Goat anti human igg peroxidase

Plasma was collected from heparinised whole blood and stored at −80°C until use. HCMV infection status was determined by HCMV IgG ELISA (BioKit). IgG antibodies to pertussis toxin (PT; NIBSC) and to formalin-inactivated whole H1N1 influenza virus (influenza A/California/7/2006(H1N1)v(NYMC-X179A); H1N1; NIBSC) were determined using in-house ELISA assays with goat anti-human IgG-peroxidase (Sigma) as the secondary antibody and SIGMAFAST™ OPD (Sigma) as the substrate. IgG concentrations were calculated by interpolation from a standard curve which was produced using anti-pertussis reference serum (NIBSC; IU/ml) or using plasma from a donor with high titres of antibodies to H1N1 influenza (IgG concentration expressed in Arbitrary ELISA Units, AEU) [28 (link)]). The pooled AB plasma used for in vitro assays contained 6.8 IU/ml IgG to PT and had an H1N1 titre of 273.8 AEU.

Levels of total IgG against TIV (Split Virion BP, Sanofi Pasteur MSD) or inactivated H1N1 were determined by ELISA as previously described (25 (link), 29 (link)). Briefly, plasma samples were applied to antigen coated ELISA plates (NuncMaxisorp™) and bound IgG detected with goat anti-human IgG-peroxidase (Sigma) as the secondary antibody and SIGMAFAST™ OPD (Sigma) as the substrate. Antibody levels were calculated as arbitrary ELISA units (AEU) with reference to values obtained for an in-house, high titre standard plasma (assigned a value of 1000 AEU). The commercial AB serum used for tissue culture was estimated to contain 413.4 AEU against TIV and 273.7 AEU against H1N1, reflecting widespread natural exposure to influenza among the blood donor community. IgG-depleted AB plasma contained 7.6 and 14.6 AEU against TIV and H1N1, respectively. HCMV infection status was determined using a commercially available ELISA kit for anti-HCMV pp65 IgG (Biokit, Spain). Nineteen of the 52 study subjects (37%) were HCMV IgG seropositive.