OBJECTIVES: To study the role of different components in normal human serum and the role of lysozyme in rapid immobilisation of Percoll purified T pallidum (Nichols). MATERIALS AND METHODS: The immobilisation of Percoll purified T pallidum was studied after pre-incubations with different serum fractions (Fr) of normal human serum (Fr 1, containing IgM; Fr 2, containing IgG and a low level of haemolytic complement, and Fr 1 (abs), depleted of IgG). A guinea-pig serum pool was used as a complement source in the immobilisation experiments. The influence was studied of removal of lysozyme from guinea-pig serum on the immobilisation reactions. Further experiments were performed, using a fluorescence technique, to detect C3b depositions on fixed treponemes and treponemes in suspension. RESULTS: Rapid immobilisation of Percoll-purified treponemes by the NHS serum fractions occurred only after preincubation with Fr 1 and Fr 2 simultaneously. This was largely dependent on the presence of a small amount of haemolytic C in Fr 2. Removal of lysozyme reduced this rapid rate of immobilisation. In fluorescence experiments it was demonstrated that C3b deposition on fixed (that is damaged) treponemes occurred upon their incubation with Fr 2 or the combination of Fr 1 and 2. However, on treponemes in suspension C3b deposition occurred only after incubation with the combination of Fr 1 and 2. CONCLUSION: The rapid immobilisation of Percoll purified treponemes by serum fractions from normal human serum requires antibodies of the IgM and IgG class, together with complement and lysozyme. Omission of one of these reactants slows immobilisation. Our experiments suggest that the reactants act in sequence: the loss of integrity of the outer membrane by an attack by IgM and C offers the opportunity for lysozyme to hydrolyse the peptidoglycan layer surrounding the cytoplasmic membrane of the treponemes, which then is accessible for attack by antibodies and C.
OBJECTIVES: To study the role of different components in normal human serum and the role of lysozyme in rapid immobilisation of Percoll purified T pallidum (Nichols). MATERIALS AND METHODS: The immobilisation of Percoll purified T pallidum was studied after pre-incubations with different serum fractions (Fr) of normal human serum (Fr 1, containing IgM; Fr 2, containing IgG and a low level of haemolytic complement, and Fr 1 (abs), depleted of IgG). A guinea-pig serum pool was used as a complement source in the immobilisation experiments. The influence was studied of removal of lysozyme from guinea-pig serum on the immobilisation reactions. Further experiments were performed, using a fluorescence technique, to detect C3b depositions on fixed treponemes and treponemes in suspension. RESULTS: Rapid immobilisation of Percoll-purified treponemes by the NHS serum fractions occurred only after preincubation with Fr 1 and Fr 2 simultaneously. This was largely dependent on the presence of a small amount of haemolytic C in Fr 2. Removal of lysozyme reduced this rapid rate of immobilisation. In fluorescence experiments it was demonstrated that C3b deposition on fixed (that is damaged) treponemes occurred upon their incubation with Fr 2 or the combination of Fr 1 and 2. However, on treponemes in suspension C3b deposition occurred only after incubation with the combination of Fr 1 and 2. CONCLUSION: The rapid immobilisation of Percoll purified treponemes by serum fractions from normal human serum requires antibodies of the IgM and IgG class, together with complement and lysozyme. Omission of one of these reactants slows immobilisation. Our experiments suggest that the reactants act in sequence: the loss of integrity of the outer membrane by an attack by IgM and C offers the opportunity for lysozyme to hydrolyse the peptidoglycan layer surrounding the cytoplasmic membrane of the treponemes, which then is accessible for attack by antibodies and C.