er of antibodies and showing a diversity of bands in the Western blot were selected to create serum pools of 5 individual sera per pool. Materials and Methods Ethical statement All human serum samples used for these studies were collected according to the general national ethical guidelines and upon consent from individual subjects. Sera from healthy individuals were collected for this 
and similar studies by Intercell with written consent given by each individual specifically for this study. Collection of sera at the Erasmus University Medical Center was approved specifically for this study by the medical ethical Committee of the Erasmus MC with patient consent given. Human sera were also collected at the Semmelweis University as approved specifically for this study by the ethical committee of Semmelweis University. All animal experiments were approved by Stockholm’s Norra djurforsokse tiska namnd and were conducted in agreement with the European Communities Council Directive 86/609/EEC and the Swedish animal protection legislation. Mice were scored and sacrificed according to the obtained ethical permission. ELISA on Ariflo web Bacterial cell lysates and recombinant proteins M. catarrhalis cells were grown in liquid medium at 37uC, 5% CO2 until a late log phase was reached. Cells were harvested by centrifugation and washed twice with PBS. Bacteria were re-suspended in PBS containing protease inhibitors and then lysed on ice by sonication and the supernatant was collected by centrifugation. ELISA plates were coated with either bacterial lysates or recombinant proteins and human serum samples were tested at 3-fold dilutions from 1:50 to 1:36,450. Highly specific Horse Radish Peroxidase conjugated goat anti-human IgG was used for signal detection. Bacterial strains and growth conditions M. catarrhalis strain RH4 was originally isolated from the blood of an infected patient and strain BBH18 was from the sputum of a COPD patient during an exacerbation. Both strains were obtained from Arne Forsgren and Kristian Riesbeck. Bacteria were grown in brain heart infusion broth at 37uC with shaking or on Columbia agar supplemented with 5% sheep blood or horse blood at 37uC. Additional M. catarrhalis strains and clinical isolates were obtained from the Pediatric department at Semmelweis University, Erasmus University Medical Center or were commercially acquired from GR Micro. The following strains from GR Micro isolated from patients with acute otitis media were used for the gene conservation studies: Australia, Belgium, Brazil, Canada, France, Germany, Hungary, Italy, Japan, 17496168 Portugal, South Africa, South Korea at 5 mg/mL and incubated overnight at 4uC. Sera were tested in duplicate at a 1:1000 dilution. Horse Radish Peroxidase conjugated anti-human IgG antibodies were used according to the manufacturer’s recommendation. ABTS was used as a substrate for HRP and the absorbance read at 405 nm. Preparation of IgGs from human serum pools Prior to library screening, human serum pools were adsorbed against E. coli cells in order to reduce background. The cell suspension was added to the serum pools and rotated overnight at 4uC. The next day, the mixture was centrifuged and the supernatants were transferred into a clean tube. The whole procedure was carried out three times for each single serum pool. Protective Moraxella 17702890 catarrhalis Antigens The E. coli adsorbed human sera were heat-inactivated at 56uC for 45 min and centrifuged to remove precipitated proteins. The su
