Immunometric Assay Interference: Incidence and Prevention Johan Bjerner, * Kjell Nustad, Lars F. Norum, Kari Hauge Olsen, and Ole P. Børmer Background: The primary aim of the study was to reduce interference in an in-house two-site, two-step immunometric assay. Methods: In the running laboratory routine, 11 261 samples were tested with a carcinoembryonic antigen (CEA) assay with bovine immunoglobulin but no mu- rine immunoglobulins in the buffer, in parallel to our routine CEA assay, using 15 mg/L heat-treated nonspe- cific murine immunoglobulin (MAK33) in the buffer and with the Fc fragments removed from the capture antibody. Results: The frequency of interference was estimated to be 4.0% (95% confidence interval, 3.3– 4.7%). The addi- tion of 15 mg/L native MAK33 had little effect (frequen- cy, 3.9%; 95% confidence interval, 3.2– 4.6%), whereas adding 15 mg/L heat-treated MAK33 reduced interfer- ence to 0.86% (0.61–1.12%), and adding 50 mg/L reduced it further to 0.06% (0 – 0.13%). Removing the Fc frag- ments by itself reduced interference to 0.10% (0.02– 0.19%). There were no statistically significant differ- ences for age (P <0.23) or gender (P <0.40) between patients with interference (n  210) and a randomly selected interference-negative control group (n  186). Interference was not constant in patients: 15 of 25 individuals positive for interference and with four or more samples screened for interference had an interfer- ence-negative sample either before or after the peak of interference. Conclusions: In a two-site, two-step immunometric as- say using mouse monoclonal antibodies, use of heat- treated nonspecific murine immunoglobulin in the buffer or removal of the Fc fragment from the capture antibody could improve performance. © 2002 American Association for Clinical Chemistry Interference is a serious problem in immunoassays. Het- erophilic antibodies and human anti-mouse antibodies (HAMAs) 1 are important sources of both positive and negative interference, particularly in two-site (sandwich) immunoassays (1–5 ). Kaplan and Levinson (6) defined interference from heterophilic antibodies as interference from human antibodies of any subclass against any part of a murine antibody, where the human antibodies are of sufficient titer and affinity to have an analytically signif- icant effect and the immunogen has not been identified. Kaplan and Levinson distinguish HAMAs, with a known immunogen, from heterophilic antibodies because they may give different types of interference. Interference from heterophilic antibodies has been documented as a tran- sient effect in case reports (7, 8), which is indicative of antigen-driven processes, although the antigens are not known. The frequency of interference from heterophilic anti- bodies has been investigated in several studies with somewhat differing results (9 –12 ). The observed fre- quency depends on the method of detection, but gender, age, smoking habits, and autoimmune or other diseases in the population studied are also suggested factors, as heterophilic antibodies have features common with rheu- matoid factors (RFs) (13 ). Furthermore, interference may be influenced by the handling of samples. We use combinations of two murine monoclonal anti- bodies in all our immunometric assays, and to avoid interference from complement (14, 15), we use only cap- ture antibodies of isotype IgG1. Our sample buffer was chosen after a flow cytometric study of interference in real time (16 ). When we recently changed assay formats from magnetic microspheres and radiolabeled tracers to micro- titer wells and europium labeling, we experienced a high frequency of interference in several tumor marker assays. This high frequency was seen both with the capture antibody directly coated on the microtiter plate and with Central Laboratory, Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway. *Author for correspondence. Fax 47-22-730725; e-mail johan.bjerner@ klinmed.uio.no. Received September 28, 2001; accepted January 10, 2002. 1 Nonstandard abbreviations: HAMA, human anti-mouse antibody; RF, rheumatoid factor; CEA, carcinoembryonic antigen; and BSA, bovine serum albumin. Clinical Chemistry 48:4 613– 621 (2002) Enzymes and Protein Markers 613