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

Abstract

Background: Canine anti-mouse antibodies are a potential source of immunoassay interference, but erroneous immunoassay results are not always easily identifiable.Anti-Müllerian hormone (AMH) is a marker for the presence of gonads in dogs, but elevated AMH concentrations in neutered dogs could also be caused by antibody interference. For other assays, a discrepant result obtained after antibody precipitation might indicate antibody interference. Objectives:We aimed to evaluate if canine anti-mouse antibodies are a source of erroneous results in the AMH assay and if antibody precipitation with polyethylene glycol (PEG) is a useful tool for detecting antibody interference in a variety of immunoassays used in the veterinary clinical laboratory.Methods: Twenty-nine positive and 25 negative samples for anti-mouse antibodies were analyzed for AMH, canine total thyroxine (TT 4 ), canine thyroid-stimulating hormone (TSH), and progesterone before and after treatment with PEG. Results that differed by more than four SDs from the intra-assay coefficients of variation were considered discrepant. Elevated AMH concentrations in neutered dogs with anti-mouse antibodies and no visible gonads present were considered evidence of interference.Results: Evidence of antibody interference was found in two samples analyzed for AMH. The presence of anti-mouse antibodies did not lead to a higher proportion of discrepant results after PEG treatment for any of the immunoassays. The overall incidence of discrepant results for healthy controls was very high (73%). Conclusions:Canine anti-mouse antibodies are a source of erroneous AMH results.Antibody precipitation with PEG is not a useful tool for detecting interference caused by such antibodies. K E Y W O R D Santibody, anti-Müllerian hormone, canine, PEG, polyethylene glycol

Prevalence of heterophilic antibodies in serum samples from horses in an equine hospital, and elimination of interference using chicken IgY

“…Evaluating the effect of heterophilic antibodies on immunoassays is challenging, as the true result most often is unknown. Reanalyzing the sample after precipitation with polyethylene glycol (PEG) can result in normalized values in humans [22], but the method does not work well for samples from dogs [8]. One assay with a known expected result for castrated animals is the AMH immunoassay.…”

“…This assay has the advantage of being very sensitive, but it is also prone to interference by heterophilic antibodies [1]. Heterophilic antibodies can cross-link capture antibodies with detection antibodies and have been shown to cause false-positive results in human medicine [2][3][4][5][6][7], for anti-Müllerian hormone (AMH) and B-type natriuretic hormone in dogs [8,9], and for equine growth hormone (eGH) in horses [10,11]. In human medicine, heterophilic antibodies can be grouped as true heterophilic antibodies, human antimouse antibodies (HAMA) and rheumatoid factors (RF) [1,12].…”

“…The frequency of interference in human serum samples has been reported to be from 0.5 to 2% to around 4% [15,16]. It will vary with the assay used but will be lower than the prevalence of heterophilic antibodies [8,16].…”

Pre-existing canine anti-IgG antibodies: implications for immunotherapy, immunogenicity testing and immunoassay analysis

“…The Fab regions of canine and murine IgG contain constant heavy and light chains with approximately 60–70% interspecies sequential homology 9 , so these antibodies may be cross-reactive autoantibodies that present as heterophilic antibodies when they bind to mouse IgG. Although canine heterophilic antibodies have received increased attention in recent years 8 , 10 – 12 , their origin and their underlying clinical significance remains elusive. The assumption is often that these antibodies originate from incidental, unknown exposures to animal antigen and that they wax and wane over time 13 , 14 , but most accounts are anecdotic as they come from individual case reports.…”

An introduction for the Veterinary Clinical Pathology Special Issue on clinical immunology

“…Electrophoresis and immunofixation techniques to characterize serum proteins are reviewed by Moore et al Harris et al validate a densitometric assay to quantify monoclonal proteins in canine sera, and Rout et al assess immunoglobulin heavy and light chain, and T‐cell receptor clonality to diagnose feline lymphoid neoplasia . Bergman et al investigate interferences from canine anti‐mouse antibodies in hormone immunoassays, and Irvine et al assess and compare two immunoassays to measure serum cardiac Troponin I in dogs and cats . Finally, Dörfelt et al and Raskin et al tackle immunocytochemical staining of previously stained smears.…”