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

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.

Subsynaptic Distribution, Lipid Raft Targeting and G Protein-Dependent Signalling of the Type 1 Cannabinoid Receptor in Synaptosomes from the Mouse Hippocampus and Frontal Cortex

“…The specificity of the detected signals was validated using cortical synaptosomes from CB1-KO animals (see Figure 1A). The molecular weight of the ~50 kDa-specific band detected by Western blot agrees with previous results where CB1-Rb-Af380 and CB1-Go-Af450 antibodies have been used [14,16,17], including some from our laboratory [5,10,15,18,19]. However, the second less intense but clearly positive ~35 kDa band detected here was not previously reported in mice.…”

“…Therefore, proper antibody testing and validation must be considered when studies using anti-CB1 antibodies are conducted. In this sense, we have recently provided robust data on the suitability for different applications of several anti-CB1 antibodies [15], highlighting the need for the fit-for-purpose (F4P) approach for validation of antibodies and the importance of choosing the platform that best fits their end-use. In this previous work, the CB1-Rb-Af380 and CB1-Go-Af450 antibodies, both raised against the carboxy-terminal 31 amino acids of the mouse CB1 receptor, provided excellent results for the recognition of the denatured CB1 receptor from brain tissue in Western blot assay.…”

“…To study the CB1 receptor protein located in the synaptosomal fraction by Western blot assays, we used three commercially available antibodies (CB1-Immunogenes, CB1-Go-Af450 and CB1-Rb-Af380) that were raised against the 31 amino acids of the extreme carboxy-terminus of the mouse CB1 receptor. These have been recently shown to be the most reliable ones for Western blot [15]. As negative control, we used brain cortical tissue of CB1-deficient mice (CB1-KO) to test the specificity of these antibodies for the CB1 receptor.…”

Up-regulation of CB1 cannabinoid receptors located at glutamatergic terminals in the medial prefrontal cortex of the obese Zucker rat

“…The CB 1 receptor immunostaining pattern in the neocortex (Figure 2) and various subcortical regions (Supplementary Results and Supplementary Figure 3) of the lean Zucker rat matched the described previously in the rat (Egertová and Elphick, 2000;Bodor et al, 2005;Deshmukh et al, 2007;Echeazarra et al, 2021). Thus, in NCx, a layer-specific immunolabelling pattern was clearly observed, being markedly denser in layers II-III, upper part of layer V (Va) and layer VI than in the rest (Figure 2A).…”

Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence

“…Facing both scenarios, we want to help the reader in the selection of the best protocol according to their specific needs. It is important to clarify that the advice given in this review does not solve other important issues associated with immunofluorescence, such as antibody specificity tests and establishment of adequate controls, for which there are detailed studies [58][59][60][61][62].…”