An ELISA for detection of complement-bound circulating immune complexes in mice

Abstract

Measurements of complement-bound circulating immune complexes (cCICs) in pre-clinical studies may provide important information about the etiology of certain pathology findings suggestive of being immune complex mediated. This article describes the development and qualification of a universal methodology to measure cCIC in mice after dosing with species foreign proteins. The assay is a sandwich enzyme-linked immunosorbent assayexclusively based on commercially available reagentsthat could detect mouse IgG bound to complement C3 independent of the test-substance present in the plasma sample. Heat-aggregated serum was used as positive control. The assay was qualified by assessment of acceptance criteria, stability of positive control, precision, and specificity. Finally, the performance of the assay was tested using plasma from mice administered either of three different proteins, i.e bovine serum albumin (BSA), a fully human monoclonal antibody, and a humanized monoclonal antibody.

Formation and glomerular deposition of immune complexes in mice administered bovine serum albumin: Evaluation of dose, frequency, and biomarkers

“…Detection of IC formation and deposition was performed by supplemental measures of BSA exposure, CIC and cCIC concentrations by ELISA, pathological changes by H&E and PAS stainings, and IC deposits by IHC. The CIC assay measures ADA bound to BSA, which in similar assay set-ups have been used to detect ADA bound to human IgG in mice and non-human primates (Stubenrauch et al 2010(Stubenrauch et al , 2012Pierog et al 2015;Boysen et al 2019a). This assay set-up was suggested to have improved drug-tolerance compared to a standard bridging ELISA for detection of monomeric ADA (Pierog et al 2015;Boysen et al 2019a).…”

“…Any CIC formed by ADA in a complex with BSA were analyzed in a sandwich ELISA, as described in Boysen et al (2019a), with modifications. In brief, rabbit anti-BSA (Thermo Fisher, Waltham, MA) diluted 1:10.000 in carbonate buffer (0.1 M; pH 9.6) was used as the coating antibody; peroxidase-conjugated…”

Formation and Glomerular Deposition of Immune Complexes in Mice Administered Human Antibodies: Evaluation of Dose, Frequency, and Biomarkers

“…[49][50][51][52][53] In contrast, exposure was not expected to interfere with measurements in the cCIC assay. 20 No measurement of aTNP-specific ADAs were included in the present studies; however, this would be interesting to include in future studies.…”

“…A cCIC sandwich ELISA was used for plasma concentration measurement of complement C3 bound to CIC (cCIC), as described previously. 20 Rabbit anti-C3 (Bioss Inc, Boston, Massachusetts) and goat anti-mIgG HRP (κ light chain–specific; Jackson ImmunoResearch) were used as binding and detecting antibodies, respectively. The cCIC signal was expressed as signal-to-noise ratio (OD sample /QC neg ) in order to normalize for plate variation.…”

“…During the studies, daily all mice were cage side monitored for clinical signs. Samples from the present studies were partly used for qualification and characterization of the immune assays 20,21 and vehicle animals partly described in a previous publication. 22…”