ANTIGLOBULINTEST-AustinCommunityCollege.pptVIP

ANTIGLOBULIN TESTandANTIBODY DETECTION AHLS 311 ANTIGLOBULIN TEST Detection of non-agglutinating Ab, especially IgG1 and IgG3 or complement components (C3d) affixed to RBCs in vivo or in vitro. Direct antiglobulin test (DAT) - detection of sensitization of RBC s (coated with Abs and/or complement components) in vivo Indirect antiglobulin test (IDAT) - detection of sensitization of RBCs in vitro ANTIGLOBULIN TEST Principle - Antihuman globulins (AHG) from immunized animals bind to human globulins either free in serum or attached to RBCs Pentameric IgM Abs are so large that, when bound to RBC Ags, the RBCs agglutinate (usually at RT) IgG Abs usually need a little help, a bridge molecule, to agglutinate RBCs AHG acts as a bridge molecule REAGENT PREPARATION Polyclonal or monoclonal sources (see Figure 4-1) Polyclonal - Animals hyperimmunized with human globulins; bled for antisera to obtain high titered, high avidity specificity to human Monoclonal - Hybridoma cells from mice; collection of culture or ascites fluid yields antisera mice hyperimmunized with human globulins prepare cell suspension from spleens; fuse with immortalized myeloma cells screen hybridoma clones for desirable specificities and affinities maintain cultures of clones in vivo or in vitro Product is highly regulated for potency REAGENT PREPARATION Polyspecific AHG Abs to human IgG, and Abs to human C3d (C3b breaks down to C3c and C3d) Advantage is that polyspecific AHG may detect complement-dependent Abs on RBCs (anti-Jka) Disadvantage - more nuisance positives Monospecific AHG Abs to human IgG only or human C3d only Fewer nuisance positives; may miss an important Ab DIRECT ANTIGLOBULIN TEST Detects in vivo sensitization of RBCs Procedure (see Color Plate 1) Wash unknown RBCs 3 X with saline (removes unbound globulins) Add AHG (binds to IgG or C3d, if any, that is bound to RBCs; forms agglutinates) Centrifuge (accelerates agglutination) Grade and record agglutination as 0 to 4+ Add Ab-coate