Immunoglobulin Levels in Serum After MV130 Therapy

Levels of specific IgA and IgG against MV130 and against each bacteria contained in the mixture (i.e., S. pneumoniae, S. epidermidis, S. aureus, M. catarrhalis, K. pneumoniae, and H. influenzae) were determined in serum samples from blood collected before starting immunotherapy with MV130 and between 4 to 41 months after initiating the treatment. Quantification was performed by ELISA following standard procedures as previously described (26 (link)). Once collected, sera samples were stored frozen at −80°C until further processing. Briefly, 96-well non-tissue culture-treated plates were pretreated with poly-L-lysine (Sigma-Aldrich) for 1 h under UV light and coated with each of the heat-inactivated whole cell bacteria or the polybacterial mixture (300 FTU/ml ~ 10⁹ bacteria/ml) overnight at 4°C, and, subsequently, incubated with human serum dilutions for 2 h at room temperature. Specific immunoglobulins were detected with biotin rat anti-human IgA and IgG (both from Sigma-Aldrich). Signal was developed by incubation with streptavidin-horseradish peroxidase (HRP) (Sigma-Aldrich). Peroxidase activity was revealed by the addition of o-phenylenediamine dihydrochloride (Sigma-Aldrich) and the reaction was stopped with HCl 1N. Plates were read on an ELISA reader at 490 nm (Triturus Elisa, Grifols).