In each cell, the top entry gives the phi coefficient between the corresponding two markers

3 Influenza A Vaccine Antigen and Narcolepsy Risk Fig. 5. Western-blot analysis of the presence of NP in Pandemrix and Arepanrix H1N1 antigen suspension. Pandemrix and Arepanrix H1N1 antigen suspensions were run under reducing conditions and NP stained with monoclonal mouse NP-antibody. doi:10.1371/journal.pone.0114361.g005 In order to confirm the co-migration of NP and HA under reducing gel conditions, we performed a western blot analysis of the viral HA in both samples under reducing and non-reducing conditions. As shown in Fig. 4D and 4E, HA shifts clearly from its original band position in the reducing gel as compared to the corresponding position in the non-reducing gel. Unmasking NP from HA under non-reducing conditions allowed us to verify the large amount of NP present in both samples. Dual bands seen of both NP and HA at the 60 kDa molecular weight marker level under reducing conditions indicate that each protein masks the epitope response from the other due to the partial overlap of the bands. 13 / 23 Influenza A Vaccine Antigen and Narcolepsy Risk Increased IgG-antibody response in narcolepsy to H1N1 viral proteins of Pandemrix We then compared the IgG-antibody reactivity in children with narcolepsy and in healthy children against H1N1 viral proteins; HA and NA the major immunogens in influenza vaccines; and NP, a viral antigen found in a significant amount in Pandemrix according to our analyses. We first measured antibodies to HA using the HI test, in which antibody response to native viral HA is detected. We found a trend for higher titers of antibodies against influenza A/California/07/2009 vaccine strain and epidemic A/Finland/814/01 whereas no differences were seen in HI titers against or A/Finland/715/00 virus strains. In order to evaluate the antigenic moieties in the vaccine derived viral proteins HA, NP, and NA, we developed a sandwich EIA, in which the protein from the H1N1 viral antigen suspension of Pandemrix is SKI II site captured with a specific monoclonal antibody coated on the polystyrene wells. These coated, antigen containing plates were then used to determine plasma IgG binding to vaccine derived protein. When the specificity of the sandwich EIA for HA, NP, and NA was analyzed using monoclonal antibodies to viral proteins as PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19682619 detection antibodies, we found that detection antibody to NA bound to HA captured from Pandemrix, and vice versa, which suggests the presence of HA-NA complexes in H1N1 antigen suspension of Pandemrix. The sandwich EIA for NP captured from Pandemrix appeared to be antigen specific. We found that the binding of antibodies PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19681941 to NP captured from Pandemrix was enhanced in children with narcolepsy when compared to healthy vaccinated children. Due to the complex formation of HA and NA, the specific antibodies against Pandemrix derived HA or NA could not be determined. Lipid-protein micelles in H1N1 antigen of Pandemrix Because polysorbate 80 and Triton X, the non-ionic lipid detergents used for the manufacturing process of the Pandemrix H1N1 antigen suspension, are able to form micelles with proteins and lipids, and viral proteins could be in a complex due to micelle formation, we studied whether the viral proteins were incorporated in lower density lipid micelles in H1N1 antigen suspension. We separated micelles with different lipid-protein composition from Pandemrix H1N1 antigen suspension using a sequential density ultracentrifugation method validated for the isolation of human HDL, LDL and VLDL lipopr