Fit-for-purpose based testing and validation of antibodies to amino- and carboxy-terminal domains of cannabinoid receptor 1

Leyre Echeazarra,Maider López De Jesús,Leire Borrega-Román,Joan Sallés,Carlos Matute,Imanol González-Burguera,Xabier Aretxabala,Miquel Saumell-Esnaola,Gontzal García Del Caño,Sergio Barrondo,Catherine Ledent,María Aranzazu Goicolea,Susana Mato

doi:10.1007/s00418-021-02025-5

Abstract

Specific and selective anti-CB1 antibodies are among the most powerful research tools to unravel the complex biological processes mediated by the CB1 receptor in both physiological and pathological conditions. However, low performance of antibodies remains a major source of inconsistency between results from different laboratories. Using a variety of techniques, including some of the most commonly accepted ones for antibody specificity testing, we identified three of five commercial antibodies against different regions of CB1 receptor as the best choice for specific end-use purposes. Specifically, an antibody against a long fragment of the extracellular amino tail of CB1 receptor (but not one against a short sequence of the extreme amino-terminus) detected strong surface staining when applied to live cells, whereas two different antibodies against an identical fragment of the extreme carboxy-terminus of CB1 receptor (but not one against an upstream peptide) showed acceptable performance on all platforms, although they behaved differently in immunohistochemical assays depending on the tissue fixation procedure used and showed different specificity in Western blot assays, which made each of them particularly suitable for one of those techniques. Our results provide a framework to interpret past and future results derived from the use of different anti-CB1 antibodies in the context of current knowledge about the CB1 receptor at the molecular level, and highlight the need for an adequate validation for specific purposes, not only before antibodies are placed on the market, but also before the decision to discontinue them is made.

Highlights

The endogenous cannabinoid system is composed of endogenous ligands, such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), the enzymes responsible for their turnover and the inhibitory G-protein-coupled receptors (GPCRs) CB1 and CB2 (Piomelli 2003; Kano et al 2009). CB1 receptor is the most abundant GPCR in the central nervous system (Herkenham 1991; Piomelli 2003) and is densely expressed in brain (Herkenham 1991; Mailleux and Vanderhaeghen 1992; Matsuda et al 1993; Dove Pettit et al 1998; Tsou et al 1998; Marsicano and Lutz 1999; Egertová and Elphick 2000; Howlett et al 2002; McPartland et al 2007)
Five commercial antibodies designed against different sequences of the CB1 receptor highly conserved across human mouse and rat were assayed for specificity
Goat polyclonal antibodies N15 and H150 were designed against two different sequences of the N-terminal region of the human CB1 receptor

Summary

Introduction

The endogenous cannabinoid system is composed of endogenous ligands (endocannabinoids), such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), the enzymes responsible for their turnover and the inhibitory G-protein-coupled receptors (GPCRs) CB1 and CB2 (Piomelli 2003; Kano et al 2009). CB1 receptor is the most abundant GPCR in the central nervous system (Herkenham 1991; Piomelli 2003) and is densely expressed in brain (Herkenham 1991; Mailleux and Vanderhaeghen 1992; Matsuda et al 1993; Dove Pettit et al 1998; Tsou et al 1998; Marsicano and Lutz 1999; Egertová and Elphick 2000; Howlett et al 2002; McPartland et al 2007). Development of reliable antibodies against GPCRs is especially challenging (Saper 2005; Jositsch et al 2009; Kirkpatrick 2009; Talmont et al 2012; Baker 2015), and serious doubts had been raised about the usefulness of a variety of anti-GPCR antibodies (O’Connell et al 2006; Rhodes and Trimmer 2006; Pradidarcheep et al 2008; Jositsch et al 2009; Michel et al 2009) All these caveats are applicable to antibodies against CB1 receptor, and proper validation is a fundamental pre-requisite before studies using these antibodies are conducted. This study emphasized the importance of temperature and detergents for the final result and proposed a new interpretation of Western blot and immunoprecipitation data based on the folding and packing state of CB1 and the detergent used

Methods

Results

Conclusion