Improvement of monoclonal antibody–immobilized granulocyte antigen assay for the detection of anti‐HNA‐1 alloantibodies

Abstract

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.

Human neutrophil antigens: Nature, clinical significance and detection

“…This assay may be performed using freshly isolated or cryopreserved granulocytes as well as by cell lines expressing granulocyte antigen. MAIGA is currently regarded as the gold standard for identification of granulocyte antibody specificity and modifications to avoid reliance on live cells, for example use of transfected human embryonic kidney cells, may permit higher throughput (Heinzl et al, 2015;Simtong et al, 2018). on Luminex would require use of a secondary conjugated antibody (duplicate testing for each isotype) which would also increase costs (Flesch, 2019;Schulz et al, 2017).…”

Autoimmune Neutropenias: Update on Clinical and Biological Features in Children and Adults

“…The other pitfall is related to the presence of autoantibodies against HLA antigens, as a consequence of previous pregnancies and/or blood transfusions, that cross‐react in the GAT and GIFT giving a false positive signal 37 . A means to circumvent this problem is the inclusion of tests identifying the specificity of the autoantibodies, such as the monoclonal antibody immobilization of granulocyte antigen (MAIGA) test 37 . In this test, several HNA epitopes (usually CD16, CD11a, CD11b, and CD177) (Figure 1) are captured by monoclonal antibodies, which allow the recognition of specific antibodies to these glycoproteins irrespective of the potential presence of antibodies to HLA.…”

“…Such putative epitopes have not been characterized yet. The other pitfall is related to the presence of autoantibodies against HLA antigens, as a consequence of previous pregnancies and/or blood transfusions, that cross‐react in the GAT and GIFT giving a false positive signal 37 . A means to circumvent this problem is the inclusion of tests identifying the specificity of the autoantibodies, such as the monoclonal antibody immobilization of granulocyte antigen (MAIGA) test 37 .…”

“…37 A means to circumvent this problem is the inclusion of tests identifying the specificity of the autoantibodies, such as the monoclonal antibody immobilization of granulocyte antigen (MAIGA) test. 37 In this test, several HNA epitopes (usually CD16, CD11a, CD11b, and CD177) (Figure 1) are captured by monoclonal antibodies, which allow the recognition of specific antibodies to these glycoproteins irrespective of the potential presence of antibodies to HLA. MAIGA, however, is a complex assay offered by specialized laboratories only.…”

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

“…In antigen-capture assays such as MAIGA, the capture antibody may compete with the alloantibody due to epitope overlap, resulting in falsenegative results. 2,3 Thus, MAIGA assays require multiple capture antibodies with different epitopes and consequently, relatively large amounts of serum, cells, and reagents. MAIGA is also a time-consuming and low-throughput assay due to procedure complexity and requirements for highly skilled personnel to obtain consistent results.…”