NRPA2 Antibody

What is Neuropilin-2 (NRP2) and what cellular functions does it regulate?

Neuropilin-2 (NRP2) is a single-pass multifunctional transmembrane glycoprotein that plays crucial roles in cell development, immunity, cancer progression, and both physiological and pathological angiogenesis. NRP2 was initially discovered as a regulator of nervous system development, functioning as a semaphorin co-receptor with plexins. Additionally, it enhances cellular responses to members of the vascular endothelial growth factor (VEGF) family. The protein contains a large extracellular N-terminal domain comprising five subdomains, a short transmembrane domain, and a small cytoplasmic domain . NRP2 is typically expressed as a homodimer but can also form heterodimers with its family counterpart, Neuropilin-1 (NRP1) .

In which cell types is NRP2 expression commonly detected?

NRP2 exhibits a diverse expression pattern across multiple cell types and tissues. It is prominently expressed in endothelial cells, neurons, pancreatic islet cells, hepatocytes, melanocytes, and osteoblasts. This wide distribution reflects its multifunctional role in various physiological processes. In pathological contexts, NRP2 has been demonstrated to mediate proliferation, survival, and migration of tumor cells, highlighting its significance in cancer biology . The broad expression profile makes NRP2 an important target for research across multiple disciplines, from developmental biology to oncology and immunology.

What are the key differences between NRP2 and NR2 antibodies in terms of target specificity?

NRP2 antibodies specifically target Neuropilin-2, a transmembrane glycoprotein involved in developmental, immune, and cancer processes. These antibodies are designed to recognize specific epitopes on NRP2, such as the extracellular domain represented by the amino acid sequence (C)REDQGGEWKHGR, corresponding to residues 747-758 of human NRP2 . In contrast, NR2 (N-methyl-D-aspartic acid receptor subunit 2) antibodies target glutamate receptor subunits that are primarily expressed in neuronal tissues. Anti-NR2 antibodies have been studied extensively in the context of autoimmune conditions like systemic lupus erythematosus (SLE), where they cross-react with DNA and potentially contribute to neuropsychiatric symptoms . The specificity of anti-NRP2 antibodies is typically confirmed through validation processes that ensure they do not cross-react with the structurally related NRP1 protein .

How can researchers validate the specificity of anti-NRP2 antibodies in experimental settings?

Validation of anti-NRP2 antibodies requires a multi-method approach to ensure specificity. Researchers should implement a combination of techniques including enzyme-linked immunoassays (ELISA), Western blot analyses, biolayer interferometry, and immunohistochemistry . A robust validation protocol should demonstrate that the antibody detects all major NRP2 isoforms while showing no cross-reactivity with the structurally similar Neuropilin-1 (NRP1) protein. When validating novel anti-NRP2 antibodies, it is recommended to first establish detection limits using recombinant NRP2 protein and then validate expression in relevant cell lines known to express NRP2, such as endothelial cells or specific tumor cell lines. Researchers should also consider validation in knockout or knockdown systems to confirm specificity. For example, comparing signal between wild-type cells and cells with CRISPR/Cas9-mediated NRP2 knockout can provide definitive evidence of antibody specificity . Additionally, researchers should verify antibody performance across multiple applications to ensure consistent target recognition under different experimental conditions.

What experimental considerations are crucial when using anti-NR2 antibodies to study effects on the blood-brain barrier (BBB)?

When investigating the effects of anti-NR2 antibodies on the BBB, researchers must carefully control several experimental variables. First, the concentration of antibodies must be standardized based on previous literature; studies have used anti-NR2 at concentrations of approximately 10 μg/ml . Transepithelial electrical resistance (TEER) measurements provide a quantitative assessment of BBB integrity, with decreases in TEER values indicating increased permeability. In reported experiments, anti-NR2 antibody treatment decreased TEER values by approximately 54.6% compared to controls .

The blood-brain barrier model should incorporate primary brain microvessel endothelial cells grown to confluence, with verification of model integrity through leak tests before experimental manipulations. When studying the mechanistic actions of anti-NR2 antibodies, it's essential to include appropriate controls, such as comparing effects against NMDA receptor agonists (e.g., glutamate) and antagonists (e.g., ifenprodil and memantine). Research has demonstrated that NMDA receptor antagonists can counteract the permeability-increasing effects of anti-NR2 antibodies when co-administered, suggesting that anti-NR2 antibodies function as receptor agonists in this context . Additional controls should include normal serum, negative cerebrospinal fluid, and negative serum to establish baseline readings and account for non-specific effects.

How do neuropsychological manifestations correlate with anti-NR2 antibody titers in systemic lupus erythematosus (SLE) patients?

In systemic lupus erythematosus (SLE), the relationship between serum anti-NR2 antibodies and neuropsychological manifestations presents a complex picture. Studies indicate that anti-NR2 antibodies have a prevalence of approximately 25-35% in SLE patients, with moderate specificity (70-80%) . The correlation between these antibodies and cognitive dysfunction has been inconsistent across research studies, with no significant correlation established between serum anti-NR2 antibody positivity and general cognitive dysfunction .

What are the functional differences between anti-P ribosomal and anti-NR2 antibodies in neuropsychiatric lupus research?

Anti-P ribosomal and anti-NR2 antibodies represent distinct autoantibody populations with different pathogenic mechanisms in neuropsychiatric lupus, despite some overlapping clinical manifestations. Anti-P antibodies have a positivity rate of 10-40% in SLE patients with high specificity (95-100%), while anti-NR2 antibodies show similar prevalence (25-35%) but lower specificity (70-80%) .

Functionally, anti-P antibodies bind to a neuronal surface protein antigen, subsequently triggering increased calcium influx and enhanced glutamatergic transmission through activation of both AMPA receptors (AMPAR) and NMDA receptors (NMDAR). This cascade leads to apoptosis in cortical and hippocampal neurons, potentially explaining memory impairment and psychotic symptoms observed in animal models . Notably, intracerebroventricular injection of anti-P antibodies in mice induces depression-like behavior .

In contrast, anti-NR2 antibodies directly target NMDA receptor subunits, cross-reacting with DNA through molecular mimicry. Their presence in cerebrospinal fluid has been associated with diffuse psychiatric manifestations of SLE, while their presence in serum correlates more specifically with depression . When conducting research on these autoantibodies, appropriate quantification methods are essential – anti-P antibodies are typically measured using commercial ELISA kits (with established cut-offs around 17 U/mL), while anti-NR2 antibodies may be detected using custom ELISA protocols with the DWEYSVWLSN peptide .

What cell-based assays are recommended for evaluating NRP2 expression using fluorescently labeled antibodies?

For evaluating NRP2 expression using fluorescently labeled antibodies such as Anti-Neuropilin-2 (NRP2) (extracellular)-FITC Antibody, flow cytometry on intact live cells represents a preferred methodological approach. Based on documented protocols, researchers should employ the following methodology:

Cell preparation should focus on maintaining intact cell membranes to preserve the extracellular epitopes targeted by the antibody. Human cell lines with known NRP2 expression, such as THP-1 acute monocytic leukemia cells, serve as appropriate positive controls . The experimental design should include:

• A negative control of unstained cells to establish autofluorescence baseline

• An isotype control (e.g., rabbit IgG isotype control-FITC) to determine non-specific binding

• The test condition using Anti-Neuropilin-2 (extracellular)-FITC Antibody at an optimized concentration (approximately 2.5 μg per sample)

For quantitative assessment, mean fluorescence intensity should be measured and compared between test and control conditions. When analyzing results, researchers should consider:

• Signal-to-background ratio to determine specific binding

• Population homogeneity/heterogeneity in NRP2 expression

• Correlation with other known markers or cellular phenotypes

This methodology allows for quantitative assessment of cell surface NRP2 expression while maintaining native protein conformation in the cellular context.

What validation techniques ensure optimal performance of anti-NRP2 antibodies in western blotting applications?

Validation of anti-NRP2 antibodies for western blotting requires a systematic approach to ensure specificity, sensitivity, and reproducibility. A comprehensive validation protocol should incorporate the following elements:

• Multi-cell line validation: Evaluate antibody performance across multiple biologically relevant cell lysates known to express endogenous NRP2 at varying levels. This approach helps confirm specificity across different cellular contexts .

• Appropriate controls: Include negative controls (cell lines or tissues with confirmed absence of NRP2) and positive controls (recombinant NRP2 protein). Where available, CRISPR/Cas9 knockout models provide definitive negative controls for validation .

• Complete blot assessment: Examine the entire blot without cropping to narrow molecular weight ranges, ensuring all potential cross-reactive bands are visible and documented. This transparency allows researchers to identify any non-specific binding .

• Loading and transfer monitoring: Employ technologies like stain-free imaging to verify consistent protein loading and efficient transfer, eliminating these variables as potential sources of inconsistency .

• Optimization of conditions: Systematically test various antibody concentrations, blocking conditions, and incubation times to determine optimal signal-to-noise ratios.

• Isoform detection verification: Confirm that the antibody detects all relevant NRP2 isoforms by analyzing samples known to express different variants.

Researchers should document these validation steps thoroughly and provide this information when reporting experimental results to ensure reproducibility across laboratories.

How should researchers design ELISA protocols to quantify anti-NR2 antibodies in clinical samples?

Designing robust ELISA protocols for quantifying anti-NR2 antibodies in clinical samples requires careful consideration of several methodological factors. Based on established research approaches, the following protocol framework is recommended:

• Antigen selection: Utilize the DWEYSVWLSN peptide, which has been validated for detection of anti-NR2 antibodies following protocols established by Putterman and Diamond . This peptide specifically represents the epitope targeted by pathogenic anti-NR2 antibodies.

• Reference standards: Establish a calibration curve using purified anti-NR2 antibodies of known concentration. If unavailable, a pooled high-positive serum sample with assigned arbitrary units can serve as a working standard.

• Control selection: Include at least 10 healthy control samples to establish background levels. Define positivity threshold as optical density (OD) values exceeding 2 standard deviations above the mean of healthy controls .

• Sample processing: Standardize sample collection, processing, and storage procedures to minimize pre-analytical variability. Serum samples should be heat-inactivated at 56°C for 30 minutes to inactivate complement.

• Detection system: For human studies, use horseradish peroxidase (HRP)-conjugated anti-human IgG as the secondary antibody, with optical density monitored at 405 nm using a standardized plate reader .

• Quality control: Include internal quality control samples (high, medium, and low anti-NR2 levels) on each plate to monitor inter-assay variability.

• Validation criteria: Establish acceptance criteria for assay performance including intra-assay CV (<10%), inter-assay CV (<15%), and minimum detectable concentration.

This methodological approach ensures reliable quantification of anti-NR2 antibodies, facilitating both research applications and potential clinical translation.

How are anti-NRP2 antibodies being utilized in sarcoidosis research and potential companion diagnostics?

The development of novel anti-NRP2 antibodies has opened significant avenues for sarcoidosis research and companion diagnostics. NRP2 has emerged as a relevant target in sarcoidosis due to the ongoing clinical trials of efzofitimod, a novel immunomodulatory molecule targeting NRP2, for pulmonary sarcoidosis treatment . Researchers utilizing anti-NRP2 antibodies in this context should consider several methodological approaches:

First, immunohistochemistry applications using validated anti-NRP2 antibodies have demonstrated high NRP2 expression in granulomas from sarcoidosis patient skin and lung biopsies . This technique provides critical insight into the tissue-specific expression patterns of NRP2 in affected organs, potentially identifying patients most likely to benefit from NRP2-targeted therapies.

For companion diagnostic development, antibodies must undergo rigorous validation across multiple platforms including enzyme-linked immunoassay, Western blot, biolayer interferometry, and immunohistochemistry . The antibody should demonstrate detection capability for all major NRP2 isoforms without cross-reactivity with the structurally similar NRP1.

Researchers can employ these antibodies to establish correlations between NRP2 expression levels and clinical parameters such as disease severity, treatment response, and prognostic indicators. This approach may help stratify patients for clinical trials and potentially predict treatment outcomes with NRP2-targeting therapies like efzofitimod.

What methodological approaches are optimal for studying anti-NR2 antibody effects on blood-brain barrier permeability?

To effectively study anti-NR2 antibody effects on blood-brain barrier (BBB) permeability, researchers should implement in vitro BBB models based on primary brain microvessel endothelial cells. The following methodological approach has demonstrated effectiveness:

• Model establishment: Primary brain microvessel endothelial cells should be isolated, cultured until confluence, and subjected to leak tests to verify barrier integrity before experimentation .

• Permeability assessment: Transepithelial electrical resistance (TEER) measurements provide the primary quantitative endpoint for evaluating BBB integrity. TEER values should be measured at baseline and at standardized intervals following antibody exposure .

• Experimental design: A comprehensive experiment should include:

  • Test condition: Anti-NR2 antibody (10 μg/ml)

  • Positive controls: Glutamate (5 mM) as a known NMDA receptor agonist

  • Negative controls: Normal serum samples

  • Mechanistic investigation: NMDA receptor antagonists (ifenprodil and memantine at 10 μg/ml) alone and in combination with anti-NR2 antibody

• Data interpretation: Expected results based on previous studies include:

  • Decreased TEER values (approximately 54.6% reduction) following anti-NR2 antibody treatment

  • Similar decreases with positive cerebrospinal fluid and positive serum (57.5% and 59.6% respectively)

  • Less pronounced decreases with negative CSF and negative serum (22% and 24.1%)

  • No significant changes with normal serum

• Pharmacological validation: Co-administration of NMDA receptor antagonists with anti-NR2 antibody should prevent TEER reductions, confirming the receptor-mediated mechanism of action .

This methodological framework enables quantitative assessment of BBB disruption by anti-NR2 antibodies and facilitates mechanistic investigations of pathophysiological processes relevant to neuropsychiatric SLE.

How can researchers correlate anti-NR2 antibody levels with neuropsychiatric manifestations in SLE patients?

A comprehensive approach to correlating anti-NR2 antibody levels with neuropsychiatric manifestations in SLE patients requires integration of serological, neuropsychological, and neuroimaging methodologies. Based on established research protocols, the following approach is recommended:

This integrated approach allows researchers to establish both direct associations between antibody levels and clinical manifestations and potential mechanistic pathways through altered functional brain connectivity.

What emerging research directions are advancing our understanding of NRP2 antibodies in cancer and inflammatory conditions?

Research on NRP2 antibodies is rapidly evolving, with several promising directions emerging in cancer and inflammatory disease contexts. The development of novel, highly specific antibodies against NRP2 has enabled more precise investigation of its role in pathophysiological processes. Current evidence highlights NRP2's significance in mediating proliferation, survival, and migration of tumor cells, suggesting therapeutic potential in oncology applications .

In inflammatory conditions, the identification of high NRP2 expression in sarcoidosis granulomas represents a significant advancement, supporting ongoing clinical trials of NRP2-targeting therapeutics like efzofitimod . These findings are driving the development of companion diagnostics using anti-NRP2 antibodies to identify patients most likely to benefit from targeted therapies.

Future research directions should explore the relationship between NRP2 expression patterns and clinical outcomes across various diseases, potentially identifying new therapeutic applications. Integration of anti-NRP2 antibodies with emerging technologies such as single-cell analysis and spatial transcriptomics will likely provide deeper insights into the cell type-specific functions of NRP2 in health and disease.