CML23 Antibody

What is ChemR23 and what detection methods are most effective for studying it?

ChemR23, also known as CMKLR1 (chemokine-like receptor 1), is a G protein-coupled receptor expressed on various human leukocytes. The receptor spans from Met1 to Leu371 (Accession # NP_004063) and functions as a receptor for chemerin .

For detection, multiple complementary techniques are recommended:

• Flow cytometry: Effective for quantifying cell surface expression levels on intact cells using specific anti-ChemR23 antibodies (like clone #84939)

• Immunocytochemistry/Immunofluorescence: Useful for visualizing cellular localization, particularly when combined with membrane markers such as wheat germ agglutinin (WGA)

• Confocal microscopy: Essential for detailed subcellular localization studies

• Competition binding assays: Valuable for functional characterization using radiolabeled ligands

For optimal results, permeabilized human neutrophils can be stained with anti-ChemR23 antibodies (with appropriate secondary antibodies like goat-anti-mouse Alexa 488) alongside membrane markers .

How is ChemR23 expression distributed across human immune cell populations?

ChemR23 shows distinct expression patterns across immune cell populations, with significant presence on:

• Neutrophils: Express detectable levels that can be modulated by inflammatory stimuli

• Natural killer cells: Show consistent expression as demonstrated by flow cytometry

• Dendritic cells: Particularly bone marrow-derived dendritic cells (BMDCs) express functional ChemR23

Expression can be verified using flow cytometry with specific antibodies. When characterizing expression, it's essential to include proper controls, such as isotype controls and ChemR23-knockout models where available, to ensure specificity of detection .

What factors affect ChemR23 expression in experimental settings?

ChemR23 expression is dynamically regulated by several factors that should be controlled in experimental designs:

• Pro-inflammatory mediators: TNF alpha (10 ng/ml, 20 min exposure), fMLF (1 μM), and IL-8 (100 ng/ml) can significantly alter ChemR23 expression levels

• Anti-inflammatory mediators: Annexin A1 (10 nM), alpha-melanocyte-stimulating hormone (alpha MSH, 10 nM), and C15 (10 pM) modify expression patterns

• Endothelial interactions: Flow over activated endothelial cells affects ChemR23 expression on neutrophils

For consistent results, researchers should standardize cell isolation procedures, cytokine concentrations, and incubation times. Statistical significance should be assessed relative to vehicle-treated or pre-flow cells with multiple independent experiments (n=3-6 recommended) .

How can I optimize antibody dilutions for ChemR23 detection in different applications?

Optimization strategies should be application-specific:

• Flow cytometry: Start with manufacturer-recommended dilutions (typically 1:100-1:200) and perform titration experiments to determine optimal signal-to-noise ratio

• Immunocytochemistry: Test a range of dilutions (1:50 to 1:500) with different fixation protocols

• Western blot: Begin with 1:1000 dilution and adjust based on signal intensity

How can ChemR23 knockout models be effectively utilized in research?

ChemR23 knockout (ChemR23−/−) models provide powerful research tools for studying receptor function through comparison with wild-type counterparts. Key applications include:

• Validation of antibody specificity: ChemR23−/− mouse neutrophils serve as essential negative controls to confirm antibody specificity in flow cytometry and immunostaining

• Functional studies: Comparing calcium flux and migration responses between wild-type and knockout cells helps establish ChemR23-dependent signaling pathways

• Competition binding assays: Using BMDCs from wild-type and ChemR23−/− mice helps distinguish specific from non-specific binding in ligand interaction studies

When using these models, researchers should confirm complete absence of ChemR23 expression using both protein detection methods and functional assays to ensure the knockout is effective.

What methodologies are most effective for studying ChemR23 signaling mechanisms?

For investigating ChemR23 signaling pathways, several complementary approaches provide robust data:

• Calcium flux assays: Load neutrophils with Fura2-AM and measure responses to specific ligands (C15 at 10 pM or chemerin at 1 nM). Include controls such as ionomycin (positive control), vehicle (negative control), and specific ChemR23 inhibitors (e.g., CCX2005 at 100 nM)

• Receptor co-localization: Co-stain for ChemR23 alongside markers of secretory vesicles (CD35), specific granules (CD66b), and azurophil granules (CD63) to determine subcellular compartmentalization

• Signaling inhibitor studies: Use specific pathway inhibitors to dissect downstream signaling components

Data representation as delta F340/F380 ratios with mean values±standard error from multiple independent experiments (n=3-6) provides statistically robust measurements .

How can researchers design ChemR23 antibodies with customized specificity profiles?

Designing antibodies with tailored specificity requires sophisticated approaches:

The development of customized ChemR23 antibodies can be approached through:

• Phage display selection: Using systematically varied antibody libraries where complementary determining regions (CDRs, particularly CDR3) are modified with different amino acid combinations

• Biophysics-informed modeling: Training computational models on experimentally selected antibodies to identify distinct binding modes associated with specific ligands

• Specificity optimization:

  • For cross-specific antibodies: Jointly minimize the energy functions associated with all desired ligands

  • For highly specific antibodies: Minimize energy functions for the target ligand while maximizing those for undesired ligands

This approach enables the generation of novel antibody sequences not present in initial libraries but predicted to have desired binding profiles, offering greater control than traditional selection methods alone .

What are the challenges in distinguishing between closely related epitopes in ChemR23 research?

Discriminating between similar epitopes presents significant challenges:

• Epitope similarity: Closely related epitopes may differ by only a few amino acids, making specificity difficult to achieve

• Co-expression: ChemR23 may be co-expressed with related receptors in the same cells

• Conformational states: Different activation states of ChemR23 may present distinct epitopes

Advanced solutions include:

• High-throughput sequencing combined with computational analysis to identify antibody variants that recognize specific epitopes

• Competition binding assays using known ligands (e.g., chemerin at 300 nM) to determine binding site overlap

• Careful validation using knockout models to confirm specificity

How can ChemR23 antibodies be used to study receptor trafficking and internalization?

Tracking ChemR23 trafficking requires specialized techniques:

• Live-cell imaging: Using fluorescently labeled antibodies to track receptor movement in real-time

• Subcellular fractionation: Combined with Western blotting to quantify receptor distribution

• Co-localization studies: Confocal microscopy with permeabilized neutrophils, staining for ChemR23 alongside wheat germ agglutinin to visualize the cell membrane

For granule association studies, researchers should stain human neutrophils for ChemR23 together with markers of secretory vesicles (CD35), specific granules (CD66b), and azurophil granules (CD63) to determine storage compartments and mobilization dynamics .

What methodologies can assess the functional consequences of ChemR23 activation or blockade?

Functional assessment requires multiple complementary approaches:

• Calcium mobilization: Measure intracellular calcium flux using Fura2-AM-loaded neutrophils stimulated with ligands like C15 (10 pM) or chemerin (1 nM). Include appropriate controls: ionomycin (positive), scrambled peptides (negative), and specific inhibitors (CCX2005, 100 nM)

• Migration assays: Quantify chemotaxis toward chemerin gradients with/without blocking antibodies

• Cell activation markers: Measure degranulation, respiratory burst, or cytokine production following receptor engagement

When reporting results, express calcium responses as delta F340/F380 ratios, and present data as mean values±standard error from multiple independent experiments to ensure reproducibility .

How do single-cell methodologies enhance ChemR23 antibody research?

Single-cell approaches offer unprecedented insights:

Recent advancements in single-cell culture (SCC) platforms enable comprehensive analysis of cell-specific responses to ChemR23 ligands. While primarily developed for other receptors, these approaches can be adapted for ChemR23 research:

• Single-cell antibody sequencing: Allows simultaneous determination of antigen-binding specificities and immunoglobulin gene sequences

• Epitope-specific responses: Enables identification of cells responding to specific ChemR23 epitopes

• Cross-reactivity studies: Facilitates analysis of antibody cross-reactivity across related receptors

These methods eliminate the need for large-scale sequencing and transfection, allowing direct screening and functional assays using culture supernatants followed by selective recombinant production of promising candidates.

What therapeutic potential exists for ChemR23-targeting antibodies?

While research is still emerging, therapeutic applications may follow similar development paths as other receptor-targeting antibodies:

The therapeutic potential of ChemR23 antibodies may parallel successful antibody therapies targeting other immune receptors. For example, anti-CD23 antibodies like Lumiliximab have demonstrated:

• Well-tolerated safety profiles in phase I clinical trials

• Sustained and dose-dependent modulation of downstream mediators

• Predictable pharmacokinetics with half-lives extending from 2 to 10 days at increasing doses

Similarly, novel investigational antibodies like CM313 (targeting CD38) show how receptor-specific antibodies can be developed for multiple immune conditions .

For ChemR23, potential therapeutic applications might target inflammatory conditions where neutrophil activation plays a key role, with careful attention to receptor expression patterns across tissues.