title: Contribution of murine innate serum inhibitors toward interference within influenza virus immune assays.
abstract: BACKGROUND
  Prior to detection of an antibody response toward influenza viruses using the hemagglutination inhibition assay (HAI), sera are routinely treated to inactivate innate inhibitors using both heat inactivation (56°C) and recombinant neuraminidase [receptor-destroying enzyme (RDE)].
  
  
  OBJECTIVE
  We revisited the contributions of innate serum inhibitors toward interference with influenza viruses in immune assays, using murine sera, with emphasis on the interactions with influenza A viruses of the H3N2 subtype.
  
  
  METHODS
  We used individual serum treatments: 56°C alone, RDE alone, or RDE + 56°C, to treat sera prior to evaluation within HAI, microneutralization, and macrophage uptake assays.
  
  
  RESULTS
  Our data demonstrate that inhibitors present within untreated murine sera interfere with the HAI assay in a manner that is different from that seen for the microneutralization assay. Specifically, the γ class inhibitor α(2) -Macroglobulin (A2-M) can inhibit H3N2 viruses within the HAI assay, but not in the microneutralization assay. Based on these findings, we used a macrophage uptake assay to demonstrate that these inhibitors can increase uptake by macrophages when the influenza viruses express an HA from a 1968 H3N2 virus isolate, but not a 1997 H3N2 isolate.
  
  
  CONCLUSIONS
  The practice of treating sera to inactivate innate inhibitors of influenza viruses prior to evaluation within immune assays has allowed us to effectively detect influenza virus-specific antibodies for decades. However, this practice has yielded an under-appreciation for the contribution of innate serum inhibitors toward host immune responses against these viruses, including contributions toward neutralization and macrophage uptake.
authors: Division of Basic Biomedical Sciences, University of South Dakota, Vermillion, SD 57069-2390, USA.
authors: Kevin T
authors: KT
authors: Cwach
authors: Heather R
authors: HR
authors: Sandbulte
authors: Joshua M
authors: JM
authors: Klonoski
authors: Victor C
authors: VC
authors: Huber
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D006385:Hemagglutination Inhibition Tests / Q000379:methods*; D006358:Hot Temperature; D053122:Influenza A Virus, H3N2 Subtype / Q000276:immunology*; D008264:Macrophages / Q000276:immunology; D051379:Mice; D009439:Neuraminidase / Q000378:metabolism; D009500:Neutralization Tests; D010587:Phagocytosis; D044967:Serum / Q000276:immunology*; D013048:Specimen Handling / Q000379:methods*
chemical_list: D000914:Antibodies, Viral; D009439:Neuraminidase