P1.28 Production of a polyclonal antibody against the vaginolysin ofgardnerella vaginalis

Introduction Bacterial vaginosis (BV) is a polymicrobial syndrome characterised by the decrease in Lactobacilli and an increase of anaerobic bacteria, mainly G. vaginalis a Gram-positive coccobacillus that is isolated in up to 98% of BV cases. This bacteria produces differents virulence factors like sialidase, succinate, biofilm formation, phospholipase C, and vaginolysin (VLY). The VLY is a protein of 56 kDa that belongs to the family of cholesterol-dependent cytolysins, the function of this protein is the cellular lysis of erythrocytes and epithelial cells through the binding to the CD59 receptor and cholesterol present in cell membranes. The production of a policlonal antibody agains VLY will allow the study of this cytolysin in the pathogenesis of Gardnerella vaginalis in the vaginal tract. Methods We use as antigen the ATCC 14018 of G. vaginalis wich was obtained from a extract of total proteins by electrophoresis. The band corresponding to the molecular weight of the VLY (56 kDa) was cut, macerated and resuspended in PBS. Two New Zealand rabbits were immunised for 6 weeks, for the first immunisation Freund’s complete adjuvant was used while for the second to fifth immunisation we use Freund’s incomplete adjuvant, in the sixth week only the antigen was used. At week 0, 3 and 6, rabbits were bled for the evaluation of the immune response by the indirect ELISA. The final bleeding was performed and the serum obtained was stored at −20°C until use. For he purification of the antibodies we use a protein A purification kit and the integrity of the immunoglobulins was verified by electrophoresis, the titration of the obtained polyclonal antibody was performed by indirect ELISA. We evaluated the antibody by Western blot, Dot blot and inhibition of hemagglutination using as antigen the ATCC 14018 of G. vaginalis. Results The first evaluation of the immune response of rabbits showed that prior to immunisation the rabbits had no antibodies against VLY, whereas during the third and sixth week of immunisation they had antibody titres of 1:250 and 1:1000 respectively. Electrophoresis of the polyclonal antibody showed the purity and integrity of the purified antibodies, this antibodies can be use at titers of 1:1000 for the subsequent assays. The western blot showed that this antibody recognised a band of approximately 56 kDa that matched with the molecular weight reported for the VLY, whereas the Dot blot showed that the antibody recognises the VLY of ATCC 14018 of G. vaginalis. To corroborate that the antibody inhibited erythrocyte lysis, hemagglutination inhibition assays were performed and we showed that the use of this antibody decreased the cell lysis in around 80%. Conclusion We produce a polyclonal antibody agains the VLY of G. vaginalis capable of inhibiting the erythrocyte lysis. This antibody will be useful in investigating the role of VLY in the pathogenesis of G. vaginalis during the development of BV.