Generation and Characterization of Anti-phenyl Sulfate Monoclonal Antibodies and a Potential Use for Phenyl Sulfate Analysis in Human Blood

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  • Kanemitsu Yoshitomi
    Laboratory of Oncology, Pharmacy Practice and Sciences, Graduate School of Pharmaceutical Sciences, Tohoku University
  • Tsukamoto Hiroki
    Laboratory of Oncology, Pharmacy Practice and Sciences, Graduate School of Pharmaceutical Sciences, Tohoku University
  • Matsumoto Yotaro
    Laboratory of Oncology, Pharmacy Practice and Sciences, Graduate School of Pharmaceutical Sciences, Tohoku University
  • Nozawa-Kumada Kanako
    Laboratory of Molecular Transformation, Graduate School of Pharmaceutical Sciences, Tohoku University
  • Kondo Yoshinori
    Laboratory of Molecular Transformation, Graduate School of Pharmaceutical Sciences, Tohoku University
  • Abe Takaaki
    Department of Medical Science, Graduate School of Biomedical Engineering, Tohoku University Department of Clinical Biology and Hormonal Regulation, Graduate School of Medicine, Tohoku University
  • Tomioka Yoshihisa
    Laboratory of Oncology, Pharmacy Practice and Sciences, Graduate School of Pharmaceutical Sciences, Tohoku University

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Abstract

<p>Patients with chronic kidney disease (CKD) have increased blood levels of phenyl sulfate (PS), a circulating uremic toxin. In this study, we produced anti-PS monoclonal antibodies (mAbs) and characterized their cross-reactivity to structural PS analogs. To induce PS-specific mAbs, we synthesized 4-mercaptophenyl sulfate with a sulfhydryl group at the para-position of PS and conjugated it to carrier proteins via bifunctional linkers. Using these PS conjugates as immunogens and as antigens for enzyme-linked immunosorbent assay (ELISA) screening, we produced by a hybridoma method two novel mAbs (YK33.1 and YKS19.2) that react with PS conjugates independent of carrier and linker structures. Although all of the PS analogs tested, with the exception of indoxyl sulfate, were cross-reactive to both mAbs in phosphate buffered saline (PBS), PS specificity for YKS19.2 was enhanced in human plasma and serum. YKS19.2 mAb was cross-reactive only with o-cresyl sulfate, which is absent in human blood. PS sensitivity for YKS19.2 mAb increased to an IC50 of 10.4 µg/mL when 0.1% Tween 20 was added in a primary competitive reaction. To explore potential clinical applications, we determined concentrations of PS in serum samples from 19 CKD patients by inhibition ELISA using YKS19.2 mAb and compared them to those found using an LC-MS/MS method. A good correlation was observed between each value (R2=0.825). Therefore, the unique antigen specificity of YKS19.2 mAb could be useful for prescreening of patients with accumulated PS or for comprehensive analysis of uremic toxins that have a PS-like structure.</p>

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