Alemtuzumab: validation of a sensitive and simple enzyme-linked immunosorbent assay

Jilani, Iman; Keating, Michael; Giles, Francis J.; O’Brien, Susan; Kantarjian, Hagop M.; Albitar, Maher

doi:10.1016/j.leukres.2004.04.003

« Previous Next »Leukemia Research
Volume 28, Issue 12 , Pages 1255-1262, December 2004

Alemtuzumab: validation of a sensitive and simple enzyme-linked immunosorbent assay

Iman Jilani
- Department of Hematopathology, Nichols Institute, Quest Diagnostics, 33608 Ortega Highway, Rm#108B, San Juan Capistrano, CA 92690-6130, USA
Michael Keating
- Department of Leukemia, MD Anderson Cancer Center, University of Texas, Houston, TX, USA
Francis J. Giles
- Department of Leukemia, MD Anderson Cancer Center, University of Texas, Houston, TX, USA
Susan O’Brien
- Department of Leukemia, MD Anderson Cancer Center, University of Texas, Houston, TX, USA
Hagop M. Kantarjian
- Department of Leukemia, MD Anderson Cancer Center, University of Texas, Houston, TX, USA
Maher Albitar
- Department of Hematopathology, Nichols Institute, Quest Diagnostics, 33608 Ortega Highway, Rm#108B, San Juan Capistrano, CA 92690-6130, USA
- Corresponding author. Tel.: +1 949 728 4784; fax: +1 949 728 4990; (mobile: +1 949 275 7564).

Received 1 December 2003; accepted 1 April 2004.

Abstract

Alemtuzumab (MabCampath^®) is a humanized rat monoclonal antibody that targets the CD52 antigen. It has been approved for the treatment of patients with resistant chronic lymphocytic leukaemia (CLL). Measuring plasma/serum levels of alemtuzumab is important for optimizing the dosing and scheduling of therapy; however, current assays in serum or plasma, based on the capture of alemtuzumab using CD52, are complicated and difficult to adapt for high throughput testing. We developed a simple sandwich enzyme-linked immunosorbent assay (ELISA) to measure alemtuzumab that takes advantage of the remaining rat sequence in alemtuzumab. Using specific anti-rat immunoglobulin (Ig) antibodies (absorbed against human Ig), alemtuzumab levels were measured in the serum and plasma of patients treated with alemtuzumab. Levels were similar between plasma and serum samples, in fresh samples and samples stored at 4°C for 24h, but were significantly lower in samples stored at room temperature for 24h. The assay was successfully used to determine serum alemtuzumab pre- and post-treatment. This assay is simple and adaptable for high throughput testing, with a limit of detection of 0.05μg/ml and a coefficient of variation of ±12.5%. No false positivity was observed in >200 samples tested. This validated assay should help optimize the dosing and scheduling of alemtuzumab therapy. The underlying principles are also applicable to the measurement of other humanized antibodies using an appropriate anti-Ig.