title: Setting of methods for analysis of mucosal antibodies in seminal and vaginal fluids of HIV seropositive subjects from Cambodian and Italian cohorts.
abstract: BACKGROUND
  Genital mucosae play a key role in protection from STD and HIV infection, due to their involvement in both horizontal and vertical disease transmission. High variability of published observations concerning IgA isolation and quantification underlies the strong requirement of specific methods able to maximize investigation on HIV-specific IgA.
  
  
  METHODS
  Genital fluids from 109 subjects, including male and female cohorts from Italy and Cambodia, were collected, aliquoted and processed with different techniques, to assess optimal conditions maximizing mucosal antibody recovery. Three sampling techniques, up to sixteen preservation conditions, six ELISA methods and four purifications protocols were compared.
  
  
  RESULTS
  The optimal method here described took advantage of Weck-Cel sampling of female mucosal fluids. Immediate processing of genital fluids, with the addition of antibiotics and EDTA, improved recovery of vaginal IgA, while the triple addition of EDTA, antibiotics and protease inhibitors provided the highest amount of seminal IgA. Due to low amount of IgA in mucosal fluids, a high sensitive sandwich ELISA assay was set; sensitivity was enhanced by milk-based overcoating buffer and by a two-step biotin-streptavidin signal amplification. Indeed, commercial antisera to detect human immunoglobulins showed weak cross-reactivity to different antibody types. Three-step affinity purification provided reproducible immunoglobulin recovery from genital specimens, while conventional immuno-affinity IgA purification was found poorly manageable. Affinity columns were suitable to isolate mucosal IgA, which are ten-fold less concentrated than IgG in genital specimens, and provided effective separation of IgA monomers, dimers, and J-chains. Jacalin-bound resin successfully separated IgA1 from IgA2 subfraction.
  
  
  CONCLUSIONS
  Specific, effective and reliable methods to study local immunity are key items in understanding host mucosal response. The sequence of methods here described is effective and reliable in analysing humoral local responses, and may provide a solid advance to identify and measure the effective mucosal responses to HIV.
authors.affiliation: Division of Immunology, Infectious Diseases and Trasplantation, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Carla
authors.initials: C
authors.lastname: Donadoni
authors.forename: Cinzia
authors.initials: C
authors.lastname: Bisighini
authors.forename: Lorenza
authors.initials: L
authors.lastname: Scotti
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Diomede
authors.forename: Marie
authors.initials: M
authors.lastname: Ngyen
authors.forename: Janin
authors.initials: J
authors.lastname: Nouhin
authors.forename: Lucia
authors.initials: L
authors.lastname: DeSantis
authors.forename: Antonella
authors.initials: A
authors.lastname: Zambon
authors.forename: Davide
authors.initials: D
authors.lastname: Ferrari
authors.forename: Giulia
authors.initials: G
authors.lastname: Gallotta
authors.forename: Giovanni
authors.initials: G
authors.lastname: Corrao
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Pancino
authors.forename: Lucia
authors.initials: L
authors.lastname: Lopalco
mesh_terms: D000328:Adult; D000906:Antibodies / Q000737:chemistry*; D001826:Body Fluids / Q000276:immunology / Q000821:virology*; D002160:Cambodia; D015331:Cohort Studies; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D015658:HIV Infections / Q000276:immunology / Q000821:virology; D006679:HIV Seropositivity / Q000378:metabolism*; D006801:Humans; D007070:Immunoglobulin A / Q000276:immunology; D007558:Italy; D008297:Male; D008875:Middle Aged; D009092:Mucous Membrane / Q000276:immunology / Q000821:virology*; D012661:Semen / Q000276:immunology / Q000821:virology*; D014621:Vagina / Q000276:immunology / Q000821:virology*
chemical_list: D000906:Antibodies; D007070:Immunoglobulin A