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We identified Alizarin Red S and other well known fluorescent dyes useful for the online detection of pyrophosphate in enzymatic assays, including the loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays. An iterative screening was used for a selected set of compounds to first secure enzyme compatibility, evaluate inorganic pyrophosphate sensitivity in the presence of manganese as quencher and optimize conditions for an online detection. Of the selected dyes, the inexpensive alizarin red S was found to selectively detect pyrophosphate under LAMP and PCR conditions and is superior with respect to its defined red-shifted spectrum, long shelf life and low toxicity. In addition, the newly identified properties may also be useful in other enzymatic assays which do not generate nucleic acids but are based on inorganic pyrophosphate. Finally, we propose that our screening method may provide a blueprint for rapid screening of compounds for detecting inorganic pyrophosphate.
Neisseria gonorrhoeae is the causative organism of gonorrhoea, a sexually transmitted disease that globally accounts for an estimated 80 to 100 million new infections per year. Increasing resistances to all common antibiotics used for N. gonorrhoeae treatment pose the risk of an untreatable disease. Further knowledge of ways of infection and host immune response are needed to understand the pathogen-host interaction and to discover new treatment alternatives against this disease. Therefore, detailed information about immunogenic proteins and their properties like epitope sites could advance further research in this area. In this work, we investigated immunogenic proteins of N. gonorrhoeae for linear epitopes by microarrays. Dominant linear epitopes were identified for eleven of the nineteen investigated proteins with three polyclonal rabbit antibodies from different immunisations. Identified linear epitopes were further examined for non-specific binding with antibodies to Escherichia coli and the closely related pathogen Neisseria meningitidis. On top of that, amino acids crucial for the antibody epitope binding were detected by microarray based alanine scans.