title: HLA antibody specification using single-antigen beads--a technical solution for the prozone effect.
abstract: BACKGROUND
  Substantial progress in human leukocyte antigen antibody specification has been made by the introduction of Luminex single-antigen bead (SAB) assays. This progress was impaired when it turned out that this method is prone to a prozone effect leading to false-negative results in the case of high antibody titers. Testing serum and ethylenediaminetetraacetic acid (EDTA) plasma of one patient in parallel, we observed the prozone effect with the serum sample only. This led us to investigate complement component 1 (C1) as the cause of the prozone in SAB testing. We also found an easy way to overcome the prozone effect.
  
  
  METHODS
  Sera with a prozone effect were tested in the SAB assay, applying different methods of serum pretreatment to explore the parameters leading to the prozone.
  
  
  RESULTS
  The prozone was not observed when EDTA plasma or serum with EDTA added were tested. Further, addition of dithiothreitol, addition of C1 inhibitor, or heat inactivation of the sera abolished the prozone effect. Adding fresh nonimmune serum to heat-inactivated sera restored the prozone effect. Only beads showing a prozone were found to be covered with C1q.
  
  
  CONCLUSIONS
  Our observations are consistent with the hypothesis that dissociation or destruction of complement C1 eliminates the prozone effect. Addition of EDTA to serum of highly immunized patients is the easiest way to avoid false-negative results in SAB testing caused by a prozone effect.
authors.affiliation: Center for Clinical Transfusion Medicine, University Hospital of Tübingen, Otfried-Müller-Strasse 4/1, Tübingen, Germany. martina.schnaidt@med.uni-tuebingen.de
authors.forename: Martina
authors.initials: M
authors.lastname: Schnaidt
authors.forename: Christof
authors.initials: C
authors.lastname: Weinstock
authors.forename: Marija
authors.initials: M
authors.lastname: Jurisic
authors.forename: Barbara
authors.initials: B
authors.lastname: Schmid-Horch
authors.forename: Andrea
authors.initials: A
authors.lastname: Ender
authors.forename: Dorothee
authors.initials: D
authors.lastname: Wernet
mesh_terms: D000937:Antigen-Antibody Reactions; D003172:Complement C1 / Q000032:analysis; D015922:Complement C1q / Q000032:analysis; D004492:Edetic Acid; D005188:False Negative Reactions; D006680:HLA Antigens / Q000276:immunology*; D006801:Humans; D007118:Immunoassay / Q000379:methods* / Q000706:statistics & numerical data; D007074:Immunoglobulin G / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood; D007518:Isoantibodies / Q000097:blood*; D016030:Kidney Transplantation / Q000276:immunology*; D012680:Sensitivity and Specificity
chemical_list: D003172:Complement C1; D006680:HLA Antigens; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007518:Isoantibodies; D015922:Complement C1q; D004492:Edetic Acid