Immunoblots demonstrate that 40 strains of influenza encompassing HA subtypes H1 to H13, as well as B strains from the Yamagata and Victoria lineage were detected when the two mAbs are combined to from a pan-HA mAb cocktail. peptide-conjugate. Immunoblots demonstrate that 40 strains of influenza encompassing HA subtypes H1 to H13, as well as B strains from the Yamagata and Victoria lineage were detected when the two mAbs are combined to from a pan-HA mAb cocktail. Quantification using this pan-HA mAbs cocktail was achieved in a dot blot assay and results correlated with concentrations measured in a hemagglutination assay with a coefficient of correlation of 0.80. A competitive ELISA was also optimised with purified viral-like particles. Regardless of the quantification method used, pan-HA antibodies can be employed to accelerate process development when strain-specific antibodies are not available, and represent a valuable tool in case of pandemics. These antibodies were also expressed in CHO cells to facilitate large-scale production using bioreactor technologies which might be required to meet industrial needs for quantification reagents. Finally, a simulation model was created to predict the binding affinity of the two anti-HA antibodies to the amino acids composing the highly conserved epitope; different probabilities of interaction between a given amino acid and the antibodies might explain the affinity of each antibody against different influenza strains. == Introduction == Influenza is a contagious disease that can lead to hospitalization, and even death for vulnerable patients. The influenza virus belongs to theOrthomyxoviridaefamily and is classified as type A, B or C. Annual influenza infections are caused by type A, and to a lesser extent by type B [1]. Two viral surface glycoproteins are used to identify the subtype: hemagglutinin (HA) and neuraminidase (NA). Type A can PKC-IN-1 further be classified into two phylogenetic groups based on HA (group 1 and 2). Disease prevention is achieved through vaccination, and the production of vaccine lots is initiated several months before the flu season based on predictions. When vaccines are well matched with the circulating strains, vaccination offers up to 83% protection [2]. Unfortunately, new strains of influenza emerge every few years and lead to a mismatch between the predicted vaccine strains and the circulating strains, which can significantly decrease the efficacy of the vaccine. Currently, sixty percent of influenza vaccines are still produced by inoculating fertilized eggs, but new production platforms have been reported or are being used such as insect-cell cultures [3], other mammalian cell lines [49] as well as plant-based vaccines [1013]. Despite the progress made in the influenza vaccine industry, the quantification remains a challenge. The main quantification method approved by regulatory agencies is the single radial immunodiffusion (SRID) assay, which is used to quantify HA. The dose of a trivalent PKC-IN-1 or quadrivalent influenza vaccine is typically 45g to 60 g HA (15g of each strain). The SRID assay has been used by the influenza vaccine community PKC-IN-1 since 1978 and a PKC-IN-1 large amount of historical data is available PKC-IN-1 [14]. The main limitation however, is that it necessitates the use of strain-specific anti-HA polyclonal antibodies and calibrating-antigens. The production of SRID reagents requires about three months which can significantly slow down the release of new vaccines, and constitutes a major limiting factor in the case of pandemics. The two main options would therefore be to generate universal SRID-reagents that could be used with all influenza strains, or to adopt an alternative quantification method that would not be limited by the availability of reagents. A number of putative quantification methods were reviewed recently [15]. To support the progress of Rabbit polyclonal to KCNV2 alternative quantification methods and to speed up process development of new vaccines, antibodies that can recognize HA from multiple strains of influenza are a valuable tool. For instance, a polyclonal antibody named Uni-1 was generated in rabbits against a highly conserved sequence found between the two subunits of HA (the fusion peptide) [16]. Thirteen influenza A subtypes, as well as an influenza B subtype were detected by Uni-1..
