BACKGROUND: Several methods exist for the detection of neutrophil antibodies; most of them, however, require fresh neutrophils. In this study, an enzyme-linked immunosorbent assay (ELISA) using recombinant HNA-1 antigens (rHNAs) was developed to detect HNA-1a, -1b, and -1c alloantibodies in serum samples. STUDY DESIGN AND METHODS: Soluble rHNA-1a, -1b, and -1c were isolated from culture supernatant of transfected insect cells. Purified rHNA antigens were immobilized on microtiter wells using antibody against V5-Tag protein. Sera were added, and bound antibodies were detected by enzyme-labeled secondary antibodies. In parallel, monoclonal antibody-immobilized granulocyte antigen (MAIGA) was performed with two different monoclonal antibodies (MoAbs) against FcγRIIIb (3G8 and BW209). RESULTS: Fifteen MAIGA-positive sera containing HNA-1a alloantibodies were tested in ELISA. Thirteen of 15 (86.7%) MAIGA-positive sera captured by MoAbs 3G8 and/or BW209 reacted specifically with rHNA-1a. Four (26.7%) HNA-1a sera showed additional reaction with rHNA-1c. When anti-HNA-1b alloantibodies were analyzed in ELISA, 13 of 15 (86.7%) showed specific positive reaction with rHNA-1b, and 12 of 15 (80.0%) cross-reacted with rHNA-1c. Two HNA-1c sera reacted specifically with rHNA-1c. Immunoprecipitation analysis of all ELISA-negative HNA-1a and -1b sera did not show any specific band indicating false-positive reaction of these sera in MAIGA assay. CONCLUSIONS: These results suggested that rapid ELISA using recombinant neutrophil antigens may provide a valuable method for rapid screening of human alloantibodies against HNA-1a, -1b, and -1c in patients with neutropenia and in blood donors.
BACKGROUND: Several methods exist for the detection of neutrophil antibodies; most of them, however, require fresh neutrophils. In this study, an enzyme-linked immunosorbent assay (ELISA) using recombinant HNA-1 antigens (rHNAs) was developed to detect HNA-1a, -1b, and -1c alloantibodies in serum samples. STUDY DESIGN AND METHODS: Soluble rHNA-1a, -1b, and -1c were isolated from culture supernatant of transfected insect cells. Purified rHNA antigens were immobilized on microtiter wells using antibody against V5-Tag protein. Sera were added, and bound antibodies were detected by enzyme-labeled secondary antibodies. In parallel, monoclonal antibody-immobilized granulocyte antigen (MAIGA) was performed with two different monoclonal antibodies (MoAbs) against FcγRIIIb (3G8 and BW209). RESULTS: Fifteen MAIGA-positive sera containing HNA-1a alloantibodies were tested in ELISA. Thirteen of 15 (86.7%) MAIGA-positive sera captured by MoAbs 3G8 and/or BW209 reacted specifically with rHNA-1a. Four (26.7%) HNA-1a sera showed additional reaction with rHNA-1c. When anti-HNA-1b alloantibodies were analyzed in ELISA, 13 of 15 (86.7%) showed specific positive reaction with rHNA-1b, and 12 of 15 (80.0%) cross-reacted with rHNA-1c. Two HNA-1c sera reacted specifically with rHNA-1c. Immunoprecipitation analysis of all ELISA-negative HNA-1a and -1b sera did not show any specific band indicating false-positive reaction of these sera in MAIGA assay. CONCLUSIONS: These results suggested that rapid ELISA using recombinant neutrophil antigens may provide a valuable method for rapid screening of human alloantibodies against HNA-1a, -1b, and -1c in patients with neutropenia and in blood donors.