A novel immunocomplex capture fluorescence assay (ICFA) using fluorescent beads and transfected cells for specific identification of human neutrophil antigen (HNA)‐1a and ‐1b antibodies

Abstract

BackgroundTo identify specific human neutrophil antigen (HNA) antibodies, assays using neutrophils such as monoclonal antibody‐specific immobilization of granulocyte antigens (MAIGA) are recommended. However, these assays are limited by labor‐intensive neutrophil preparation and varying antigen expression levels.MethodsWe evaluated a newly developed immunocomplex capture fluorescence assay (ICFA) for identifying HNA‐1 antibodies and compared it to MAIGA and LABScreen Multi (LABM), which utilizes recombinant HNA‐coated Luminex beads. For ICFA, HNA‐1a or HNA‐1b transfected cells replaced neutrophils. Cells incubated with serum were lysed, and immune complexes were captured using five CD16 monoclonal antibody‐conjugated Luminex beads. Nine antisera with known specificity and 26 samples suspected of containing HNA antibodies were analyzed by ICFA and MAIGA using neutrophils or transfected cells (ICFA‐N or ICFA‐T, and MAIGA‐N or MAIGA‐T, respectively).ResultsICFA‐T and MAIGA‐N accurately determined the specificity of all antibodies in the nine antiserum samples. The ICFA‐T detection limit was 2048‐fold for anti–HNA‐1a and 256‐fold for anti‐HNA‐1b; the limits of MAIGA‐T, MAIGA‐N, and LABM were 32‐, 4 ~ 64‐, and 128‐fold for anti‐HNA‐1a and 64‐, 16 ~ 64‐, and 32‐fold for anti–HNA‐1b, respectively. Twelve and 7 of the remaining 26 samples tested negative and positive, respectively, in both ICFA‐T and MAIGA‐N. Antibody specificity against HNA‐1a or HNA‐1b determined using ICFA‐T agreed with that determined using MAIGA‐N and LABM. Another seven samples tested positive in ICFA‐T but negative in MAIGA‐N.ConclusionThe novel ICFA is highly sensitive and exhibits specificity similar to MAIGA and LABM for detecting HNA‐1 antibodies.