Elsevier

Journal of Immunological Methods

Volume 451, December 2017, Pages 118-121
Journal of Immunological Methods

Technical note
Detection and quantification of rituximab in the human urine

https://doi.org/10.1016/j.jim.2017.09.001Get rights and content

Abstract

B cell depletion by rituximab treatment might be inefficient in patients suffering from nephrotic syndrome. Due to the impaired glomerular filtration barrier a significant portion of the therapeutic antibody might be lost into the urinary space. In order to determine the amount of rituximab in the urine of such patients, CD20 + Daudi cells were stained with the patients' urine followed by a fluorochrome-labeled secondary antibody. Mean fluorescence intensity of that way labeled Daudi cells was determined by flow cytometry. Control samples with defined rituximab concentrations were used to create standard curves. The analyses revealed that all nephelometric IgG + urine samples tested also manifested rituximab at concentrations between 100 and 46,707 μg/L. The flow cytometry-based approach is an easy and reliable method to assess rituximab in patients' urine samples for monitoring individual rituximab treatment courses in all patients co-presenting impaired renal filtration. Presence of such antibodies in the urine could be considered as criteria to modify the formulation or modality of rituximab delivery in order to prevent the loss of the therapeutic antibodies and thereby ensuring efficacy of the therapy.

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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