Abstract
Hydrogen sulfide (H2S) is endogenously produced by enzymes and via reactive persulfide/polysulfide degradation; it participates in a variety of biological processes under physiological and pathological conditions. H2S levels in biological fluids, such as plasma and serum, are correlated with the severity of various diseases. Therefore, development of a simple and selective H2S measurement method would be advantageous. This study aimed to generate antibodies specifically recognizing H2S derivatives and develop a colorimetric immunoassay for measuring H2S in biological samples. We used N-ethylmaleimide (NEM) as an H2S detection agent that forms a stable bis-S-adduct (NEM-S-NEM). We also prepared bis-S-heteroadduct with 3-maleimidopropionic acid, which, in conjugation with bovine serum albumin, was to immunize Japanese white rabbits and Wistar rats to enable generation of polyclonal and monoclonal antibodies, respectively. The generated antibodies were evaluated by competitive enzyme-linked immunosorbent assay. We could obtain two stable hybridoma cell lines producing monoclonal antibodies specific for NEM-S-NEM. By immunoassay with the monoclonal antibody, the H2S level in mouse plasma was determined as 0.2 μM, which was identical to the level detected by mass spectrometry. Taken together, these monoclonal antibodies can be a useful tool for a simple and highly selective immunoassay to detect H2S in biological samples.
Highlights
Hydrogen sulfide (H2S), which was first documented as a highly hazardous chemical with a distinct smell of a rotten egg roughly 300 years ago, is a well-established gaseous molecule with numerous biological activities [1]
It has been reported that H2S is endogenously generated in mammalian cells and tissues from cysteine or cysteine derivatives via reactions catalyzed by cystathionine β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase [1,6,7], as well as by degradation of persulfides/polysulfides [8,9,10]
Kakinohana et al reported that inhaling H2S at 80 ppm after ischemia/reperfusion could prevent motor neuron degeneration in the ventral horn of the lumbar spinal cord via persulfidation of nuclear factor-κB (NF-κB) and p65 [11]
Summary
Hydrogen sulfide (H2S), which was first documented as a highly hazardous chemical with a distinct smell of a rotten egg roughly 300 years ago, is a well-established gaseous molecule with numerous biological activities [1]. Studies have reported H2S participation in a variety of biological processes, including antioxidative stress response [2,3], protection against ischemia-reperfusion injury [4], and the development/progression of neurodegenerative diseases [5], under physiological and pathological conditions. Kakinohana et al reported that inhaling H2S at 80 ppm after ischemia/reperfusion could prevent motor neuron degeneration in the ventral horn of the lumbar spinal cord via persulfidation of nuclear factor-κB (NF-κB) and p65 [11]. The chemical mechanisms associated with the biological activities of H2S are not fully elucidated
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