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
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.
Section snippets
Cell culture
IMR-32 was purchased from CellBank Australia and was grown in 25cm2 canted neck cell culture flasks in Eagle's Minimum Essential Media (EMEM) + 2 nM Glutathione + 1% Non-Essential Amino Acids + antibiotics (penicillin/streptomycin), supplemented with 10% Fetal Calf Serum (FCS). Cells were cultured at 37 °C, 5% CO2 and at 95% humidity. Media was replenished every 48–72 h, with cells either being harvested for staining or split when approximating 80% confluence by the addition of 1 mL of 0.25%
Results
All commercially available antibodies and fluorescent conotoxins failed to stain the gnACHR on IMR-32 cells appropriately, except for mab35 as seen in Fig. 3a. On live (unfixed) cells, mab35 stained 43.2% of cells as compared to the unstained cells, with all other commercial antibodies staining less than 10% of cells above the unstained cells, as seen in Figs. 3a and 4a. Three antibodies showed no meaningful staining as compared to the unstained cells (Orb159820, 102–16,035 and clone 1H11F2).
Discussion
Mab35 has previously been validated for identifying α3 containing ACHRs found on the surface of IMR-32(Vernino et al., 1998). We were able to support the use of this antibody also to fluorescently label the gnACHRs of IMR-32 in a flow cytometric assay. Specificity was confirmed, as when gnACHRs were depleted from the plasma membrane surface (via immunomodulation with human sera containing anti-gnACHR antibodies), staining by mab35 was minimal on live cells (Fig. 3, Fig. 4). Whilst some staining
Conclusions
This work helps to validate the use of mab35 for the flow cytometric evaluation of gnACHR on IMR-32, and confirms that the epitope on gnACHR that is recognized by mab35 is still stable after paraformaldehyde fixation.
Declaration of competing interest
The authors declare no conflict of interest. Funding of this work was provided through the Department of Immunology, Royal Prince Alfred Hospital. Author work declaration: Study design, implementation and manuscript preparation, NU. Radioimmunoprecipitation studies and manuscript review, BL. Manuscript review, SA.
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