GNRHR Antibody

What is GNRHR and what cellular functions does it regulate?

GNRHR belongs to the G-protein coupled receptor 1 family and functions as a receptor for GnRH that mediates the stimulation of gonadotropic hormones, specifically luteinizing hormone (LH) and follicle-stimulating hormone (FSH) . This receptor mediates its action by association with G-proteins that activate a phosphatidylinositol-calcium second messenger system . GNRHR plays a crucial role in reproductive function, and dysregulation has been implicated in various reproductive disorders. Alternative isoforms, such as isoform 2, may act as inhibitors of GnRH-R signaling .

What applications are GNRHR antibodies validated for in current research?

Based on extensive validation data, GNRHR antibodies can be used in multiple applications with varying dilution requirements:

It is recommended to optimize dilutions for each specific experimental system .

What are the key differences between antibodies targeting GnRH versus GnRHR?

Anti-GnRH antibodies target the gonadotropin-releasing hormone ligand itself, while anti-GnRHR antibodies recognize the receptor protein. This distinction is critical because:

• Anti-GnRHR antibodies are used to detect receptor expression in tissues and cells, helping to determine which tissues might respond to GnRH signaling .

• Anti-GnRH antibodies have been found as autoantibodies in certain pathological conditions, such as gastrointestinal dysmotility .

• The detection methods and experimental applications differ: GnRHR antibodies are primarily used for protein localization and quantification, while GnRH antibodies may be used to neutralize the hormone in functional studies.

How should researchers optimize antigen retrieval protocols for GNRHR immunohistochemistry?

For optimal immunohistochemical detection of GNRHR:

• Primary recommendation: Use TE buffer at pH 9.0 for antigen retrieval .

• Alternative method: Citrate buffer at pH 6.0 can also be effective .

• Incubation parameters: For primary antibody incubation, use 1:100-1:500 dilution (depending on the specific antibody) overnight at 4°C in a humidified chamber .

• Detection systems: Avidin-biotin-peroxidase kits with diaminobenzidine as the chromogen have shown reliable results .

• Include appropriate positive controls: Pituitary tissue is recommended as a positive control for GNRHR expression .

How can researchers map epitopes of GnRHR-activating autoantibodies?

Epitope mapping for GnRHR autoantibodies requires a systematic approach:

• Design overlapping peptides spanning the target region (e.g., the second extracellular loop of GnRHR).

• Test peptide reactivity using sera from antibody-positive patients compared to antibody-negative controls.

• Identify specific sequences recognized by autoantibodies.

A successful example from PCOS research identified two peptide sequences (FSQCVTHC and HCSFSQWW) that were recognized by all PCOS sera but not by control sera . The location of these epitopes can be visualized in 3D structure using protein databases (e.g., Protein Data Bank) and molecular visualization software like Jmol .

What methodologies are effective for developing GnRHR-targeted imaging probes?

The development of GnRHR-targeted imaging probes involves:

• Selection of a specific GnRHR ligand (e.g., GnRH peptide or antagonist like Cetrorelix).

• Conjugation with appropriate fluorophores:

  • For microscopy: FITC conjugation using an Acp (amino caproic acid) linker

  • For in vivo imaging: Near-infrared fluorophores like ICG

The synthesis process typically involves:

• Adding the peptide with N,N-diisopropylethylamine in ultra-dry DMF under nitrogen protection.

• Adding the fluorophore-NHS ester (e.g., ICG-NHS).

• Purification by precipitation followed by HPLC.

• Confirmation of the final product by analytical HPLC and MALDI mass spectroscopy .

What experimental approaches are used to characterize GnRHR mutations in clinical samples?

For characterizing GNRHR mutations in clinical research:

• Genetic screening: Sequence the GNRHR gene in patient cohorts to identify rare sequence variants (RSVs).

• Frequency analysis: Compare allele frequencies between patient cohorts and population databases (e.g., gnomAD) to determine statistical significance.

• Functional analysis: Assess the impact of mutations on receptor function through in vitro assays.

• Correlation studies: Analyze the relationship between specific mutations and clinical manifestations or treatment outcomes.

For example, a study on Chinese IHH patients identified a heterozygous GNRHR RSV with a prevalence of 2.6% (4/153) in the cohort, significantly higher than in the East Asian general population .

What are the methodological considerations for studying GnRHR autoantibodies in disease states?

When investigating GnRHR autoantibodies in conditions like PCOS or gastrointestinal dysmotility:

• Detection methods:

  • ELISA-based assays using purified GnRHR protein or peptides

  • Cell-based assays to assess functional activity of autoantibodies

• Correlation analysis:

  • Measure inflammatory markers (e.g., CRP) and immune activation markers (e.g., sCD40)

  • Analyze correlation between antibody titers and clinical symptoms

• Neutralization studies:

  • Design peptide inhibitors (e.g., retro-inverso peptides) that mimic identified epitopes

  • Test inhibitor efficacy in blocking autoantibody-induced effects in vitro

• Tissue analysis:

  • Examine GnRHR expression in target tissues

  • Assess potential neuronal damage in tissues affected by autoantibodies

How should researchers design experiments to validate GNRHR antibody specificity?

A comprehensive validation strategy should include:

• Multiple detection methods:

  • Western blot to confirm molecular weight (expected: 38 kDa calculated; observed: 60-70 kDa)

  • Immunohistochemistry in tissues with known expression patterns

  • Flow cytometry with appropriate controls

• Essential controls:

  • Positive tissue controls: Pituitary for high expression

  • Negative controls: Equivalent concentration of non-specific IgG

  • Peptide competition assays to confirm specificity

• Cross-validation with different antibodies:

  • Compare results using antibodies targeting different epitopes

  • Use both polyclonal and monoclonal antibodies when available

What methodological approaches help resolve discrepancies in GNRHR expression data?

When faced with conflicting GNRHR expression data:

• Quantitative analysis:

  • Use qRT-PCR with validated primers:
    Forward primer: GGC TGC CTC TTC ATC CCC CT
    Reverse primer: CGT TCT CAG CCG AGC TCT TGG G
    (Expected product: 147 bp)

  • Normalize to appropriate housekeeping genes (e.g., β-actin)

• Protein-level confirmation:

  • Use multiple antibodies targeting different epitopes

  • Compare results from different detection methods (Western blot, IHC, flow cytometry)

• Consider tissue/cell heterogeneity:

  • Single-cell analysis techniques may reveal subpopulations with different expression levels

  • Spatial analysis in tissues may show regional expression differences

• Compare with transcriptome data:

  • Analyze FPKM-UQ values from TCGA or other databases

  • Consider potential post-transcriptional regulation affecting protein levels

What factors should be considered when analyzing the therapeutic potential of GNRHR-targeting peptides?

When evaluating peptides that target GNRHR for therapeutic applications:

• Peptide stability:

  • Standard peptides are susceptible to proteolytic degradation

  • Retro-inverso peptides (using D-amino acids in reversed sequence) offer increased biological half-life

• Target specificity:

  • Confirm binding to the intended epitope

  • Evaluate potential off-target effects

• Functional efficacy:

  • Use appropriate cell-based bioassays (e.g., GeneBLAzer GnRHR cell-based β-lactamase reporter assay)

  • Compare efficacy to established GnRH analogues or antagonists

• Delivery considerations:

  • Assess different administration routes (oral, parenteral)

  • Evaluate biodistribution using imaging approaches

How are GNRHR antibodies being utilized in cancer research and diagnostics?

GNRHR antibodies are emerging as valuable tools for cancer research:

• Targeted imaging:

  • GnRHR-targeted near-infrared imaging probes can effectively distinguish peritoneal metastases from adjacent normal tissue in ovarian cancer models

  • These approaches leverage the differential expression of GnRHR in cancer versus normal tissues

• Expression profiling:

  • GnRHR expression varies across cancer types, with significant expression in ovarian, breast, endometrial, and prostate cancers

  • Transcriptome profiling data from TCGA can be used to identify high-expressing cancer types

• Potential therapeutic applications:

  • Detection of GnRHR in tumors may predict response to GnRH analog therapy

  • GnRHR antibodies could potentially be used for targeted drug delivery

What is the current understanding of GnRHR autoantibodies in autoimmune conditions?

Emerging research on GnRHR autoantibodies reveals:

• Disease associations:

  • Polycystic ovary syndrome (PCOS): GnRHR-activating autoantibodies (GnRHR-AAb) target the second extracellular loop of GnRHR

  • Gastrointestinal dysmotility: Anti-GnRH antibodies correlate with sCD40 levels

• Pathogenic mechanisms:

  • Prolonged uncontrolled activation of GnRHR by autoantibodies may disrupt pulsatile GnRH signaling

  • In PCOS, this could contribute to increased LH and testosterone levels

  • In gastrointestinal dysfunction, these antibodies may affect enteric neuron viability

• Therapeutic implications:

  • Retro-inverso peptides designed to target GnRHR-AAb can effectively block autoantibody-stimulated GnRHR activation in vitro

  • This approach may offer new treatment options for autoantibody-associated conditions

How can researchers reconcile the observed molecular weight differences in GNRHR detection?

The discrepancy between calculated (38 kDa) and observed (60-70 kDa) molecular weights of GNRHR can be addressed through:

• Post-translational modifications analysis:

  • Glycosylation status assessment

  • Phosphorylation site mapping

• Technical approaches:

  • Deglycosylation experiments prior to Western blotting

  • Mass spectrometry to identify actual protein composition

  • Use of multiple antibodies targeting different epitopes

• Expression system considerations:

  • Native tissue vs. recombinant expression systems

  • Cell-type specific modifications

Understanding these differences is crucial for accurate interpretation of experimental results and avoiding false negatives or positives in GNRHR detection.

What criteria should guide researchers in selecting the appropriate GNRHR antibody for specific applications?

When selecting a GNRHR antibody, researchers should consider:

• Target epitope location:

  • Antibodies targeting the second extracellular loop (ECL2) of GnRHR are useful for detecting functional receptors on cell surfaces

  • Antibodies against internal domains may be better for total protein detection

• Validated applications:

  • Check if the antibody has been validated for your specific application (WB, IHC, IF, FC)

  • Review published citations using the specific antibody

• Host species and clonality:

  • Polyclonal antibodies offer broader epitope recognition

  • Monoclonal antibodies provide higher specificity for a single epitope

  • Consider host species compatibility with your experimental system

• Sample type compatibility:

  • Confirm reactivity with your species of interest (human, mouse, rat, etc.)

  • Verify effectiveness in your tissue or cell type of interest