Technical noteDetection and quantification of rituximab in the human urine
Introduction
The chimeric monoclonal antibody rituximab is used for depleting B lymphocytes in a wide variety of diseases (Gürcan et al., 2009, Keating, 2010, Plosker and Figgitt, 2003). The depleting capacity of the treatment is in general excellent (Wilk et al., 2009). However, in our clinic B cell depletion was incomplete in some patients suffering from nephrotic syndrome as assessed by monitoring B cell numbers in peripheral blood by flow cytometry (Stahl et al., 2017). Suspecting the insufficiency of the B cell depletion was due to loss of the therapeutic antibody via the impaired glomerular filtration barrier into the urinary space, we screened urine collected from nephrotic patients (Table 1) for the presence of rituximab. In order to determine the concentration of the suspected monoclonal antibody in urine samples in an easy and reliable assay, we followed a flow cytometric approach.
Section snippets
Urine samples
Spot urine samples from healthy controls (HC) and patients (PTS) after rituximab infusion (spot or 24-hour urine) were collected and kept frozen at − 20 °C until analysed.
Determination of total IgG in the human urine
For the general detection of antibodies in the human urine, total IgG levels were determined by nephelometry using a BN ProSpec analyzer (Siemens, Erlangen, Germany).
Cell culture
The CD20+ Daudi cell line was originally established from a Burkitt lymphoma (Klein et al., 1968). CD20 is constitutively and homogeneously expressed at high
Results
Daudi cells tolerated the presence of human urine well up to the maximal chosen concentration of 50% in the media for 30 min without any signs of damage as assessed by flow cytometry by checking dead and apoptotic cells. There was no difference between samples collected from healthy donors (HC) or patients (PTS) suffering from nephrotic syndrome. Viability of ungated Daudi cells was always around 90% (data not shown) confirming that the cells were unaffected by the urine and hence suitable for
Discussion
This study shows that the detection of the specific antibody rituximab in the human urine by flow cytometry is feasible. The MFI of Daudi cells labeled with primary rituximab and secondary PE-tagged mouse anti human IgG corresponds well with the amount of rituximab present in the urine samples. By generating standard curves, rituximab can also be quantified with high sensitivity. For this study samples of spot urine and 24 h collections were used. In the future, the contents of total IgG and
Conclusion
The here reported flow cytometric approach is a reliable and easy-to-perform assay for the detection and quantification of rituximab in human urine samples. The information obtained this way might support monitoring and, if required, adapting the therapeutic regimens in order to improve efficacy and cost effectiveness of the biologica therapy. Although developed specifically for detecting rituximab in the urine of nephrotic patients, the assay can be adapted to other antibody-based therapies in
Disclosure
All authors declare that there is no conflict of interests regarding the publication of this paper.
Acknowledgements
R. Jacobs and T. Langer-Jacobus were supported by a grant from the Deutsche Forschungsgemeinschaft (DFG): SFB738/A5. M. Schiffer is supported by German Federal Ministry for Education and Research (BMBF) Grant 01GM1518A.
Authorship
Roland Jacobs conceived and designed the work, acquired data, interpreted results and drafted the manuscript
Thais Langer-Jacobus acquired data and revised the manuscript
Michelle Duong, acquired data and revised the manuscript
Klaus Stahl, acquired data and revised the manuscript
Hermann Haller, revised the manuscript, approved the final version
Reinhold E. Schmidt, revised the manuscript, approved the final version
Mario Schiffer, conceived the work, played an important role in interpreting the
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Both authors contributed equally to this study.