Rapid enzyme-linked immunosorbent assay for the detection of antibodies against human neutrophil antigens -1a, -1b, and -1c


  • This work was supported by the German Research Foundation TRR84 and grant from the Excellence Cluster Cardiopulmonary System (to SS and BB).

Sentot Santoso, PhD, Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University, Langhansstrasse 7, D-35385 Giessen, Germany; e-mail: Sentot.Santoso@immunologie.med.uni-giessen.de.


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.