LEPR Antibody

What is the LEPR protein and why is it a significant research target?

LEPR (also known as CD295, Fa, Obr, LEP-R, OB receptor, or OB-R) functions as the receptor for the hormone leptin, which is primarily produced by adipocytes. The biological significance of LEPR extends far beyond simple receptor-ligand interactions, encompassing critical roles in energy homeostasis and multiple physiological systems. In the hypothalamus, LEPR mediates leptin's effects as an appetite-regulating factor that decreases food intake and increases energy consumption by inducing anorexinogenic factors and suppressing orexigenic neuropeptides, while also regulating bone mass and secretion of hypothalamo-pituitary-adrenal hormones . In peripheral tissues, LEPR signaling increases basal metabolism, influences reproductive function, regulates pancreatic beta-cell function and insulin secretion, promotes angiogenesis, and affects both innate and adaptive immunity . The control of energy homeostasis and melanocortin production through LEPR involves STAT3 signaling, which stimulates POMC and represses AgRP transcription, while separate signaling pathways regulate NPY and control fertility, growth, and glucose homeostasis . These diverse functions make LEPR a compelling target for researchers studying metabolic disorders, obesity, diabetes, and inflammatory conditions.

What are the key structural characteristics of LEPR that influence antibody selection?

LEPR belongs to the gp130 family of cytokine receptors that stimulate gene transcription via activation of cytosolic STAT proteins . When selecting antibodies for LEPR research, understanding the structural variations of this receptor is essential for experimental design and result interpretation. LEPR exists in multiple isoforms with varying signaling capabilities and tissue distribution. Isoform A may transport leptin across the blood-brain barrier, binds leptin, and mediates leptin endocytosis, but does not induce phosphorylation or activate STAT3 . Conversely, isoform E acts as an antagonist to isoform A and isoform B-mediated leptin binding and endocytosis . These structural differences directly impact antibody binding and experimental outcomes. When selecting an anti-LEPR antibody, researchers should consider the specific epitope targeted, as antibodies directed against the extracellular domain might detect all isoforms, while those targeting the intracellular domain may selectively recognize signaling-competent isoforms. The calculated molecular weight of LEPR is approximately 132 kDa, which serves as an important reference point for validating antibody specificity through techniques like Western blotting .

What are the primary applications for LEPR antibodies in research?

LEPR antibodies serve diverse research applications depending on their specific characteristics and the experimental questions being addressed. The most commonly employed techniques include Western Blotting (WB) for protein expression quantification, Immunohistochemistry (IHC) for tissue localization studies, Immunocytochemistry (ICC) for cellular distribution analysis, Flow Cytometry for cell population studies, and Immunoprecipitation (IP) for protein-protein interaction investigations . These antibodies have demonstrated reactivity with human, mouse, and rat samples, making them versatile tools for comparative species studies . The selection of application should align with research objectives; for instance, Western blotting would be appropriate for quantifying total LEPR expression changes in response to experimental manipulations, while immunohistochemistry would better serve investigations of LEPR distribution across different cell types within a tissue section. Importantly, validation across multiple applications enhances confidence in experimental findings, particularly when studying complex metabolic disorders where LEPR signaling may be altered in multiple tissues simultaneously.

How should researchers validate LEPR antibody specificity before experimental use?

Proper validation of LEPR antibodies is essential to ensure experimental reliability and reproducibility. A comprehensive validation approach should include multiple complementary techniques. First, Western blotting should be performed to confirm that the antibody detects a protein of the expected molecular weight (approximately 132 kDa for full-length LEPR) . Researchers should include positive controls (tissues known to express high LEPR levels such as hypothalamus) and negative controls (tissues with minimal LEPR expression or LEPR knockout samples when available). Second, comparing staining patterns across multiple antibodies targeting different LEPR epitopes can enhance confidence in specificity. Third, blocking peptide experiments, where the antibody is pre-incubated with the immunizing peptide, should abolish specific signals if the antibody is truly selective for its target. Fourth, RNA interference or CRISPR-based knockdown/knockout of LEPR should result in corresponding reduction or elimination of antibody signal. Finally, immunoprecipitation followed by mass spectrometry can provide definitive evidence of antibody specificity. Throughout these validation processes, researchers should maintain consistent experimental conditions, including sample preparation methods, antibody dilutions (typically 1:500-1:1,000 for Western blotting and 1:50-1:100 for Flow Cytometry with the antibodies described) , and detection systems to ensure reproducible results.

What are the optimal fixation and antigen retrieval methods for LEPR immunohistochemistry?

Successful immunohistochemical detection of LEPR requires careful consideration of fixation and antigen retrieval methods to preserve epitope accessibility while maintaining tissue morphology. For formalin-fixed paraffin-embedded (FFPE) tissues, 10% neutral buffered formalin fixation for 24-48 hours is typically suitable, followed by standard paraffin embedding procedures. Paraformaldehyde fixation has been successfully employed for immunocytochemistry of LEPR in cell lines such as MCF-7, where nuclear counterstaining with DAPI provides contextual cellular information . For antigen retrieval in FFPE tissues, heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 9.0) for 20-30 minutes has proven effective for many LEPR antibodies. For frozen sections, brief fixation with 4% paraformaldehyde or acetone may be sufficient, with minimal or no antigen retrieval required. Regardless of the specific protocol, optimization is essential for each antibody and tissue type combination. Researchers should systematically test different fixation durations, antigen retrieval methods, and antibody concentrations using appropriate positive control tissues to establish optimal conditions. Well-executed IHC should yield clear membrane and/or cytoplasmic staining patterns depending on the LEPR domain targeted by the antibody, as demonstrated in the immunohistochemical analysis of rat heart tissue using anti-Leptin Receptor antibody (A00350-4) .

What dilution ranges and incubation conditions are recommended for different applications of LEPR antibodies?

Optimal antibody dilutions and incubation conditions vary by application and specific antibody characteristics. For Western blotting applications, LEPR antibodies typically perform well at dilutions ranging from 1:500 to 1:1,000 . Flow cytometry applications generally require higher antibody concentrations, with recommended dilutions between 1:50 and 1:100 . For immunohistochemistry and immunocytochemistry, dilutions may vary widely based on the antibody, detection system, and sample type, but typically fall within the 1:100 to 1:500 range. Primary antibody incubations are generally performed overnight at 4°C to maximize specific binding while minimizing background, although some antibodies may perform adequately with shorter incubations (2-4 hours) at room temperature. Secondary antibody incubations typically range from 30 minutes to 2 hours at room temperature. Researchers should note that these parameters represent starting points that must be optimized for each experimental system. For polyclonal antibodies like the rabbit polyclonal anti-LEPR (ab5593), which has applications in IP, WB, and IHC-Fr, systematic titration experiments comparing multiple dilutions under standardized conditions will identify the optimal working concentration that maximizes specific signal while minimizing background . Temperature, duration, buffer composition, blocking conditions, and washing steps should all be systematically optimized and then strictly maintained across experiments to ensure reproducibility.

How can LEPR antibodies be used to investigate leptin resistance mechanisms?

Leptin resistance, a condition where elevated leptin levels fail to produce the expected physiological responses, is central to obesity pathophysiology and represents a complex research area where LEPR antibodies play crucial investigative roles. Researchers can employ LEPR antibodies to quantify receptor expression levels via Western blotting across different tissues in lean versus obese models, potentially revealing receptor downregulation as a resistance mechanism. Immunoprecipitation with LEPR antibodies followed by phosphotyrosine detection can assess receptor activation status, providing insights into signaling defects even when receptor levels appear normal. Double immunofluorescence staining using LEPR antibodies alongside antibodies against downstream signaling molecules (like phosphorylated STAT3) can visualize spatial disruptions in signaling cascades within specific cell populations. Flow cytometry with LEPR antibodies can identify shifts in receptor surface expression versus internalization rates in response to leptin exposure. For mechanistic studies, researchers can use LEPR antibodies to investigate how specific post-translational modifications (such as glycosylation patterns) might differ between leptin-sensitive and leptin-resistant states. When designing these experiments, careful consideration of antibody specificity for different LEPR isoforms is essential, as changes in isoform expression ratios (particularly the expression of antagonistic isoforms like isoform E relative to signaling-competent isoforms) might contribute to resistance mechanisms .

What considerations are important when using LEPR antibodies to study different LEPR isoforms?

The existence of multiple LEPR isoforms with distinct functional properties necessitates careful experimental design when using antibodies for isoform-specific research. Researchers must first determine whether their scientific question requires discrimination between isoforms or detection of all isoforms collectively. For isoform-specific detection, antibodies targeting unique C-terminal sequences are essential, as the isoforms primarily differ in their intracellular domains. When studying isoform A, which may transport leptin across the blood-brain barrier and mediates leptin endocytosis but does not activate STAT3 signaling, researchers should select antibodies targeting its unique C-terminal sequence . Similarly, for studies focused on isoform E, which antagonizes isoform A and B-mediated leptin binding and endocytosis, antibodies directed against its distinctive C-terminal region are required . When interpreting Western blot results, researchers should carefully evaluate banding patterns, as molecular weight differences between isoforms can be subtle. For functional studies comparing isoforms, immunoprecipitation with isoform-specific antibodies followed by activity assays can reveal distinct signaling capacities. When designing primers for RT-PCR validation of isoform expression, researchers should consult the primer sequences provided in the literature, such as the exonic primers used for Lepr expression analysis (5′-CCTCTGCCCCCACTGAAAGACA and 5′-GGGTCACTGTCACTCTGAAGTGCAA) . This complementary molecular validation strengthens antibody-based findings in isoform-specific research.

How can researchers apply LEPR antibodies in studies of LEPR mutations and polymorphisms?

LEPR antibodies provide powerful tools for investigating how genetic variations in the LEPR gene affect receptor expression, localization, and functionality. When studying specific mutations like the R223 variant, researchers can employ site-directed mutagenesis to create expression constructs (as demonstrated by the two-stage PCR method described in the literature) for cellular studies . After transfecting cells with these constructs, anti-LEPR antibodies can assess whether mutations alter total receptor expression levels (via Western blotting), cellular localization (via immunocytochemistry), or surface expression (via flow cytometry). Co-immunoprecipitation experiments using LEPR antibodies can reveal whether mutations disrupt interactions with signaling partners. For functional studies, researchers can use phospho-specific antibodies targeting LEPR downstream effectors (such as antibodies against Tyr705 phosphorylated STAT3) to assess signaling capacity of mutant receptors following leptin stimulation . When studying naturally occurring polymorphisms in human populations, researchers can isolate primary cells from individuals with different LEPR genotypes and use LEPR antibodies to compare receptor characteristics. In these applications, antibody selection should consider whether the mutation or polymorphism being studied affects the epitope recognized by the antibody, as epitope alterations could lead to misleading changes in antibody binding independent of actual receptor expression or localization changes.

How are LEPR antibodies contributing to the development of therapeutic interventions?

The development of therapeutic antibodies targeting LEPR represents a significant frontier in treating disorders characterized by leptin signaling dysregulation. The fully human monoclonal antibody REGN4461 (mibavademab) exemplifies this approach, as it activates human LEPR in both the presence and absence of leptin . This agonistic antibody has demonstrated remarkable preclinical efficacy in leptin-deficient mouse models, where it normalized body weight, food intake, blood glucose, and insulin sensitivity . In mouse models of generalized lipodystrophy, REGN4461 treatment alleviated hyperphagia, hyperglycemia, insulin resistance, dyslipidemia, and hepatic steatosis, suggesting therapeutic potential for metabolic disorders associated with leptin deficiency . Phase 1 clinical trials have shown that REGN4461 is well-tolerated with an acceptable safety profile, and treatment of individuals with overweight or obesity resulted in decreased body weight over 12 weeks specifically in those with low circulating leptin concentrations (<8 ng/ml) . Interestingly, compassionate-use treatment of a patient with atypical partial lipodystrophy and a history of undetectable leptin concentrations was associated with notable improvements in circulating triglycerides and hepatic steatosis . These translational findings reveal how agonistic LEPR antibodies may provide clinical benefit in disorders associated with relatively low leptin concentrations, opening new therapeutic avenues where conventional recombinant leptin therapy might be ineffective due to neutralizing antibodies or other limitations.

What methodological approaches can researchers use to evaluate potential LEPR-targeting therapeutic antibodies?

Evaluating therapeutic antibodies targeting LEPR requires comprehensive methodological approaches spanning in vitro, in vivo, and clinical assessments. For initial in vitro characterization, researchers should establish binding affinity and specificity using techniques like surface plasmon resonance (SPR) or bioluminescence resonance energy transfer (BRET). Functional assays measuring STAT3 phosphorylation in LEPR-expressing cell lines can quantify agonistic or antagonistic activity. Cell-based assays measuring gene expression changes (particularly POMC and AgRP) provide insights into downstream signaling effects . For mechanistic studies, competition binding assays with labeled leptin can determine whether the antibody competes with or enhances endogenous ligand binding. In vivo evaluation should begin with pharmacokinetic studies determining antibody half-life and tissue distribution. Efficacy assessment in relevant disease models is crucial, as demonstrated with REGN4461 in both leptin knockout mice and a mouse model of generalized lipodystrophy, where normalization of body weight, food intake, glucose parameters, and liver steatosis served as key endpoints . For safety assessment, researchers should monitor for immune reactions, off-target effects, and compensatory physiological responses. When advancing to clinical studies, careful patient stratification based on leptin levels and metabolic parameters is essential, as REGN4461 showed efficacy only in individuals with low baseline leptin concentrations (<8 ng/ml) . Comprehensive biomarker analysis should include changes in body weight, food intake, glucose homeostasis, lipid profiles, and hepatic fat content, with long-term monitoring for sustained efficacy and potential development of neutralizing antibodies.

What are common challenges in LEPR antibody experiments and how can they be addressed?

Researchers frequently encounter several technical challenges when working with LEPR antibodies across various applications. One common issue is inconsistent or weak signal in Western blotting despite adequate LEPR expression. This may result from inefficient protein extraction due to LEPR's membrane localization; researchers should optimize lysis conditions by incorporating stronger detergents (like SDS or NP-40) and avoiding excessive heating that might cause receptor aggregation. For immunohistochemistry applications, high background staining often occurs due to LEPR's expression in multiple cell types within tissues. This can be mitigated through careful titration of primary antibody (typically starting at 1:100-1:500 dilutions), extended blocking steps with species-appropriate serum, and inclusion of detergents like Triton X-100 in washing buffers to reduce non-specific binding . Another frequent challenge is discrepancies between transcript and protein expression levels, which may reflect post-transcriptional regulation; researchers should validate findings using both RT-PCR and protein detection methods. When studying LEPR in obesity models, the presence of excessive adipose tissue can interfere with antibody penetration and create artifacts; extended incubation times and thorough washing steps can help overcome this limitation. Cross-reactivity with related cytokine receptors from the gp130 family might occur with some antibodies; verification using LEPR knockout tissues or cells as negative controls is strongly recommended. Finally, for flow cytometry applications, cell permeabilization conditions must be carefully optimized to maintain LEPR epitope integrity while enabling antibody access to intracellular domains when studying receptor internalization or total cellular expression .

What quality control measures should be implemented when using LEPR antibodies?

Implementing rigorous quality control measures for LEPR antibody experiments is essential for generating reliable and reproducible results. Researchers should first verify antibody specifications through manufacturer documentation, including the immunogen sequence (such as "synthetic peptide corresponding to a sequence at the C-terminus of human Leptin Receptor") , clonality, host species, and validated applications. Prior to main experiments, researchers should conduct preliminary validation studies comparing the selected antibody's performance against published literature, using positive control tissues known to express LEPR (hypothalamus, liver, or adipose tissue) and negative controls (ideally LEPR knockout samples or tissues with minimal LEPR expression). Lot-to-lot variation in antibody performance necessitates consistent use of the same antibody lot throughout a study when possible, with re-validation when changing lots. Standard curves using recombinant LEPR protein or lysates from LEPR-overexpressing cells should be generated for quantitative applications like Western blotting. For IHC applications, tissue processing and staining should incorporate standardized protocols with timed incubations and consistent reagent preparations . Researchers should maintain detailed records of antibody handling, including freeze-thaw cycles (which should be minimized as recommended in storage guidelines for products like A00350) , dilution preparations, and storage conditions (typically -20°C for long-term storage and 4°C for up to one month for frequent use) . When publishing results, comprehensive methodology sections should include antibody catalog numbers, dilutions, incubation conditions, and validation approaches to facilitate reproduction by other laboratories.

How might emerging technologies enhance LEPR antibody applications?

Emerging technologies are poised to revolutionize LEPR antibody applications across basic research and clinical domains. Single-cell antibody-based techniques, including mass cytometry (CyTOF) and imaging mass cytometry, can enable simultaneous detection of LEPR alongside dozens of other proteins at single-cell resolution, revealing heterogeneity in LEPR expression and signaling across diverse cell populations within complex tissues. Proximity ligation assays utilizing LEPR antibodies can visualize protein-protein interactions in situ, potentially uncovering novel LEPR signaling complexes and regulatory mechanisms. The integration of LEPR antibodies with optogenetic or chemogenetic systems could enable precise temporal control of LEPR pathway activation, allowing researchers to dissect the kinetics of downstream signaling events. CRISPR-based genetic screens combined with LEPR antibody-based phenotyping may identify novel regulators of LEPR trafficking, turnover, and signaling. For therapeutic applications, advances in antibody engineering such as bispecific antibodies simultaneously targeting LEPR and other metabolic receptors could enhance efficacy in complex metabolic disorders. The development of antibody-drug conjugates targeting LEPR-expressing cells might enable selective delivery of therapeutic payloads to specific cell populations. Innovations in intrabody development—antibodies designed to function within living cells—could enable visualization and manipulation of LEPR in real-time. Finally, the application of artificial intelligence algorithms to analyze large datasets from high-content imaging with LEPR antibodies may reveal subtle patterns in receptor localization and trafficking that correlate with disease states or treatment responses.

What are promising research areas where LEPR antibodies may provide new insights?

LEPR antibodies are positioned to drive discoveries across several frontier research areas with significant translational potential. In neuroendocrine research, cell-type specific analysis of LEPR expression and signaling in hypothalamic circuits using multiplexed immunohistochemistry may reveal previously unrecognized heterogeneity in leptin responsiveness that contributes to feeding behavior regulation. The application of LEPR antibodies to study the receptor's role in neurodevelopment could illuminate how early life metabolic perturbations influence brain development and predispose to later metabolic disorders. In immunometabolism, investigating how LEPR signaling in different immune cell populations affects their metabolic programming and inflammatory responses may identify novel therapeutic targets for inflammatory disorders. The potential role of LEPR in cancer biology represents another promising research direction, with antibody-based studies exploring how leptin signaling influences tumor cell proliferation, migration, and therapy resistance. For regenerative medicine, examining LEPR expression and function in stem cell populations might reveal roles in cell fate decisions and tissue repair processes. In the emerging field of gut-brain axis research, antibody-based mapping of LEPR expression along this axis could identify novel sites of leptin action influencing both systemic metabolism and neurocognitive functions. Finally, exploration of potential cross-talk between LEPR and other metabolic receptors using co-localization and co-immunoprecipitation approaches may reveal integrated signaling networks that collectively regulate energy homeostasis, potentially explaining why single-receptor therapeutic approaches often show limited efficacy in complex metabolic disorders.