR, next-generation sequencing, or amplification-based miRNA assays. Antibody-based detection systems allow an easy onset handling compared to PCR or sequencing and can be considered as alternative methods to support miRNA diagnostic in the future. In this study, we describe the generation of a camelid heavy-chain-only antibody specifically recognizing miRNAs to establish an antibody-based detection method. The generation of nucleic acid-specific binders is a challenge. We selected camelid binders via phage display, expressed them as VHH as well as full-length antibodies, and characterized the binding to several miRNAs from a signature specific for dilated cardiomyopathy. The described workflow can be used to create miRNA-specific binders and establish antibody-based detection methods to provide an additional way to analyze disease-specific miRNA signatures.

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Section: Methodsmentioning

confidence: 99%

“…VHH sequences were amplified by two-step PCR using the primers listed in Table 1 . The amplification of the camelid VHH repertoire was performed as previously described [ 44 , 46 , 47 ]. PCR products with a size of 400 bp were used for the construction of the naïve phage library.…”

Section: Methodsmentioning

confidence: 99%

Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA

Czarnecka

Weichelt

Rödiger

et al. 2022

IJMS

Self Cite

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“…A naïve camelid VHH library was generated as previously described by Schlör et. al [11]. Briefly, RNA was isolated from peripheral blood mononuclear cells (PBMC; NucleoSpin RNA Mini Plus, Macherey-Nagel GmbH & Co.KG, Düren, Germany).…”

Section: Library Construction and Vhh-biopanningmentioning

confidence: 99%

“…Camelid single domain antibodies were selected by phage display out of a naïve camelid library as previously described [11]. We could identify around 15 monophages with a specific ELISA signal of more than 0.5 when recombinant full length SARS-CoV-2 Spike protein was used for coating (Fig.…”

Section: Development Of Hcabsmentioning

confidence: 99%

SARS-CoV-2 neutralizing camelid heavy-chain-only antibodies as powerful tools for diagnostic and therapeutic applications

Schloer¹,

Hirschberg

Amor³

et al. 2022

Preprint

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The ongoing COVID-19 pandemic situation caused by SARS-CoV-2 and variants of concern such as B.1.617.2 (Delta) and recently, B.1.1.529 (Omicron) is posing multiple challenges to humanity. The rapid evolution of the virus requires the adaptation of diagnostic and therapeutic applications. In this study, we describe camelid heavy-chain-only antibodies (hcAb) as useful for novel in vitro diagnostic assays and for therapeutic applications due to their neutralizing capacity. Five antibody candidates were selected out of a naive camelid library by phage display and expressed as full-length IgG2 antibodies. The antibodies were characterized by Western blot, enzyme-linked immunosorbent assays, surface plasmon resonance with regard to their specificity to the recombinant SARS-CoV-2 Spike protein, and to SARS-CoV-2 virus-like particles. Neutralization assays were performed with authentic SARS-CoV-2 and pseudotyped viruses (wildtype and Omicron). All antibodies efficiently detect recombinant SARS-CoV-2 Spike protein and SARS-CoV-2 virus-like particles in different ELISA setups. The best combination was shown with hcAb B10 as catcher antibody and HRP-conjugated hcAb A7.2 as the detection antibody. Further, four out of five antibodies potently neutralized authentic wildtype SARS-CoV-2 and particles pseudotyped with the SARS-CoV-2 Spike proteins of the wildtype and Omicron variant, sublineage BA.1 at concentrations between 0.1 and 0.35 ng/mL (ND50). Collectively, we report novel camelid hcAbs suitable for diagnostics and potential therapy.

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“…For example, carboxymethyl dextran (CMD)-coated gold SPR sensor increases the number of immobilized biomolecules. Biomolecules immobilized on the CMD surface should carry amine, carboxyl, aldehyde or thiol groups [91][92][93][94][95][96][97]. The most commonly used surface chemistry to immobilize biomolecule on CMD sensor is amine coupling process using mixture of EDC and NHS.…”

Section: Immobilization Of Biomolecules On Zno Layermentioning

confidence: 99%

ZnO for performance enhancement of surface plasmon resonance biosensor: a review

Mei

Menon

Hegde³

2020

Mater. Res. Express

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This paper reviews Kretschmann-based SPR sensor utilizing ZnO thin films and nanostructures for performance enhancement. The advancement in surface plasmon resonance technology relies on low-cost, high sensitivity and high selectivity sensor. Metal oxide has been incorporated in SPR sensor to be used for detection of biological and chemical compounds. ZnO as one of the metal oxides is an attractive material due to its unique physical and optical properties. Numerous techniques for fabrication and characterization of ZnO on SPR gold substrate have been studied. The mechanism for gas and biomolecules detection depends on their interaction with ZnO surface, which is mainly attributed to the high isoelectric point of ZnO. There are several types of ZnO nanostructures which have been employed for SPR application based on the Kretschmann configuration. In future, the thin film and nanostructures of ZnO have potential applications for miniature design, robust, high sensitivity, and low-cost portable type of SPR biosensor to be used for on-site testing in real-time and label-free manner.

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Generation and validation of murine monoclonal and camelid recombinant single domain antibodies specific for human pancreatic glycoprotein 2

Cited by 5 publications

References 25 publications

Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA

Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA

SARS-CoV-2 neutralizing camelid heavy-chain-only antibodies as powerful tools for diagnostic and therapeutic applications

ZnO for performance enhancement of surface plasmon resonance biosensor: a review

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