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  • Mapping of Recognition Sites of Monoclonal Antibodies Responsible for the Inhibition of Pneumolysin Functional Activity.

Mapping of Recognition Sites of Monoclonal Antibodies Responsible for the Inhibition of Pneumolysin Functional Activity.

Biomolecules (2020-07-12)
Indre Kucinskaite-Kodze, Martynas Simanavicius, Justas Dapkunas, Milda Pleckaityte, Aurelija Zvirbliene
ABSTRACT

The pathogenicity of many bacteria, including Streptococcus pneumoniae, depends on pore-forming toxins (PFTs) that cause host cell lysis by forming large pores in cholesterol-containing cell membranes. Therefore, PFTs-neutralising antibodies may provide useful tools for reducing S. pneumoniae pathogenic effects. This study aimed at the development and characterisation of monoclonal antibodies (MAbs) with neutralising activity to S. pneumoniae PFT pneumolysin (PLY). Five out of 10 produced MAbs were able to neutralise the cytolytic activity of PLY on a lung epithelial cell line. Epitope mapping with a series of recombinant overlapping PLY fragments revealed that neutralising MAbs are directed against PLY loops L1 and L3 within domain 4. The epitopes of MAbs 3A9, 6E5 and 12F11 located at L1 loop (aa 454-471) were crucial for PLY binding to the immobilised cholesterol. In contrast, the MAb 12D10 recognising L3 (aa 403-423) and the MAb 3F3 against the conformational epitope did not interfere with PLY-cholesterol interaction. Due to conformation-dependent binding, the approach to use overlapping peptides for fine epitope mapping of the neutralising MAbs was unsuccessful. Therefore, the epitopes recognised by the MAbs were analysed using computational methods. This study provides new data on PLY sites involved in functional activity.

MATERIALS
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Brand
Product Description

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Polyethylene glycol/ dimethyl sulfoxide solution, Hybri-Max, average mol wt 1,450, 50 % (w/v), 0.2 μm filtered, BioReagent, suitable for hybridoma
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Alkaline Phosphatase Yellow (pNPP) Liquid Substrate System for ELISA, ready to use solution
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HAT Media Supplement (50×) Hybri-Max, γ-irradiated, lyophilized powder, BioXtra, suitable for hybridoma