Abstract
Over the past years, neutrophil extracellular traps (NETs) were shown to contribute to states of acute and chronic inflammatory disease. They are composed of expelled chromatin and decorated by neutrophil-derived proteins. Therefore, the analysis of DNA complexes with myeloperoxidase (MPO) by ELISA has become an attractive tool to measure NET formation in in vitro and in vivo samples. When we used a published MPO-DNA ELISA protocol and included an isotype control for the anti-MPO coating antibody, we observed high assay specificity for in vitro prepared NET samples, whereas the specificity for in vivo plasma samples was low. In addition, the assay failed to detect in vitro generated MPO-DNA complexes when spiked into plasma. Therefore, we set out to improve the specificity of the MPO-DNA ELISA for plasma samples. We found that the use of Fab fragments or immunoglobulins from different species or reversal of the antibody pair led to either a high background or a low dynamic range of detection that did not improve the specificity for plasma samples. Also, the use of higher plasma dilutions or pre-clearing of plasma immunoglobulins were ineffective. Finally, we found that a commercial reagent designed to block human anti-mouse antibodies and multivalent substances increased the detection window between the MPO antibody and isotype control for highly diluted plasma. We applied this modified ELISA protocol to analyze MPO-DNA complexes in human blood samples of acute and chronic inflammatory conditions. While markers of neutrophil activation and NET formation such as MPO, elastase and citrullinated histone H3 correlated significantly, we observed no correlation with the levels of MPO-DNA complexes. Therefore, we conclude that ELISA measurements of MPO-DNA complexes in human plasma are highly questionable regarding specificity of NET detection. In general, plasma analyses by ELISA should more frequently include isotype controls for antibodies to demonstrate target specificity.
Highlights
Neutrophil extracellular trap (NET) formation is a type of cell death distinct from apoptosis and necrosis [1, 2] that extends the classical neutrophil defense strategies of phagocytosis and degranulation [3]
Our efforts to improve the MPO-DNA enzyme-linked immunosorbent assay (ELISA) protocol for plasma samples led to a detectable decrease in unspecific signals for isotype control which we further deducted from corresponding plasma MPO-DNA measurements to best reflect specific signals
The essential lack of correlation between measured MPO-DNA levels and other neutrophil activation or NET parameters in this set of plasma samples leads us to question the suitability of the ELISA to reliably quantitate circulating MPO-DNA complexes reflecting in vivo generated NETs
Summary
Neutrophil extracellular trap (NET) formation is a type of cell death distinct from apoptosis and necrosis [1, 2] that extends the classical neutrophil defense strategies of phagocytosis and degranulation [3]. NETs have been originally characterized as web-like extracellular structures composed of nuclear DNA, histones and neutrophil granule proteins resulting in a high local concentration of antimicrobial substances to entrap and destroy pathogens [4]. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2)-triggered ROS stimulate the nuclear translocation of two neutrophil granule proteins, neutrophil elastase (NE) and myeloperoxidase (MPO). NE and MPO are major mediators of histone degradation and chromatin decondensation and promote NET formation [6, 7]. Decondensation of nuclear DNA and subsequent NET release can as well be induced by peptidylarginine deiminase 4 (PADI4)-driven histone citrullination [8]
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