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Clinical and Diagnostic Laboratory Immunology, March 2004, p. 325-329, Vol. 11, No. 2
1071-412X/04/$08.00+0     DOI: 10.1128/CDLI.11.2.325-329.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Heterophile Antibody Interference in a Multiplexed Fluorescent Microsphere Immunoassay for Quantitation of Cytokines in Human Serum

Thomas B. Martins,1* Brian M. Pasi,1 Christine M. Litwin,1,2 and Harry R. Hill1,2

Associated Regional and University Pathologists Institute for Clinical and Experimental Pathology,1 Department of Pathology, Pediatrics and Medicine, University of Utah School of Medicine, Salt Lake City, Utah2

Received 19 June 2003/ Returned for modification 25 November 2003/ Accepted 19 December 2003

While modern immunoassays provide sensitive and specific means for the quantitation of cytokines in biological fluids, heterophile antibodies are still a well-recognized cause of interference in the measurement of cytokines in these assays. We have developed a multiplexed fluorescent microsphere immunoassay for the simultaneous quantification of 10 cytokines in only 75 µl of serum. During the development of this multiplexed assay, the amount of assay interference due to heterophile antibodies was also determined, and methods for detecting heterophile interference and minimizing its effect were incorporated into the assay. Heterophile antibodies resulted in significantly elevated cytokine values compared to those of normal blood bank samples. These falsely elevated values, and thus the components of the assay the heterophile antibodies were binding to, were identified through the use of internal controls. This information was then used to design assay-specific blockers and absorbents that were shown to significantly reduce falsely elevated cytokine values while not affecting the standard and control values. The fluorescent multiplexed microsphere-based immunoassay can be used to quantitate multiple cytokines from a single sample and should be a useful tool in furthering our understanding of the role of cytokines in disease processes.

* Corresponding author. Mailing address: ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108. Phone: (801) 583-2787, ext. 2158. Fax: (801) 584-5109. E-mail: martintb{at}aruplab.com.

Clinical and Diagnostic Laboratory Immunology, March 2004, p. 325-329, Vol. 11, No. 2
1071-412X/04/$08.00+0     DOI: 10.1128/CDLI.11.2.325-329.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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