A Human Erythrocyte-based Haemolysis Assay for the Evaluation of Human Complement Activity.

The complement system consists of at least 50 proteins that serve as one of the first lines of defence against foreign, or damaged, cells and invading microorganisms. Its dysregulation underlies the pathophysiology of many different diseases, which makes functional assays of complement activity crucial; they are, however, underutilised. Standard haemolysis assays for the analysis of complement function employ sensitised non-human erythrocytes (e.g. from the sheep, guinea-pig or rabbit), the use of which raises animal welfare concerns. To provide an alternative to the use of such animal-derived products for complement function assays, we developed a method that employs modified human erythrocytes to evaluate the activity of complement pathways. Human erythrocytes were subjected to various chemical and/or proteolytic treatments involving 2,4,6-trinitrobenzene sulphonate (TNBS) and pancreatin. Haemolysis assays demonstrated that sequential treatment with TNBS and pancreatin resulted in significantly greater complement-mediated haemolysis, as compared to TNBS or pancreatin treatment alone. Evidence that lysis of the modified erythrocytes was complement-mediated was provided by the chelation and subsequent restoration of calcium in the plasma. Thus, such modified human erythrocytes could be used as an alternative to animal-derived erythrocytes in haemolysis assays, in order to evaluate complement activity in human plasma during, for example, the screening of patients for complement deficiencies and other abnormalities in a clinical setting.

Development and Characterization of Polymeric Peptides for Antibody Tagging of Bacterial Targets.

BACKGROUND: Microbe-Binding Peptides (MBPs) are currently being investigated to address the problem of antimicrobial resistance. Strategies enhancing their antimicrobial activity have been developed, including peptide dimerization. Here, we present an alternative approach based on peptide polymerization, yielding hapten-labelled polymeric MBPs that mediate tagging of bacteria with anti-hapten antibodies, for enhanced immune recognition by host phagocytes. METHODS: C-terminally amidated analogs of the bacterial-binding peptide IIGGR were synthesized, with or without addition of cysteine residues at both N- and C-termini. Peptides were subjected to oxidizing conditions in a dimethyl-sulfoxide/water solvent system, and polymerization was demonstrated using SDS-PAGE. Peptides were then N-terminally labelled with a trinitrophenyl (TNP) group using trinitrobenzene sulfonate (TNBS). Binding to representative bacteria was demonstrated by ELISA using anti-TNP antibodies and was quantified as half-maximal effective concentration (EC50). Minimum Inhibitory Concentration (MIC) and concentration yielding 50% hemolysis (H50) were estimated. Neutrophil phagocytic index was determined for TNP-labelled polymeric bacterial- binding peptide (Pbac) with anti-TNP antibodies and/or serum complement. RESULTS: Polydisperse Pbac was synthesized. EC50 was lower for Pbac than for the corresponding monomeric form (Mbac), for both Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922. MIC and H50 were >250µg/mL for both Pbac and Mbac. A complement-independent increase in neutrophil phagocytic index was observed for E. coli treated with TNP-labelled Pbac in conjunction with anti-TNP antibodies. CONCLUSION: Our data suggest that hapten-labelled polymeric bacterial-binding peptides may easily be produced from even crude synthetic oligopeptide precursors, and that such bacterial-binding peptides in conjunction with cognate anti-hapten antibodies can enhance immune recognition of bacteria by host phagocytes.