A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests

Klausberger, Miriam; Duerkop, Mark; Haslacher, Helmuth; Wozniak‐Knopp, Gordana; Cserjan‐Puschmann, Monika; Perkmann, Thomas; Lingg, Nico; Aguilar, Patrícia Pereira; Laurent, Elisabeth; Vos, Jelle De; Hofner, Manuela; Holzer, Barbara; Stadler, Maria; Manhart, Gabriele; Vierlinger, Klemens; Egger, Margot; Milchram, Lisa; Gludovacz, Elisabeth; Marx, Nicolas; Köppl, Christoph; Tauer, Christopher; Beck, Jürgen; Maresch, Daniel; Grünwald‐Gruber, Clemens; Strobl, Florian; Satzer, Peter; Stadlmayr, Gerhard; Vavra, Ulrike; Huber, Jasmin; Wahrmann, Markus; Eskandary, Farsad; Breyer, Marie Kathrin; Sieghart, Daniela; Quehenberger, Peter; Leitner, Gerda; Straßl, Robert; Egger, Alexander; Irsara, Christian; Griesmacher, Andrea; Hoermann, Gregor; Weiß, Günter; Bellmann‐Weiler, Rosa; Loeffler‐Ragg, Judith; Borth, Nicole; Strasser, Richard; Jungbauer, Alois; Hahn, Rainer; Mairhofer, Juergen; Hartmann, Boris; Binder, Nikolaus B.; Striedner, Gerald; Mach, Lukas; Weinhäusel, Andreas; Dieplinger, Benjamin; Grebien, Florian; Gerner, Wilhelm; Binder, Christoph J.; Grabherr, Reingard

doi:10.1016/j.ebiom.2021.103348

Cited by 36 publications

(64 citation statements)

References 77 publications

(117 reference statements)

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“…ELISA with ACE2-Fc and the conformation-dependent RBD antibody CR3022 showed that RBD-215 binds equally well like HEK293-produced RBD to ACE2-Fc and CR3022 ( Figures 4E,F ). Biolayer interferometry analysis revealed that RBD-215 has an affinity (K d of 30 ± 2 nM, n = 8) for CR3022 ( Figure 4G ) that is comparable to recombinant RBD produced in mammalian or insect cells ( Klausberger et al, 2021 ).…”

Section: Rbd Is Present As a Homodimer And Non-functional After Purification From N Benthamianamentioning

confidence: 89%

“…SDS-PAGE under non-reducing conditions followed by immunoblotting revealed a major band of approximately 50 kDa for plant-produced RBD, indicating the presence of dimers. Homodimer formation for plant and mammalian-cell produced RBD was recently confirmed by size-exclusion chromatography and is likely promoted by incorrectly formed disulfide bonds ( Klausberger et al, 2021 ).…”

Section: Rbd Is Present As a Homodimer And Non-functional After Purification From N Benthamianamentioning

confidence: 93%

“…The assays with pre-COVID-19 sera and sera from SARS-CoV-2 infected individuals (AIT cohorts) were carried out as described in detail recently (Klausberger et al, 2021).…”

Section: Luminex Assaymentioning

confidence: 99%

“…ELISA 2.5 µg/ml of monoclonal antibody CR3022 (Absolute Antibody) or 3 µg/ml of HEK293-produced human ACE2-Fc (Klausberger et al, 2021) were coated (50 µl/well) in bicarbonate buffer or PBS, respectively, onto NUNC MaxiSorp 96 well plates (Thermo Fisher Scientific) overnight at 4 • C. Plates were washed three times with PBS supplemented with 0.1% (v/v) Tween 20 (PBST) and subsequently blocked for 1 h with 1% (w/v) BSA in PBST.…”

Section: Biolayer Interferometry Measurementsmentioning

confidence: 99%

“…Here, we investigated the role of N-glycosylation for expression and function of the receptor binding domain (RBD) from the SARS-CoV-2 spike protein. Recombinant RBD can be used for vaccination approaches ( Yang et al, 2020 ) or for serological assays to determine the presence and quality of an immune response against SARS-CoV-2 ( Stadlbauer et al, 2020 ; Klausberger et al, 2021 ). The SARS-CoV-2 RBD has two N-glycosylation sites (N331 and N343) that are fully glycosylated when expressed in heterologous expression systems ( Antonopoulos et al, 2020 ; Shajahan et al, 2020 ; Watanabe et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

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N-Glycosylation of the SARS-CoV-2 Receptor Binding Domain Is Important for Functional Expression in Plants

et al. 2021

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Nicotiana benthamiana is used worldwide as production host for recombinant proteins. Many recombinant proteins such as monoclonal antibodies, growth factors or viral antigens require posttranslational modifications like glycosylation for their function. Here, we transiently expressed different variants of the glycosylated receptor binding domain (RBD) from the SARS-CoV-2 spike protein in N. benthamiana. We characterized the impact of variations in RBD-length and posttranslational modifications on protein expression, yield and functionality. We found that a truncated RBD variant (RBD-215) consisting of amino acids Arg319-Leu533 can be efficiently expressed as a secreted soluble protein. Purified RBD-215 was mainly present as a monomer and showed binding to the conformation-dependent antibody CR3022, the cellular receptor angiotensin converting enzyme 2 (ACE2) and to antibodies present in convalescent sera. Expression of RBD-215 in glycoengineered ΔXT/FT plants resulted in the generation of complex N-glycans on both N-glycosylation sites. While site-directed mutagenesis showed that the N-glycans are important for proper RBD folding, differences in N-glycan processing had no effect on protein expression and function.

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Section: Rbd Is Present As a Homodimer And Non-functional After Purification From N Benthamianamentioning

confidence: 89%

Section: Rbd Is Present As a Homodimer And Non-functional After Purification From N Benthamianamentioning

confidence: 93%

“…The assays with pre-COVID-19 sera and sera from SARS-CoV-2 infected individuals (AIT cohorts) were carried out as described in detail recently (Klausberger et al, 2021).…”

Section: Luminex Assaymentioning

confidence: 99%

Section: Biolayer Interferometry Measurementsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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N-Glycosylation of the SARS-CoV-2 Receptor Binding Domain Is Important for Functional Expression in Plants

et al. 2021

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Designed SARS‐CoV‐2 receptor binding domain variants form stable monomers

Klausberger

Kienzl

Stadlmayr

et al. 2022

Biotechnology Journal

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The receptor binding domain (RBD) of the SARS‐CoV‐2 spike (S)‐protein is a prime target of virus‐neutralizing antibodies present in convalescent sera of COVID‐19 patients and thus is considered a key antigen for immunosurveillance studies and vaccine development. Although recombinant expression of RBD has been achieved in several eukaryotic systems, mammalian cells have proven particularly useful. The authors aimed to optimize RBD produced in HEK293‐6E cells towards a stable homogeneous preparation and addressed its O‐glycosylation as well as the unpaired cysteine residue 538 in the widely used RBD (319‐541) sequence. The authors found that an intact O‐glycosylation site at T323 is highly relevant for the expression and maintenance of RBD as a monomer. Furthermore, it was shown that deletion or substitution of the unpaired cysteine residue C538 reduces the intrinsic propensity of RBD to form oligomeric aggregates, concomitant with an increased yield of the monomeric form of the protein. Bead‐based and enzyme‐linked immunosorbent assays utilizing these optimized RBD variants displayed excellent performance with respect to the specific detection of even low levels of SARS‐CoV‐2 antibodies in convalescent sera. Hence, these RBD variants could be instrumental for the further development of serological SARS‐CoV‐2 tests and inform the design of RBD‐based vaccine candidates.

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Accessing Antibody Reactivities in Serum or Plasma to (Auto-)antigens Using Multiplexed Bead-Based Protein Immunoassays

Huber

Schönthaler

Hofner

et al. 2023

Methods in Molecular Biology

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A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests

Cited by 36 publications

References 77 publications

N-Glycosylation of the SARS-CoV-2 Receptor Binding Domain Is Important for Functional Expression in Plants

N-Glycosylation of the SARS-CoV-2 Receptor Binding Domain Is Important for Functional Expression in Plants

Designed SARS‐CoV‐2 receptor binding domain variants form stable monomers

Accessing Antibody Reactivities in Serum or Plasma to (Auto-)antigens Using Multiplexed Bead-Based Protein Immunoassays

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