Improvement of monoclonal antibody-immobilized granulocyte antigen assay for the detection of anti-HNA-1 alloantibodies.

BACKGROUND: Currently, the gold standard for the identification of antibodies against human neutrophil antigens (HNAs) is the monoclonal antibody-immobilized granulocyte antigen (MAIGA) assay. However, the handling of this assay is laborious and therefore cumbersome for the rapid screening of neutrophil antibodies.

STUDY DESIGN AND METHODS: In this study, we simplified the performance of the conventional MAIGA procedure and approved it for the identification of anti-HNA-1 with HNA-1-typed neutrophils and stable transfected (HEK293) cell line expressing HNA-1a, -1b, and -1c.

RESULTS: In contrast to the conventional MAIGA, all working steps including the incubation of antibodies with cells, washings, cell lysis, and subsequent antibody detection could be performed on microtiter plates. This modification accelerates the work schedule of MAIGA and reduces pipetting errors. Comparison between both assay performances using neutrophils as target showed concordant results. Subsequently, stable mammalian cell lines were tested. In comparison to neutrophils, cell lines were stable for a longer period of time (>4 weeks) and results obtained with these cell lines showed better interassay precision. Analysis of different FcγRIIIb capture monoclonal antibodies (MoAbs) for the MAIGA assay showed that MoAb LNK16 is superior for the detection of anti-HNA-1a, -1b, and -1c, whereas MoAb 3G8 showed false-negative results, caused by competitive inhibition of anti-HNA-1c alloantibodies.

CONCLUSION: The modification of MAIGA and the use of transfected HEK293 cells improve the detection of anti-HNA-1 alloantibodies that may allow screening analysis in large cohort of samples.