Antigen-free control wells in an ELISA set-up for the determination of autoantibodies against G protein-coupled receptors—a requisite for correct data evaluation

Abstract

First functional acting autoantibodies against G protein-coupled receptors such as the beta2-adrenoceptor in e.g. asthmatic patients have already been discovered in the early 1980s of the last century using assays that show their functional activity. Today, almost 40 years later, the measurement of such autoantibodies is still a challenge. Bioassays able to show the functional activity of such autoantibodies against G protein-coupled receptors are still the ne plus ultra for their detection and also classification when additionally exploiting specific receptor blockers for the neutralisation of the effect. Bioassays based on living cells make specific demands on the laboratories and are, therefore not suitable for every routine laboratory. Routine diagnostics, therefore, ideally requires different assays based on e.g. solid-phase technology, such as enzyme-linked immunosorbent assay (ELISA) technology. Here, endeavours are going on, using either the exact epitopes of such autoantibodies, if known, for trapping the autoantibodies, or the complete receptor in biological or artificial membranes that are immobilised onto a plastic carrier (ELISA principle). Here, we question and discuss the outcome of such tests, especially, if no controls such as the non-coated plastic carrier or the corresponding receptor-free membrane coat is offered as control in parallel, in light of the manifold experiences already collected with even non-agonistic acting autoantibodies by Güven et al. (J Immunol Methods 403:26–36, 2014).

P. falciparum and P. vivax Orthologous Coiled-Coil Candidates for a Potential Cross-Protective Vaccine

“…When testing each plasma sample without coating or in presence of the coiled coil peptides, some plasma gave a background signal equal or higher than the actual antigen-antibody reaction (Tables 4A,B, right panels). This phenomenon has been already reported in the literature (38)(39)(40)(41) and the term serum-specific background noise (SSBN) has been coined for this observation known to affect 4-32% of sera, especially when tested individuals had recent or ongoing bacterial infections at the time of blood sampling. In presence of SSBN, subtraction of the aspecific signal from the antigen-specific signal is suggested before data analysis, in order to increase assay sensitivity (38,40).…”

Antagonistic Autoantibodies to Insulin-Like Growth Factor-1 Receptor Associate with Poor Physical Strength

“…In our experience, this type of assay design is difficult to conduct for membrane proteins. Due to the low concentrations of natural aAb and the presence of other membrane constituents, the signal-to-noise ratio is typically high, as reported before, e.g., during TSHR-aAb assay development [19][20][21], or in relation to aAb to cardiac receptors of the G-protein coupled receptor family [22,23]. Nevertheless, additional studies comparing different detection methods are needed to highlight the particular strengths and weaknesses of different approaches and test their potential suitability for the diagnosis of aAb-related conditions.…”

Autoimmunity to the Follicle-Stimulating Hormone Receptor (FSHR) and Luteinizing Hormone Receptor (LHR) in Polycystic Ovarian Syndrome

“…A similar discrepancy between peptide sequences and full-length proteins or biological assays also exists with other GPCR, e.g., in the field of cardiac receptors, where no consensus has been reached yet, as biological and peptide-based ELISA assays give very incongruent results [49,50]. The issue of excessive noise due to the nature of the antigens as GPCR questioned early findings that lacked appropriate negative and positive controls [51]. In PCOS, the hypothesis of a primary role for another centrally important GPCR, i.e., the GnRHR, was tested by different techniques [52,53].…”