Immunometric Assay Interference

Abstract

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 murine immunoglobulins in the buffer, in parallel to our routine CEA assay, using 15 mg/L heat-treated nonspecific 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 addition of 15 mg/L native MAK33 had little effect (frequency, 3.9%; 95% confidence interval, 3.2–4.6%), whereas adding 15 mg/L heat-treated MAK33 reduced interference to 0.86% (0.61–1.12%), and adding 50 mg/L reduced it further to 0.06% (0–0.13%). Removing the Fc fragments by itself reduced interference to 0.10% (0.02–0.19%). There were no statistically significant differences 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 interference-negative sample either before or after the peak of interference. Conclusions: In a two-site, two-step immunometric assay 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.

Investigation of interference from canine anti‐mouse antibodies in hormone immunoassays

“…The frequency of interference in canine samples for the AMH Gen II assay is most likely considerably lower than the 9% reported in the screening of canine anti‐mouse antibodies since only two of the seven evaluated samples caused erroneous results. This data are consistent with larger cohort (>10 000 samples) studies of human samples submitted for immunoassay analysis, which estimate the frequency of interference to 0.03%‐4% …”

“…Increasing the MAK33 concentration to 1.0 mg/mL decreased the AMH concentrations by 57% for serum 1, and normalized the result from 5.71 to < 0.714 pmol/L in serum 2. Although the mechanism is not fully understood, aggregated antibodies have previously been shown to be superior blockers compared with native IgG . In the same study, it was also seen that 1.0 mg/mL heat‐aggregated MAK33 failed to normalize mouse IgG concentrations in only 1 out of 76 human patient samples with heterophilic antibodies.…”

Unusual results from immunoassays and the role of the clinical endocrinologist

“…14 Some studies have shown that interfering endogenous antibodies are present in up to 30 -40% of patient samples 77 but estimates vary widely. 96,97 A recently published report, however, suggests that interfering antibodies of various types only produce clinically discrepant results in about 0·5% of immunoassays, 14 although others have reported higher 16,98 and lower percentages. 87 Even with such low prevalence, the significance of interference must be appreciated within the many millions of immunoassay tests that are performed every year.…”

Avian IgY antibodies and their recombinant equivalents in research, diagnostics and therapy

“…Antigen Effect Authors Passive protection in rainbow trouts Lee et al [116] estimates of the prevalence of assay interference by heterophilic antibodies range from 1 to 80% [161e166]. A more thorough analysis of over 11,000 sera in an anti-CEA assay format revealed that about 4% of the results were potentially false [167]. Approaches to eliminate heterophilic antibody interference include the removal or inactivation of interfering immunoglobulins, e.g.…”

“…Approaches to eliminate heterophilic antibody interference include the removal or inactivation of interfering immunoglobulins, e.g. by precipitation with PEG [168], the use of various buffer additives [167], or the modification of assay antibodies by proteolytically removing of Fc fragments [167,169]. It is unlikely, however, that a single method can resolve these problems [170].…”