High-sensitivity assessment of phagocytosis by persistent association-based normalization

Neergaard, Therese de; Sundwall, Martin; Nordenfelt, Pontus

doi:10.1101/827568

Cited by 2 publications

(3 citation statements)

References 28 publications

(28 reference statements)

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“…The phagocytosis assay was performed and analyzed using the PAN method as described (de Neergaard et al, 2019). Briefly, phagocytosis was performed with 100 000 THP-1 cells in a final volume of 150 µl with different multiplicity of prey (MOP) from 0-400 at 37°C for 30 min with gentle shaking conditions.…”

Section: Phagocytosis Assaymentioning

confidence: 99%

“…The data was then further analyzed using the PAN method (de Neergaard et al, 2019) in Prism 9.3.1 (GraphPad Software). Inbuilt non-linear analysis tool "Agonist vs. response -Variable slope (four parameters)" was used to create curves and determine MOP50, corresponding to the MOP, which evoked half of the maximal response.…”

Section: Analysis Of Flow Cytometry Datamentioning

confidence: 99%

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Invasive streptococcal infection can lead to the generation of cross-strain opsonic antibodies

Neergaard

Bläckberg

Ivarsson

et al. 2022

Preprint

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Introduction: The human pathogen Streptococcus pyogenes causes substantial morbidity and mortality. It is unclear if antibodies developed after infections with this pathogen are opsonic and if they are strain-specific or more broadly protective. Here, we quantified the opsonic antibody response following invasive S. pyogenes infection. Materials and Methods: Four patients with S. pyogenes bacteremia between 2018-2020 at Skåne University Hospital in Lund, Sweden, were prospectively enrolled. Acute and convalescent sera were obtained, and the S. pyogenes isolates were genome-sequenced (emm118, emm85, and two emm1). Quantitative antibody binding and phagocytosis assays were used to evaluate isolate-dependent opsonic antibody function in response to infection. Results: Antibody binding increased modestly against the infecting isolate and across emm types in convalescent compared to acute sera for all patients. For two patients, phagocytosis increased in convalescent serum for both the infecting isolate and across types. The increase was only across types for one patient, and one had no improvement. No correlation to the clinical outcomes was observed. Conclusions: Invasive S. pyogenes infections result in a modestly increased antibody binding with differential opsonic capacity, both non-functional binding and broadly opsonic binding across types. These findings question the dogma that an invasive infection should lead to a strong type-specific antibody increase rather than a more modest but broadly reactive response, as seen in these patients. Furthermore, our results indicate that an increase in antibody titers might not be indicative of an opsonic response and highlight the importance of evaluating antibody function in S. pyogenes infections.

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Section: Phagocytosis Assaymentioning

confidence: 99%

Section: Analysis Of Flow Cytometry Datamentioning

confidence: 99%

Invasive streptococcal infection can lead to the generation of cross-strain opsonic antibodies

Neergaard

Bläckberg

Ivarsson

et al. 2022

Preprint

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“…The phagocytosis experiments are performed according to a standardised assay based on normalisation of persistent association of prey and phagocyte (6). Association measured with Xolair-coated bacteria serves as a baseline of non-Fcmediated association.…”

Section: Model Fitting and Titration Simulations Of Monoclonal And Pomentioning

confidence: 99%

A predictive model of antibody binding in the presence of IgG-interacting bacterial surface proteins

Ahnlide

Neergaard

Sundwall

et al. 2020

Preprint

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Many bacteria can interfere with how antibodies bind to their surfaces. This bacterial antibody targeting makes it challenging to predict the immunological function of bacteria-associated antibodies. The M and M-like proteins of group A streptococci exhibit IgGFc-binding regions, which they use to reverse IgG binding orientation depending on the host environment. Unraveling the mechanism behind these binding characteristics may identify conditions under which bound IgG can drive an efficient immune response. Here, we have developed a biophysical model for describing these complex protein-antibody interactions. We show how the model can be used as a tool for studying various IgG samples' behavior by performing in silico simulations and correlating this data with experimental measurements. Besides its use for mechanistic understanding, this model could potentially be used as a tool to predict the effect, as well as aid in the development of antibody treatments. We illustrate this by simulating how IgG binding in serum is altered as specified amounts of monoclonal or pooled IgG is added. Phagocytosis experiments link this altered antibody binding to a physiological function and demonstrate that it is possible to predict the effect of an IgG treatment with our model. Our study gives a mechanistic understanding of bacterial antibody targeting and provides a tool for predicting the effect of antibody treatments.

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High-sensitivity assessment of phagocytosis by persistent association-based normalization

Cited by 2 publications

References 28 publications

Invasive streptococcal infection can lead to the generation of cross-strain opsonic antibodies

Invasive streptococcal infection can lead to the generation of cross-strain opsonic antibodies

A predictive model of antibody binding in the presence of IgG-interacting bacterial surface proteins

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