Evaluation of commercially available antibodies and fluorescent conotoxins for the detection of surface ganglionic acetylcholine receptor on the neuroblastoma cell line, IMR-32 by flow cytometry

Abstract

Commercially available antibodies that bind to the human muscle acetylcholine receptor (ACHR) have been validated previously for flow cytometric use (Keefe et al., 2009; Leite et al., 2008; Lozier et al., 2015). Despite a multitude of commercially available antibodies to other nicotinic ACHRs, validation in a wide variety of immunoassay formats is lacking; when studied, a large proportion of these antibodies have been deemed not fit for most research purposes (Garg and Loring, 2017).

We have recently described a flow cytometric immunomodulation assay for the diagnosis of Autoimmune Autonomic Ganglionopathy (AAG) (Urriola et al., 2021) that utilises the monoclonal antibody mab35(Urriola et al., 2021) which is specific for ganglionic ACHR (gnACHR) that contain α3 subunits (Vernino et al., 1998). Other fluorescent ligands for α3-gnACHR have not been validated for flow cytometric use.

We investigated 7 commercially sourced antibodies and 3 synthetic fluorescent novel conotoxins purported to specifically bind to the extracellular domains of the gnACHR, and compared the results to staining by mab35, using flow cytometry with the neuroblastoma cell line IMR-32. We also evaluated the degree of non-specific binding by depleting the cell membrane of the relevant acetylcholine receptor with a pre-incubation step involving the serum from a patient with Autoimmune Autonomic Ganglionopathy containing pathogenic antibodies to the ganglionic acetylcholine receptor. None of the assessed conotoxins, and only one antibody (mab35) was found to perform adequately in flow cytometric staining of the native ganglionic acetylcholine receptor.

Introduction

Acetylcholine receptors (ACHRs) are complex pentameric membrane-bound calcium ion channels present on the surface of mammalian cells. Antibody recognition of these receptors by commercially available antibodies or pathological autoantibodies in sera from human diseases may require intact, correctly assembled quaternary receptor structure. Cholinergic autonomic transmission is mediated, in part, by the ganglionic ACHR (gnACHR), which is composed of alpha3 (α3)-containing ACHR subunits, most often in combination with beta4 (β4) subunits.

Recent work in the field of novel synthetic conotoxins have identified a range of peptides that may potentially serve as fluorescent ligands to gnACHR, the most promising of which include TP-2212-59, TxID[9sa] and RegIIa that are able to selectively bind to rat α3β4 gnACHRs (Chang et al., 2014; Cuny et al., 2018; Ren et al., 2019; Wu et al., 2017). Fluorescent analogues of these conotoxins are yet to be validated on human gnACHRs.

The neuroblastoma cell line, IMR-32, has previously been characterised as a rich source of surface-expressed, fully formed, conformationally correct gnACHR (Nelson et al., 2001). Monoclonal antibody mab35 has previously been used in identifying muscle-type ACHR (Leite et al., 2008), for correctly identifying radiolabelled gnACHR on IMR-32 via immunoprecipitation, and has been used to validate a radioimmunoprecipitation assay to detect autoantibodies against gnACHR in the rare disorder Autoimmune Autonomic Ganglionopathy (AAG) (Vernino et al., 2000; Vernino et al., 1998). We have recently described a flow cytometric assay to detect immunomodulation of gnACHR exerted by cognate autoantibodies in patients with AAG utilising mab35 (Urriola et al., 2021).

Whereas there are many commercially available antibodies that reportedly bind to α3β4 gnACHRs or their subunits, there is a paucity of evidence that they are suitable for use in flow cytometry. We investigated the suitability of 7 commercially sourced primary antibodies and three novel fluorescently-labelled conotoxins to the gnACHR using IMR-32 as a model cell line, utilising mab35 as a comparator.