G (guanine nucleotide-binding) protein-coupled receptors (GPCRs) represent the largest family human membrane receptors involved in regulating varied cellular and metabolic functions, both in health and disease. UniProt lists 825 human genes as of Nov 2019 [1]. Also known as seven transmembrane helical proteins (7TMs), GPCRs transduce cellular signals arising from a huge repertoire of ligands ranging from small ions, neurotransmitters, lipid metabolites, catecholamines, hormones, light photons, odors, and foods.

GPCRs are the most valued drug discovery targets accounting for ~34% of all FDA-approved drugs [2], due to their wide range of therapeutic applicability in diseases including cancer, diabetes, autoimmunity, hypertension, and inflammation. However, only a small proportion of GPCRs are targeted by current anti-GPCR drugs, of which the majority belong to the aminergic class of receptors. However, the emergence of novel high-throughput technologies such as next-generation sequencing and omics-based methods, and improvements in protein isolation, purification and expression systems have led to the identification of several novel orphan GPCRs, whose structural, functional and physiological role is yet to be explored.

Techniques required to characterize GPCRs, including orphan receptors, are limited. Even though small chemical moieties and radio-isotope labeled ligands have been increasingly used for targeting GPCRs, these molecules cannot selectively bind and modulate target receptors and regulate their specific intracellular signaling mechanisms. The only practicable alternative for undertaking such functional experiments is to use anti-GPCR antibodies. However, raising high-quality antibodies against GPCRs is a challenge. For example, Sando R et al tagged Lphn3 with N-terminal hemagglutinin epitopes in conditional knockout Lphn3 mice to investigate Lphn3 expression due to the lack of suitable antibodies [3]. Table 1 lists the top suppliers of the research antibodies against 99 commonly studied GPCRs. In this article, we will review the problems associated with the successful generation of anti-GPCR antibodies. Further, we will also summarize strategies successfully used by researchers to circumvent these problems.

GPCRs are predominantly transmembrane proteins embedded within the plasma membrane. Structurally, GPCRs are comprised of an extracellular N-terminal ligand binding domain, followed by a 7TM helix segment and an intracellular C-terminal G-protein binding domain [4]. Such a transmembrane structure is complicated to isolate and to crystallize into a pure and homogenous GPCR protein in its native conformation.

GPCRs are largely hydrophobic in nature. For this reason, obtaining GPCRs in a solubilized and stable form is difficult. Purified GPCR proteins are unstable unless they are solubilized in a detergent or combined with lipids such as glycerol [5].

Another major problem with isolation and crystallization of several GPCRs, excluding rhodopsin, is their abysmally low cell surface expression. Transcription and translation of GPCR proteins are tightly regulated due to their physiological effect on diverse cellular processes. As a result, the amount of GPCR present inside the cell is meager, which makes it highly difficult to isolate them from whole cell extracts [6, 7].

Immunogenicity of the antigen is critical to elicit a targeted and stable humoral and cellular immune response in vivo and is in turn dependent on the protein structure. For GPCRs, the majority of the protein is either embedded within the lipid membrane bi-layer or exists intracellularly. The only accessible regions that are exposed and can be used for generating immunogenic peptides are the extracellular N-terminus region and the transmembrane loop portions. Development of functional antibodies against GPCRs with a large extracellular region has been widely reported. However, antibodies against GPCRs with a small N-terminal region is a challenge and often require multiple trials owing to unavailability of immunogenic regions.

GPCRs exist in multiple conformational states, can bind to multiple ligands, undergo multiple post-translational modifications, and form oligomers with other proteins [8-10]. Such heterogeneity in structure and ligand-binding activities present additional challenges in the development of functional anti-GPCR antibodies.

GPCR protein expression, native conformational structure, and biological function are regulated by several physiological and pathological factors intrinsic to human health and disease. GPCR heterogeneity is also influenced by drug treatments [11]. While polymorphic receptors exhibit a high degree of structural homology, they also dimerize or oligomerize generating additional receptors with novel functions. Oligomerization provides an additional layer of receptor diversity, and profoundly alters the ligand-binding properties compared to monomers. As a result, it is a challenge to develop reliable anti-GPCR antibodies that can be used as tools to study the structure, ligand-binding, and other associated properties of these proteins.

There are also some general concerns associated with some antibodies, including anti-GPCR antibodies irrespective of whether they are developed in-house or purchased from a vendor.

Complications associated with antigen specificity were well-documented by Michel et al [12]. There are two widely used strategies used to determine antibody specificity to the target antigen. Strategy 1 involves immunostaining using lysates from different cells or tissues that express a specific protein. Strategy 2 involves incubating antibodies with specific GPCR peptides used to raise these antibodies. However, the available literature shows both these strategies are insufficient in determining antigen specificity of the antibody [13-15].

This is described as the ability of an antibody to detect a specific GPCR antigen with high positivity and avoid false-negatives.

Reproducibility is a major concern associated with commercial antibodies, including those targeting GPCR proteins. Lot-to-lot variations in antibodies, irrespective of being polyclonal and monoclonal, are routinely observed in the laboratory [16]. This non-reproducible behavior was aptly demonstrated in a recent study by Grimsey et al, who tested multiple lots of antibodies targeting cannabinoid CB1 receptor obtained from 2 different companies. All the antibodies were screened using a combination of immunological methods including western blotting, immunohistochemistry, immunocytochemistry, and receptor autoradiography. Tested antibodies demonstrated poor specificity and high cross-reactivity [17]. Polyclonal antibodies tend to be less reproducible than monoclonals; however, it appears many anti-GPCR antibodies are polyclonals. For example, Zeng Q et al used only polyclonal antibodies to investigate N-methyl-D-aspartate receptors during breast cancer metastasis [18].

Problems associated with the use of unreliable antibodies in biomedical research are enormous. Most importantly, published data generated using invalidated antibodies can never be replicated by others as they are either false-negatives or false-positives. Several strategies are suggested to avoid such problems and successfully develop highly specific anti-GPCR antibodies. Diana Pauly and Katja Hanack published one such step-by-step validation procedure, which aptly summarizes the diligence researchers should undertake while selecting the right antibody [16].

Researchers should put more emphasis on identifying and characterizing the right antigenic epitope on a GPCR to ensure maximum immunogenicity and minimal antibody cross-reactivity. Generation of anti-GPCR antibodies, either monoclonal or polyclonal, is critically dependent on the starting material used for generating an immunogenic response. This is even more relevant for GPCRs considering their low surface abundance and their conserved structure [19-22]. Researchers have used different types of antigens based on the project budget and downstream application. Choice of antigen is also dependent on the structure and function of the target GPCR. For instance, peptides might successfully mimic GPCRs with linear epitopes such as protease-activated receptor 4 [23], while immunization with cell lysates expressing recombinant protein be a suitable approach against other GPCRs such as CXCR4 or CCR5 receptors [24-26]. Pros and cons associated with each of these strategies are listed below.

Synthetic linear peptides are a predominant choice as they are easy to design and inexpensive to synthesize. Additionally, synthetic peptides can be readily conjugated with adjuvants for immunization. Synthetic peptides are also used due to unavailability of full-length GPCR proteins in their native conformation and the highly conserved sequence similarity between humans and the host to be used for immunization [15, 27-29]. Use of synthetic GPCR peptides has a huge potential as was demonstrated by Heimann et al [30]. In this study, synthetic peptides were generated against 34 GPCRs and used as immunogens for the generation of PAbs that showed high receptor specificity, apart from detecting the ligand-induced conformational state of each GPCR. Designing synthetic peptides also helps researchers to specifically select epitopes that can enhance an immune response. T Tanaka et al generated a rat IgG1/kappa monoclonal antibody against mouse CCR2 using N-terminal peptide immunization [31].

Over the years, immunization with purified native recombinant proteins produced using either cellular or cell-free systems has remained a popular choice of antigen for immunization. Expression systems used for producing recombinant GPCR proteins can be broadly categorized as non-mammalian (bacterial, yeast, and insect) and mammalian cells.

Recombinant GPCR proteins expressed in bacterial (Escherichia coli) or yeast (Pichia pastoris) are extensively used for generating anti-GPCR antibodies. Non-mammalian systems are easy to cultivate and offer better scalability compared to mammalian cell lines or cell-free systems [32]. However, it is postulated that proteins expressed using non-mammalian systems may not be suitable for generating antibodies that can recognize biologically active GPCRs that exist in a tertiary or quaternary conformational state. This may not be accurate as was shown by Talmont et al, where histidine and c-myc-tagged three full-length human GPCR proteins (neuropeptide FF receptor type 2, κ opioid receptor and μ opioid receptor) were expressed in Pichia pastoris. These recombinant proteins were then immunized into Balb/c mice following SDS denaturation to raise highly-specific antibodies recognizing native receptors [32]. Mumaw et al used a similar strategy to develop a functional MAb targeting protease-activated receptor 4 (PAR4). In this study, recombinant MBP-tagged PAR4 was expressed in E. coli, and used for immunization in conjugation with Freund’s complete adjuvant [19].

Mammalian cell lines such as HEK (human embryonic kidney) or CHO (Chinese hamster ovary) are highly suitable for heterologous expression of functionally active GPCRs proteins. These systems are advantageous owing to their ability to synthesize proteins with intact post-translational modifications (PTMs) and the presence of a lipid bilayer membrane that facilitates the optimal surface presentation of the receptor [33]. Generally, GPCRs can be expressed either transiently or stably in these mammalian systems. While transient receptor expression is ideal for rapid protein production, gene-of-interest is permanently integrated into the cellular DNA in stable cell lines leading to stable expression of target GPCR. Both of these methods have been used successfully to produce full-length GPCR proteins for immunization and generate antibodies that can selectively recognize endogenous receptors, including their conformational change upon ligand-binding [34, 35].

In certain situations, using mammalian cells might not be the right choice for expressing specific GPCR proteins. These include constitutively expressed receptors that possess a biologically compensatory mechanism, or GPCRs that exhibit promiscuity and possess multiple isoforms. A feasible alternative is using insect cell lines such as Sf9 [36, 37]. A study by Akermoun et al analyzing the expression of 16 human GPCRs expression in 3 insect cell lines infected with baculovirus showed that insect expression systems offered versatility and are suitable for achieving higher protein production. Albeit, production amounts were different for each GPCR and were dependent on the growth conditions [38]. Anti-GPCR antibodies raised by immunizing recombinant full-length protein expressed in insect cells demonstrate a high degree of specificity and can even recognize native receptors, as shown by Yamaguchi et al [39].

Plasmid DNAs containing genes expressing protein-of-interest can also be used as an antigen for generating an immune response [40-42]. Even though it is the least preferred choice as an immunogen, genetic immunization offers tremendous potential due to its ability to generate strong antibody responses in the host against the target antigen. For example, Kaptein et al made antibodies against two virus-specific GPCRs by immunizing New Zealand rabbits with plasmid DNAs cloned with the gene-of-interest expressing target proteins. In this study, in vivo electroporation method was used to inject plasmid DNA intramuscularly into the anterior tibia. ELISA and western blot analysis revealed the presence of receptor-specific antibodies and confirmed the immunogenicity of the antigen. Similar studies were undertaken by Allard et al to generate highly-selective antibodies against the endothelin-B receptor, which also behaved as a potent antagonist [42]. Additional studies are required to expand the potential of plasmid DNA as an antigen further.

The mammalian immune system recognizes heterologous foreign proteins or peptides and generates a range of innate and adaptive immune responses, prominent among which is the production of antibodies. The same mechanism is exploited for the generation of PAbs and MAbs against human GPCRs in animals such as rabbits and mice.

PAbs are described as a pool of antibodies produced by multiple B-cell clones, where each clone produces an antibody specific to a different epitope of the antigen. Anti-GPCR polyclonal antibodies are raised by immunizing animals (e.g., rabbits, guinea pigs or goats) with a target GPCR protein. Serum obtained from these immunized animals is affinity-purified against the intended target antigen and validated for its specificity either using indirect ELISA or immunoblotting [43]. In contrast, MAbs are generated using hybridoma technology, which is based on the fusion of B-lymphocytes with myeloma cells resulting in the generation of hybridomas [44], or phage display technology.

Adopting the right immunization strategy is important to allow maximal antigen exposure and generate an optimal immune response to an antigen. Also, the immunization strategy needs to be optimized for each antigen. The most commonly used antigen-delivery method is to conjugate the antigen with a carrier molecule such as keyhole limpet hemocyanin (KLH) and combine it with an adjuvant-like alum or Freund’s complete adjuvant. Used often for delivering small peptides, the carrier molecule encapsulates the antigen and increases its chance to be presented to the immune system, while adjuvant enhances the immunogenicity of the antigen [45, 46]. Adjuvants used in experimental antibody production are diverse and can be broadly categorized into emulsions, aluminum salts or microparticles. Each of these types has distinct advantages and disadvantages and should be used with caution. For instance, Freund’s adjuvants contain crude mineral oil that acts as an immunostimulant and is used to create water-in-oil emulsions. While they are apt for sustained antigen release, they are highly toxic and are associated with granuloma formation. Aluminum salts are suitable for rapid antigen delivery. However, they generate weak antibody titers and require repetitive immunizations. Researchers have also used microparticles such as liposomal formulations or immune-stimulating complexes (ISCOM) for antigen delivery. For example, Zhao Y et al used lipid A-containing liposomes mixed with antigen proteins to generate monoclonal antibodies against AMPA receptors [47]. Encapsulated within a polymeric matrix, microparticles are suitable for long-term antigen delivery and provide continuous antigenic stimulation in the experimental animals [48]. Ma Y et al, for example, immunized a bactrian camel with human apelin receptor nanodiscs to obtain a single-domain antibody phage library and screened the library with apelin receptor proteoliposomes to obtain positive clones [49].

Care should be also be exercised while delivering the antigen to the animal as administration routes affect the immune responses. Antigens, particularly peptide-adjuvant emulsions are usually administered through subcutaneous immunization. This is the most preferred choice as it is safe and easy to inject. Also, a large volume of antigen can be injected through this route. Intraperitoneal route also introduces antigen to the lymphatic system through draining lymph nodes and can be used to inject large volumes of antigen, like the subcutaneous route. This route is primarily used when cells are used as an antigen. Intravenous administration delivers antigens directly to the secondary lymphoid organs. Buffer-solutions containing recombinant proteins, water-in-oil emulsions of antigens or liposomal preparations can be delivered through this route. Whole cells should not be injected as they can cause embolism and death. Apart from these, there are other less preferred choices such as intradermal, intramuscular, or intrasplenic injections that are used only for specific antigenic preparations or conditions. For example, intrasplenic immunization for generating antibodies is a single-dose method used only when the antigen is available in extremely low amounts. This procedure leads to the generation of IgM antibodies. An exciting alternative in antigen delivery methods is repetitive immunization multiple site immunization (RIMMS). In this protocol, an antigen is administered at multiple immunization sites in low quantities.

Additionally, booster doses are injected at shorter intervals. RIMMS has a shorter immunization schedule, can generate larger antigen-specific B-cell repertoire compared to conventional immunization protocols and are useful against glycosylated antigens. Braithwaite et al successfully used this strategy to generate an active antibody to formyl peptide receptor 1, which functioned as a potent receptor antagonist [50].

Antigen administration strategy also depends on the properties of the antigen. The difficulty of raising antibodies to “problematic antigens” with a conserved structure such as GPCRs is well-known as these receptors have a limited number of antigenic epitopes. Hrabovska et al adopted a novel and effective strategy for generating antibodies to such conserved antigens that showed high antigen specificity and sensitivity on multiple platforms. In their study, they used knock-out mice and immunized them with a full-length recombinant protein [51]. Another unique immunization strategy is “subtractive immunization,” which can be very promising against epitopes selective for GPCR heteromers. In this approach, an immunosuppressive or tolerogenic environment is created within the host towards unwanted epitopes before antigen immunization. Immunosuppression allows less antigenic epitopes to become immunodominant and elicit a strong immune response [35]. The uniqueness of GPCR heteromers as drug targets and the utility of subtractive immunization to developing heteromer-selective antibodies was discussed very well by Gomes et al in their review [35].

These are rapid and robust methods for antibody isolation and development, which have shown tremendous promise in the last decade [52]. Antibody phage display method involves the expression of antibody fragments on the surface of filamentous M13-bacteriophage, a non-lytic phage that infects Escherichia coli. Full-length antibodies are comprised of 2 regions, namely fragment-constant (Fc) and fragment antigen-binding (Fab). While the Fc region of an antibody is made up of constant domains of heavy and light chains (CH and CL), the Fab region is made of both variable domains (VH and VL) and constant regions. Antibody genes, after undergoing V(D)J recombination, are isolated from B-cells, variable domains are amplified using a PCR reaction, and the Fab regions are cloned into a filamentous bacteriophage genome usually fused to pIII genes encoding membrane-embedded minor coat G3 protein. E. coli cells are infected with these phages, which results in the production of functional phage particles displaying antibody-fused G3Ps. Antibody-producing clones are enriched by selecting them against immobilized antigens using a technique called “biopanning,” and the antibody activity is commonly determined by a technique called “enzyme-linked immunosorbent assay” or ELISA [52, 53].

Antibody phage display is remarkably flexible as it can be used to express different antibody fragments, including Fab region, fragment-variable (Fv) domains, single-chain fragment variable (ScFv) and bivalent antibodies [20, 54].

Easy-to-execute methodology and suitability for generating biologically-active antibodies against less understood and difficult to isolate antigens, antibody phage display is perfectly suited for generating anti-GPCR antibodies, for example, against human CCR1 , a protein important in rheumatoid arthritis, multiple sclerosis and osteolytic bone disease [55]. Antibodies to CXCR2 is one such example where antibody phage display was successfully used against a challenging GPCR as demonstrated by Rossant et al [20]. In their study, ScFv developed from native phage library was screened for CXCR2-specificity using CXCR2-coated magnetic liposomes. Following multiple rounds of screening and selection, a highly specific anti-CXCR2 ScFv was isolated. The antibody was purified through recombinant expression in E. coli and validated on cell lines expressing CXCR2 [20]. Another example where phage display was highly useful was published by Tohidkia et al, where they used this strategy to raise ScFv against cholecystokinin-B/gastrin receptor [56]. In this study, antibody clones were screened from a semi-synthetic phage display library using a biotinylated synthetic peptide generated from the 2nd extracellular loop of the receptor. Antibody specificity to the antigen was determined by ELISA and western blotting [56].

Ribosomes are crucially involved in protein synthesis and facilitating mRNA translation. In vitro ribosome display method exploits this function for rapid screening of high-affinity antibodies and their antigenic regions. Over the years, research showed that a single phage display library is insufficient to represent the entire human antibody repertoire owing to its inherent difficulty in producing and selecting larger libraries (>1011 clones). The ribosomal display method is an effective alternative as ribosomal libraries can encode >1012 scFv, which can be easily selected in vitro [57]. It is based on a simple principle of associating phenotype with its corresponding genotype. The process involves the generation of protein-ribosome-mRNA complexes, where a synthesized protein is stalled within the ribosomal tunnel while the mRNA is being translated. At this phase, nascent single-chain antibody fragment remains attached to its corresponding mRNA, which is selected based on the affinity to selected ligands that are immobilized either on magnetic beads or a solid phase. Translation is halted randomly either by deleting the stop codon in the gene or by using antibiotics such as rifampicin or chloramphenicol. Antibody transcripts trapped inside the ribosomes are then recovered and amplified using in situ RT-PCR [58]. One of the biggest advantages of this in vitro display system is that the platform is completely cell-free. This enables researchers to introduce any desired modification inside the antibody gene without any cellular interference. The only rate-limiting criteria that define the optimum functionality of this system is the presence of a ribonuclease-free environment and utilization of RNase-free buffers [58, 59].

Ribosomal display method has multiple advantages:

• Valuable in isolating functional antibody fragments from unstable hybridomas.
• Aptly suitable for generating properly-folded full-length functional GPCRs, which can then be used for generating anti-GPCR antibodies through conventional hybridoma technology [60].
• Useful as a drug screening tool as it allows selection of a larger repertoire of high-affinity GPCR agonists and antagonists owing to its huge library size [60].

Ravn et al used ribosome display technique to generate antibodies against glucose-dependent insulinotropic polypeptide receptor (GIPr), a class B GPCR that binds to GIP on pancreatic beta cells and stimulates the production of insulin [61]. Available literature suggests the ribosome display of antibody fragments can become a system of choice for developing therapeutic human antibodies against GPCRs. However, this is yet to become a certainty.

Antibodies, in particular, MAbs, are large multimeric glycoproteins that are capable of binding only to GPCRs expressed on the surface of cell membranes. However, their large size and lack of membrane penetration capability interfere with their effective binding to either the intracellular regions of the proteins or GPCRs expressed on intracellular membranes. While ScFv was identified as an alternative, research suggests their efficacy against intracellular proteins remains questionable. In the last few years, nanobodies have emerged as a plausible solution and have made exciting progress in understanding GPCR biology.

Derived primarily from heavy-chain antibodies produced by camels and other members of the Camelidae family, nanobodies are small-sized antibodies (~15kDa) comprised of only a single-domain obtained from the variable region [62, 63]. Owing to their small size (~15kDa), compact spheroid-like shape, and their ability to expose a convex paratope, nanobodies can penetrate easily through the cell membranes and bind to antigenic regions/clefts that are inaccessible to conventional antibodies [62, 64, 65]. Nanobodies demonstrate extreme stability compared to human ScFv. Other advantages include

• Low production costs – Nanobodies can be easily produced using any recombinant expression system (can be bacterial, yeast or mammalian). Average expenditure incurred on their production is extremely low as recombinant expression systems can easily produce these antibodies in high quantities [65].
• Highly soluble in aqueous solutions. They are extremely stable even under harsh climate, chemical or pH conditions.
• Recombinant nanobodies do not form aggregates. Therefore, the isolation and purification of these antibodies are a straight-forward procedure [66].
• Nanobodies are monomeric Abs that don’t undergo any post-translational modification.
• Nanobodies are also resistant to destruction from digestive enzymes routinely found inside gastrointestinal tracts. This allows these antibodies to be delivered as an oral formulation [67].
• Nanobodies have a short half-life, due to which they are rapidly cleared from the body [68, 69].

Anti-GPCR nanobodies have wide applicability in drug discovery and research as they combine the advantages offered by small-sized synthetic drugs and monoclonal antibodies.

Further, antigen specificity and affinity of anti-GPCR nanobodies are suggested almost identical to conventional MAbs. Currently, nanobodies are used as tools for stabilizing GPCRs in their active-state conformation by binding to their intracellular regions and assist in protein crystallization [66, 70]. Further studies are warranted to better elucidate their therapeutic potential in various chronic and acute conditions.

Irrespective of whether anti-GPCR antibodies are purchased from a commercial supplier or developed in-house, they are invaluable as research tools. Therefore, researchers should diligently establish a stringent and standardized antibody validation protocol to identify the right antibody and avoid the occurrence of antibody-related problems in the data. As described by Pauly et al, the first-step in antibody selection is to perform a database search for published results that have validated the antibody specificity for a specific application. However, it is important to ascertain that the antibody is obtained from the same lot that was prior validated in these articles. This is mandatory for Pabs. If available, MAbs should be preferred as they have minimal background signal-to-noise ratio and lower cross-reactivity. Once the antibody is identified or generated, researchers can use a combination of approaches (named as “5 pillars for antibody validation”) mentioned below to ascertain the quality and specificity of the antibodies for further use [71].

In this approach, antibodies are tested for their specificity on tissues or cells derived from animals or cells genetically engineered to be deficient in target GPCR. An antibody is specific to target antigen if it stains negative in both these conditions [12, 72]. Stojanovic et al used this method to validate commercially available antibodies to dopamine receptors DRD1 and DRD2 by western blotting and immunohistochemistry. They demonstrated that only 2 out of 9 antibodies tested positive and immunoprecipitated their corresponding receptors [73]. An alternative approach in genetic testing is overexpression of protein-of-interest in cells that do not express the protein in normal conditions. Garg and Loring used this approach for rapid screening of 7 commercial antibodies to Alpha7 nicotinic acetylcholine receptors (α7 nAChRs). Using gel-shift assays, they showed that only 2 out of 7 antibodies specifically recognized nAChRs and confirmed their results by immunofluorescence [72].

This method involves the quantification of a target protein using antibody-independent methods like targeted proteomics. Simultaneously, an antibody-dependent method is used for relative quantification of the same protein across samples. Both these methods should correlate strongly to validate the antibody specificity.

This method involves testing target protein using two different antibodies that recognize two independent epitopes within the same application. Antigen expression pattern generated by these two independent antibodies should correlate. Cernecka et al used this strategy to identify the right antibody to β3-Adrenoceptors. In this study, two antibodies raised against the N- and C-termini of the receptor were concordantly used in immunocytochemistry and immunohistochemistry [14].

In this method, target-of-interest is tagged to an unrelated protein and is overexpressed in cells. Target protein specificity is evaluated by staining separately with antibodies targeting the protein and the tag respectively. Similar correlation in both conditions will validate the desired antibody. Garg and Loring also used this methodology in their study, where antibodies to α7 nAChRs were screened. In their research, green fluorescent protein (GFP) was tagged to nAChR and probed with anti-GFP antibody to confirm the results [72]. A similar approach was successfully used by Burghi et al to validate the specificity of antibodies to Mas receptor (MasR). In their study, human c-Myc-tagged MasR was overexpressed in HEK293T and tested by immunofluorescence by probing simultaneously by anti-c-Myc and anti-MasR antibodies. 3 out of 4 antibodies showed similar staining pattern in both conditions.

Interestingly, genetic testing using tissues MasR-deficient animals failed to identify the right antibody [74].

This method is a combination of immunoprecipitation and mass spectrometry. An antibody is used to affinity-capture the antigen, whose sequence is verified by mass spectrometry.

This strategy might be applicable when validating antibodies for immunohistochemistry, as demonstrated by Pyke et al In this study, antigen specificity of MAb raised against glucagon-like peptide one receptor (GLP-1R) was determined by testing it on multiple human and primate tissues. Interestingly, GLP-1R localization was different in each of these tissues, which underpins the utility of using antibodies as a research tool. For instance, GLP-1R expression was detected in the smooth muscle cells of vas afferens in primate kidney, whereas non–β-endocrine islet cells and acinar cells from primate pancreas had a weak receptor expression. The most abundant expression was observed on the islet cells, both in humans and monkeys [75].

Finally, it is essential to identify proper positive and negative control to avoid cross-reactivity and non-specific staining, irrespective of whatever method is used for validation.

Anti-GPCR antibodies have played a momentous role in advancing our knowledge on the structure and function of GPCRs, both in health and disease. Currently, anti-GPCR antibodies targeting at least 37 different GPCRs are in different phases of clinical trials and are being evaluated for their therapeutic efficacy [76]. Even though GPCRs present several challenges as targets for antibody development, it is important to explore technologies that can help generate biologically active high-affinity antibodies against these unique GPCRs. This will help us gain in-depth insights into GPCR biology due to their clinical relevance.