Kasamatsu Shingo, Kakihana Yuki, Koga Taisei, Yoshioka Hisashi, Ihara Hideshi
Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Osaka 599-8531, Japan.
Antioxidants (Basel). 2020 Nov 21;9(11):1160. doi: 10.3390/antiox9111160.
Hydrogen sulfide (HS) is endogenously produced by enzymes and via reactive persulfide/polysulfide degradation; it participates in a variety of biological processes under physiological and pathological conditions. HS levels in biological fluids, such as plasma and serum, are correlated with the severity of various diseases. Therefore, development of a simple and selective HS measurement method would be advantageous. This study aimed to generate antibodies specifically recognizing HS derivatives and develop a colorimetric immunoassay for measuring HS in biological samples. We used -ethylmaleimide (NEM) as an HS 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 HS 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 HS in biological samples.
硫化氢（HS）由酶内源性产生，并通过活性过硫化物/多硫化物降解产生；它在生理和病理条件下参与多种生物过程。生物体液（如血浆和血清）中的HS水平与各种疾病的严重程度相关。因此，开发一种简单且选择性的HS测量方法将是有益的。本研究旨在生成特异性识别HS衍生物的抗体，并开发一种用于测量生物样品中HS的比色免疫测定法。我们使用N - 乙基马来酰亚胺（NEM）作为HS检测剂，它形成稳定的双 - S - 加合物（NEM - S - NEM）。我们还制备了与3 - 马来酰亚丙基酸的双 - S - 杂加合物，该杂加合物与牛血清白蛋白偶联后分别用于免疫日本白兔和Wistar大鼠，以产生多克隆抗体和单克隆抗体。通过竞争性酶联免疫吸附测定法对产生的抗体进行评估。我们能够获得两个稳定的杂交瘤细胞系，产生对NEM - S - NEM具有特异性的单克隆抗体。通过用单克隆抗体进行免疫测定，小鼠血浆中的HS水平测定为0.2μM，这与通过质谱检测到的水平相同。综上所述，这些单克隆抗体可成为用于简单且高度选择性免疫测定以检测生物样品中HS的有用工具。
