Development of a method for selective assessment of complement microbicidal activity in human blood serum

Background. The ability of blood serum to suppress viability of microorganisms is primarily implemented by the presence of cationic factors (e.g. lysozyme, p-lysine, etc.) and complement system proteins. The complement itself is only able to cause lysis of some gram-negative bacteria, while cationic factors are predominantly active against gram-positive ones. The study of the contribution of individual antimicrobial components of blood serum and their combined action to its microbicidal activity would help to expand the understanding of the molecular mechanisms of innate immunity. The aim of the study. Development of a method for selective depletion of cationic factors from serum while preserving the functional activity of the complement system. Materials and methods. Pooled blood serum from healthy donors was used as the starting material. The depletion of cationic molecules from blood serum was performed by its incubation with a suspension of carboxymethylcellulose. In order to find the optimal conditions for binding of cationic proteins and peptides, either the NaCI concentration in the sample or the volume of the applied polymer suspension were varied. Removal of cationic factors from the serum was evaluated by the residual activity of lysozyme. The retention of complement was evaluated by serum hemolytic activity against rabbit red blood cells. The bactericidal activity of native and depleted serum against the gram-positive bacterium Listeria monocytogenes EGD was also compared. Results. Incubation of serum with carboxymethylcellulose suspension taken in egual volume in the presence of 0.1 M NaCI allowed effective removal of cationic polypeptides without evident changes in complement functional activity. Antimicrobial activity of the serum depleted of cationic factors was remarkably lower compared with the native blood serum. Conclusions. A method for selective removal of cationic factors from blood serum was developed. The described procedure allows evaluation of the complement microbicidal activity independently on the action of antimicrobial serum cationic polypeptides.

innate immunity, complement system, lysozyme, fi-lysin