Characterization of canine anti-mouse antibodies highlights that multiple strategies are needed to combat immunoassay interference

Abstract

Immunoassays are widely used for detection and quantification of analytes in biological samples, but are vulnerable to analytical errors caused by interfering sample substances. Of particular interest are endogenous anti-animal antibodies that may bind to the immunoassay antibodies and cause erroneous test results. This phenomenon is a hazard to patient safety in both human and veterinary medicine. Here, we demonstrate that anti-mouse antibodies in dogs bind selectively to different regions of the murine IgG molecule, cross-react with IgG from different species, and consist of all major antibody classes present in canine serum (IgA, IgG and IgM). The antibody characteristics varied among individuals and their prevalence differed between two dog breeds. The selective binding to different IgG regions suggests that the antibodies might not originate from immunization through exposure to mice or other species. These findings show that canine anti-mouse antibodies are highly heterogeneous in nature and therefore require a combination of strategies to be counteracted.

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

“…It is possible that LLPCs continuously produce the antibodies after initial contact with mice before the first sampling, but if that is the case, all of the dog owners must have overlooked the contact. Moreover, the heterophilic antibodies exhibit a prolonged selective binding to either Fc- or F(ab′) 2 -fragments, while immunization to mouse IgG results in reactivity against both of these fragments 8 .…”

“…Heterophilic antibodies against mouse IgG were detected using a previously described method 8 . Briefly, microtiter plates were coated with 2 μg/mL mouse IgG, 50 μL of neat serum samples were added to the wells, and an HRP-conjugated monoclonal mouse IgG1k (MyBioSource, MBS592181) diluted 1:200 was used for detection.…”

“…According to a recent theory, anti-F(ab′) 2 -autoantibodies can form in response to in vivo cleavage of IgG by proteolytic enzymes associated with cancer and autoimmune diseases 6 , 7 . We have shown that Bernese mountain dogs, a breed well-known for its high prevalence of cancer, also show a high frequency of antibodies targeting F(ab′) 2 -fragments of mouse IgG 8 . 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.…”

“…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.…”

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

“…Heterophilic antibodies are a heterogeneous group, and multiple strategies are required to eliminate their effect on assay results [17]. One approach is taking advantage of the fact that heterophilic antibodies against mammalian IgG do not cross-react with chicken IgY.…”