DARC Recombinant Monoclonal Antibody

What is DARC and why is it significant in research?

DARC (Duffy Antigen Receptor for Chemokines), also known as ACKR1 (Atypical chemokine receptor 1), CD234, or Fy glycoprotein, is a seven-transmembrane glycoprotein with multiple significant biological functions. Unlike conventional chemokine receptors, DARC lacks the DRY motif required for signal transduction, functioning instead as a "silent" or "decoy" receptor. This atypical chemokine receptor controls chemokine levels and localization through high-affinity binding that results in chemokine sequestration, degradation, or transcytosis rather than traditional signal transduction cascades .

DARC exhibits a promiscuous chemokine-binding profile, interacting with inflammatory chemokines of both CXC and CC subfamilies but not with homeostatic chemokines. It serves as a receptor for multiple chemokines including CCL2, CCL5, CCL7, CCL11, CCL13, CCL14, CCL17, CXCL5, CXCL6, IL8/CXCL8, CXCL11, and others . Additionally, DARC functions as the primary receptor for the malaria parasites Plasmodium vivax and P. knowlesi, making it crucial in malaria research .

DARC is primarily expressed on red blood cells (erythrocytes), venular endothelial cells, and Purkinje cells. Its role varies based on cellular location: on erythrocytes, it serves as a chemokine reservoir and regulator of plasma chemokine levels, while on endothelial cells, it facilitates chemokine transcytosis through vessel walls to promote leukocyte extravasation .

The significance of DARC extends to multiple research areas, including immunology, inflammation, infectious diseases, cancer biology, and transfusion medicine, as it carries the Duffy blood group antigens (Fy^a and Fy^b) .

What are the key structural features of DARC that affect antibody selection?

DARC's structure contains several important features that significantly influence antibody binding and selection for research applications:

• Post-translational modifications: DARC contains three N-linked oligosaccharide chains located at Asn-16, 27, and 33, which are primarily triantennary complex type terminated with sialic acid residues . Additionally, tyrosine residues at positions 30 and 41 are sulfated, with sulfation of Tyr-41 increasing binding of P. vivax Duffy Binding Protein approximately 1000-fold . These modifications can either enhance or obstruct antibody access to epitopes.

• Extracellular domains: DARC has multiple extracellular domains that contain distinct epitopes. The first extracellular domain (ECD1) contains the linear Fy6 epitope, while the Fy3 epitope spans fragments of extracellular domains 1 and 3, forming a conformational epitope . The positioning of these domains affects antibody accessibility in experimental conditions.

• Key epitopes: Several epitopes on DARC are recognized by antibodies:

  • The anti-Fy6 epitope is located in the first extracellular domain, with the core sequence 22FEDVW26

  • The Fy^a/Fy^b blood group polymorphism is determined by a Gly/Asp variation at position 42

  • The Fy3 conformational epitope comprises parts of extracellular domains 1 and 3

• Polymorphic residues: DARC exhibits significant polymorphism, including the Fy^a/Fy^b variants (Gly42Asp) that define the Duffy blood group system and the -67T>C promoter mutation that results in the Fy(a-b-) phenotype with no DARC expression on erythrocytes, common in African populations .

Understanding these structural features is essential for selecting appropriate antibodies for specific research applications and interpreting binding results accurately. For instance, antibodies targeting the Fy6 epitope may have altered binding properties depending on the sulfation status of nearby Tyr-30, while antibodies recognizing conformational epitopes may be affected by protein folding under different experimental conditions .

What are the main applications of DARC recombinant monoclonal antibodies?

DARC recombinant monoclonal antibodies serve diverse research applications across multiple disciplines:

• Flow cytometry analysis: DARC antibodies are widely used to detect and quantify DARC expression on red blood cells and other cell types. APC-conjugated anti-DARC antibodies can be utilized for flow cytometric analysis, providing clear separation between DARC-positive populations and controls . This application is particularly valuable for studying DARC expression patterns in different cell populations and under various physiological or pathological conditions.

• Immunofluorescence studies: DARC antibodies are effective tools for immunofluorescence and immunocytochemistry applications, typically used at dilutions of 1:50-1:200 . These techniques allow visualization of DARC distribution in tissues and cellular localization, enabling detailed analysis of DARC expression in different anatomical contexts.

• Malaria research: Since DARC serves as the receptor for Plasmodium vivax and P. knowlesi, DARC antibodies are invaluable tools for studying malaria parasite invasion mechanisms . They can be used to block potential binding sites for P. vivax Duffy Binding Protein (PvDBP) and quantify inhibition of parasite invasion into erythrocytes.

• Chemokine regulation studies: DARC antibodies facilitate investigation of DARC's role in chemokine binding, transport, and presentation. Researchers can use these antibodies to study how DARC on endothelial cells supports transcytosis of chemokines or how erythrocyte DARC functions as a chemokine sink .

• Inflammation research: As DARC plays a crucial role in leukocyte recruitment during inflammation, DARC antibodies can be used to track expression changes during inflammatory processes and investigate DARC's contribution to inflammatory diseases .

• Blood group typing: DARC antibodies that recognize the Fy^a/Fy^b polymorphisms are used in blood group typing and transfusion medicine research .

• Enzyme-linked immunosorbent assay (ELISA): DARC antibodies can be used in ELISA applications for quantitative analysis of DARC in research samples .

The versatility of DARC recombinant monoclonal antibodies makes them essential tools across immunology, infectious disease research, and hematology, providing insights into both normal physiology and pathological conditions.

How should researchers select the appropriate DARC antibody for their experimental needs?

Selecting the appropriate DARC antibody requires careful consideration of several key factors:

• Target epitope specificity: Different antibodies target distinct epitopes on DARC. Anti-Fy6 antibodies (like 2C3) target the linear epitope in the first extracellular domain with the core sequence 22FEDVW26 . If investigating DARC polymorphisms, researchers should select antibodies that specifically recognize Fy^a/Fy^b variants or choose those binding to conserved regions unaffected by polymorphisms.

• Clone characteristics: Consider the clone's proven applications and validation data. For example, clone EPR5205 is a recombinant monoclonal antibody that offers advantages including high batch-to-batch consistency and reproducibility . The clone 358307 has been specifically validated for flow cytometry applications with human red blood cells .

• Host species and format: DARC antibodies are available from different host species (rabbit, mouse) and in different formats (whole IgG, Fab fragments). Select based on:

  • Compatibility with other antibodies in multi-color panels

  • Host species that minimizes background in your sample type

  • Format appropriate for your application (e.g., Fab fragments for better tissue penetration)

• Conjugation options: Consider the detection system and instrumentation available:

  • For flow cytometry: APC-conjugated antibodies work well with red lasers and provide high brightness with minimal spectral overlap

  • For immunofluorescence: Choose fluorophores appropriate for your microscopy setup and that avoid tissue autofluorescence

  • For complex multi-parameter analyses: Select conjugates that minimize spectral overlap with other markers

• Validation for specific applications: Verify that the antibody has been validated for your intended application. The DARC monoclonal antibody CAB23374, for example, has been specifically validated for immunofluorescence and ELISA applications , while FAB4139A has demonstrated efficacy in flow cytometry .

• Reactivity with species of interest: Confirm that the antibody recognizes DARC in your species of interest. Many DARC antibodies are human-specific, with varying cross-reactivity to other species. Human DARC shares 60% amino acid sequence identity with mouse DARC, which may affect cross-reactivity .

• Performance in specific sample types: Consider whether the antibody has been validated in your specific sample type (fresh cells, fixed tissues, etc.) and whether sample preparation requirements are compatible with your experimental design.

By systematically evaluating these factors, researchers can select DARC antibodies that will provide optimal performance in their specific experimental context, ensuring reliable and reproducible results.

What are the optimal conditions for storing and handling DARC antibodies?

Proper storage and handling of DARC antibodies is crucial for maintaining their functionality and specificity. The following guidelines should be followed:

• Storage temperature requirements:

  • For APC-conjugated DARC antibodies: Store at 2-8°C (refrigerated) and do not freeze . These conjugated antibodies are particularly sensitive to freeze-thaw damage.

  • For unconjugated antibodies: Store at -20°C according to manufacturer recommendations .

  • Always follow the specific temperature guidelines provided by the manufacturer for each product.

• Light protection:

  • APC-conjugated and other fluorescently labeled antibodies must be protected from light during storage and handling .