RH15 Antibody

What is LRRC15 and why is it a significant research target?

LRRC15 (also known as LIB or Leucine-rich repeat protein induced by beta-amyloid homolog) is a transmembrane protein with significant roles in multiple biological processes. Its importance stems from its modulation of SARS-CoV-2 infection through interaction with viral spike proteins and its high expression in several cancer types, particularly osteosarcoma. LRRC15 doesn't function as a viral entry receptor but instead sequesters virions and antagonizes SARS-CoV-2 infection of ACE2(+) cells when expressed on nearby cells . In oncology research, LRRC15 has emerged as a promising therapeutic target due to its high expression on tumor cell surfaces, making it valuable for antibody-drug conjugate development .

What are the validated biological samples compatible with LRRC15 antibodies?

LRRC15 antibodies have been validated for detection in both human and mouse samples. For human samples, commercially available antibodies have demonstrated efficacy in multiple cell lines including:

• U-87 MG and U-118 MG human glioblastoma cells

• MCF7 human breast adenocarcinoma cells

• HeLa human cervical adenocarcinoma cells

• Human skin tissue samples

The reactivity with mouse samples has also been confirmed, suggesting cross-species conservation of relevant epitopes . When designing experiments, researchers should verify the specific validation status for their particular application, as some antibodies may be more thoroughly tested for certain techniques than others.

What experimental techniques are validated for LRRC15 antibody applications?

Current research validates LRRC15 antibody applications in multiple experimental techniques:

• Flow cytometry: LRRC15 antibodies reliably detect cell surface expression in multiple cell types with high specificity at dilutions around 1/50 (1μg). Signal detection typically employs secondary antibodies such as goat anti-rabbit IgG conjugated to fluorophores (e.g., Alexa Fluor 647) .

• Western blotting: LRRC15 can be detected in cell lysates and tissue samples, showing characteristic bands at approximately 70-80 kDa and 104 kDa under reducing conditions. Optimal antibody concentrations range from 2-20 μg/mL depending on sample type and detection system .

• Simple Western automated protein analysis: LRRC15 detection has been validated using this platform with similar concentration parameters as traditional Western blotting .

How can researchers optimize LRRC15 detection in cell lines with variable expression levels?

LRRC15 expression varies significantly between cell lines, presenting challenges for consistent detection. To optimize detection:

• TGF-β treatment: LRRC15 expression is significantly upregulated by TGF-β treatment. For low-expressing cell lines, pre-treatment with 20ng/ml TGF-β for 48 hours can substantially increase detectable LRRC15 levels. Flow cytometry data shows marked enhancement of LRRC15 signal in U-87 MG cells following this protocol .

• Cell line selection: For positive controls, U-118 MG and U-87 MG glioblastoma cells demonstrate high expression levels, while MCF7 cells show comparatively lower expression .

• Antibody titration: For flow cytometry applications, begin with 1:50 dilution (approximately 1μg) and adjust based on signal-to-noise ratio. Include appropriate isotype controls (e.g., rabbit monoclonal IgG) to distinguish specific from non-specific binding .

• Sample preparation: For Western blotting, thorough cell lysis and proper protein denaturation are critical for consistent LRRC15 detection, particularly given its membrane localization .

What are the optimal fixation and permeabilization protocols for LRRC15 antibody in immunological assays?

For optimal LRRC15 detection in immunological assays, researchers should consider:

For flow cytometry:

• Surface staining typically requires minimal fixation (1-2% paraformaldehyde) to preserve epitope accessibility

• When detecting both intracellular and membrane-bound LRRC15, a sequential approach works best:

  • Fix cells with 2-4% paraformaldehyde for 10-15 minutes at room temperature

  • Permeabilize with 0.1% saponin or 0.1-0.3% Triton X-100 in PBS with 1% BSA

  • Block with 5-10% normal serum from the same species as the secondary antibody

  • Incubate with primary LRRC15 antibody at optimized concentration (typically 1-5 μg/mL)

  • Detect with fluorophore-conjugated secondary antibody

For Western blotting and immunohistochemistry:

• Standard RIPA or NP-40-based lysis buffers are suitable for protein extraction

• Heat denaturation at 95°C for 5 minutes in reducing sample buffer improves detection

• For tissue sections, antigen retrieval methods (heat-induced in citrate buffer, pH 6.0) significantly enhance LRRC15 detection

How can specificity of LRRC15 antibody binding be validated in experimental systems?

Validating LRRC15 antibody specificity requires multi-faceted approaches:

• Positive and negative controls: Compare staining patterns between known high-expressing (U-118 MG) and low-expressing (MCF7) cell lines. The differential detection pattern confirms antibody specificity .

• Isotype controls: Always include appropriate isotype controls (e.g., rabbit monoclonal IgG for rabbit-derived LRRC15 antibodies) to establish baseline non-specific binding .

• Molecular weight verification: In Western blotting, LRRC15 should appear at specific molecular weights (approximately 70-80 kDa and 104 kDa bands under reducing conditions), consistent with its predicted size and post-translational modifications .

• Expression manipulation: Validate antibody specificity by artificially modulating LRRC15 expression:

  • Through TGF-β stimulation, which should increase detectable signal

  • Using siRNA knockdown or CRISPR-mediated knockout, which should eliminate specific signal

  • With overexpression systems, which should enhance signal intensity in proportion to expression level

• Cross-reactivity assessment: Test the antibody against recombinant LRRC15 and structurally similar proteins to confirm specificity within the leucine-rich repeat protein family.

How can LRRC15 antibodies be leveraged for therapeutic development in cancer research?

LRRC15 antibodies show significant promise in cancer therapeutics, particularly through antibody-drug conjugate (ADC) development:

• ADC development strategy: LRRC15 antibodies have been successfully conjugated to different cytotoxic payloads:

  • LRRC15-PNU (conjugated to anthracycline derivative PNU-159682)

  • LRRC15-MMAE (conjugated to tubulin inhibitor monomethyl auristatin E)

  Comparative studies in osteosarcoma models indicate superior efficacy of LRRC15-PNU conjugates, with dose-dependent growth inhibition in high LRRC15-expressing cell lines. This therapeutic approach achieved 40-100% cure rates in xenograft models .

• Patient stratification biomarkers: LRRC15 expression analysis using validated antibodies can identify patients most likely to respond to LRRC15-targeted therapies. Flow cytometry protocols using 1:50 antibody dilutions on tumor biopsy samples can reliably quantify expression levels .

• Combination therapy approaches: Research indicates potential for sensitizing low LRRC15-expressing tumors through TGF-β pathway modulation prior to ADC administration, expanding the therapeutic window .

• Clinical translation: The LRRC15-directed ADC ABBV-085 has progressed through preclinical studies to Phase 1 clinical trials (NCT02565758), showing partial responses in 40% of undifferentiated pleomorphic sarcoma patients and 20% of osteosarcoma patients .

What methodological approaches can distinguish LRRC15's role in SARS-CoV-2 infection from other cellular interactions?

LRRC15's complex role in SARS-CoV-2 infection necessitates sophisticated experimental designs:

• Co-culture experimental systems: Since LRRC15 doesn't function as a direct viral entry receptor but instead influences infection in trans, researchers should design co-culture systems with:

  • LRRC15-expressing cells (detected and quantified via antibody staining)

  • ACE2-positive target cells

  • Appropriate controls including LRRC15-knockout cells

  These systems allow assessment of LRRC15's ability to sequester virions and antagonize infection of neighboring cells .

• Binding interaction characterization: Use LRRC15 antibodies to:

  • Perform co-immunoprecipitation assays to isolate LRRC15-spike protein complexes

  • Identify binding domains through competition assays with domain-specific antibodies

  • Map interaction interfaces using epitope binning approaches

• Quantitative infection assays: Measure infection rates in cells with varying LRRC15 expression levels (manipulated via TGF-β stimulation or gene editing) and correlate with antibody-detected LRRC15 surface levels to establish dose-response relationships .

How can researchers overcome antibody cross-reactivity challenges when studying LRRC15 in complex tissue microenvironments?

When investigating LRRC15 in heterogeneous tissues with potential cross-reactivity concerns:

• Dual-staining approaches: Combine LRRC15 antibody staining with lineage-specific markers to distinguish true LRRC15 expression among diverse cell populations. This is particularly important in tumor microenvironments where LRRC15 may be expressed by both malignant cells and stromal components.

• Absorption controls: Pre-absorb LRRC15 antibodies with recombinant LRRC15 protein before tissue staining. Elimination of staining after absorption confirms specificity.

• Multiparametric analysis: In flow cytometry, use polychromatic panels including:

  • LRRC15 antibody (typically detected in far-red channels)

  • Cell type-specific markers

  • Viability dyes

  • Functional markers

  This approach allows accurate identification of LRRC15-positive cell populations within complex samples .

• Spectral unmixing: When using fluorescence-based detection in tissues with high autofluorescence (like bone in osteosarcoma studies), employ spectral unmixing algorithms to distinguish specific LRRC15 antibody signal from background.

How should researchers interpret variable LRRC15 antibody staining patterns across different cell lines and tissues?

When encountering variable LRRC15 staining patterns:

• Expression heterogeneity assessment: LRRC15 expression naturally varies across cell types and can be dynamically regulated. Flow cytometry data shows marked differences between cell lines like U-118 MG (high expression) versus MCF7 (low expression) .

• Microenvironmental influences: TGF-β in the tumor microenvironment significantly upregulates LRRC15 expression. Experimental data demonstrates that treating U-87 MG cells with 20ng/ml TGF-β for 48 hours dramatically increases LRRC15 detection by flow cytometry .

• Technical versus biological variation: Distinguish technical artifacts from true biological differences by:

  • Using consistent staining protocols across samples

  • Including known positive (U-118 MG) and negative controls in each experiment

  • Considering fixation effects (overfixation can mask LRRC15 epitopes)

  • Validating with multiple detection methods (e.g., flow cytometry and Western blot)

• Context-specific regulation: The presence of specific signaling molecules, cell differentiation states, and tissue microenvironments can all affect LRRC15 expression levels. Integrate these contextual factors when interpreting staining variations.

What experimental controls are essential when using LRRC15 antibodies for investigating virus-host interactions?

For virus-host interaction studies involving LRRC15, implement these critical controls:

• Baseline expression controls: Document pre-infection LRRC15 levels in target cells using calibrated antibody staining protocols to establish reference points for post-infection comparisons.

• Specificity controls:

  • Isotype-matched irrelevant antibodies to establish background staining levels

  • LRRC15 knockout or knockdown cells to confirm signal specificity

  • Competitive blocking with recombinant LRRC15 protein to validate antibody binding sites

• Functional validation controls:

  • LRRC15-neutralizing antibodies to confirm functional relevance of observed interactions

  • Domain-specific antibodies to map interaction regions

  • Shedding inhibitors to distinguish membrane-bound from soluble LRRC15 contributions

• Cell system controls:

  • ACE2-positive versus ACE2-negative cells in SARS-CoV-2 studies

  • Co-culture systems with varying ratios of LRRC15-expressing and target cells

  • Mock-infected controls to distinguish infection-specific from stress-related LRRC15 regulation

How can researchers address batch-to-batch variability in LRRC15 antibody performance?

Managing antibody variability requires systematic approaches:

• Standardized validation protocol: Establish a reference testing protocol using:

  • Known high-expressing cell line (U-118 MG or U-87 MG)

  • Standardized staining protocol with fixed antibody concentrations (1:50 dilution for flow cytometry)

  • Quantitative readout metrics (median fluorescence intensity ratios relative to isotype control)

  • Consistent instrument settings and analysis parameters

• Internal calibration standards: Maintain frozen aliquots of standardized cell preparations and reference antibody lots for side-by-side comparison with new batches.

• Parallel testing approach: When transitioning to a new antibody batch:

  • Run parallel experiments with old and new batches

  • Generate titration curves to identify optimal concentrations for the new batch

  • Document detailed performance characteristics including signal-to-noise ratio, non-specific binding, and detection sensitivity

• Recombinant antibody advantages: Consider using recombinant monoclonal antibodies like those described in the search results, which offer greater batch-to-batch consistency compared to traditional hybridoma-derived antibodies .

How might LRRC15 antibodies be adapted for multimodal imaging applications in cancer research?

Expanding LRRC15 antibody applications to multimodal imaging offers exciting research opportunities:

• Fluorescence-guided surgery: Conjugate LRRC15 antibodies to near-infrared fluorophores for real-time visualization of LRRC15-expressing tumors during surgical resection, particularly relevant for osteosarcoma where complete tumor removal is challenging.

• PET/SPECT imaging: Develop radiolabeled LRRC15 antibodies (using isotopes like 89Zr, 111In, or 64Cu) for non-invasive whole-body imaging to:

  • Detect primary and metastatic LRRC15-positive tumors

  • Monitor treatment response to LRRC15-targeted therapies

  • Stratify patients for LRRC15-directed treatments based on tumor expression levels

• Multiplexed tissue imaging: Adapt LRRC15 antibodies for multiplexed imaging technologies (e.g., imaging mass cytometry or cyclic immunofluorescence) to simultaneously visualize LRRC15 expression alongside dozens of other markers in the tumor microenvironment, revealing spatial relationships between LRRC15-positive cells and other cellular components.

• Theranostic applications: Develop dual-function conjugates that combine imaging capabilities with therapeutic payloads, building on the established efficacy of LRRC15-targeted antibody-drug conjugates in preclinical models .

What methodological approaches could enhance the development of next-generation LRRC15 antibodies for neutralizing SARS-CoV-2 variants?

As SARS-CoV-2 variants continue to emerge, next-generation LRRC15 antibody development requires:

• Epitope mapping strategies: Employ techniques including:

  • Hydrogen-deuterium exchange mass spectrometry to identify LRRC15 regions involved in spike binding

  • Cryo-EM structural analysis of LRRC15-spike complexes

  • Directed evolution of antibodies targeting conserved binding interfaces

• Variant spike protein panels: Develop screening platforms with spike proteins from multiple SARS-CoV-2 variants to identify LRRC15 antibodies maintaining activity across diversified strains.

• Functional screening assays: Design high-throughput screening systems that measure:

  • Direct LRRC15-spike binding inhibition

  • Trans-inhibition of viral entry in co-culture systems

  • Virion sequestration efficiency

• Cocktail approaches: Develop synergistic antibody combinations targeting different LRRC15 epitopes involved in variant spike recognition to create broader protection against viral escape .

How can single-cell analysis technologies be integrated with LRRC15 antibodies to better understand heterogeneous expression patterns in tumor microenvironments?

Integrating LRRC15 antibodies with single-cell technologies offers unprecedented resolution:

• Single-cell RNA-sequencing with protein detection: Combine LRRC15 antibody staining with techniques like CITE-seq to simultaneously measure LRRC15 protein expression and transcriptome-wide gene expression in thousands of individual cells, revealing:

  • Correlations between LRRC15 protein levels and transcriptional states

  • Cell type-specific expression patterns in heterogeneous tumors

  • Associations with therapeutic resistance markers

• Spatial transcriptomics integration: Combine LRRC15 immunofluorescence with spatial transcriptomics to map LRRC15 protein expression within its spatial tissue context alongside transcriptome-wide data.

• Mass cytometry (CyTOF) panels: Develop metal-conjugated LRRC15 antibodies for integration into CyTOF panels enabling simultaneous measurement of 40+ proteins on single cells, allowing comprehensive phenotyping of LRRC15-positive populations.

• Functionalized cell sorting: Use LRRC15 antibodies to isolate live LRRC15-positive cells for downstream functional assays, including:

  • Drug sensitivity testing

  • Invasion/migration capabilities

  • Single-cell cloning for heterogeneity assessment

  • Xenograft establishment to evaluate in vivo behavior