CD164 Antibody, FITC conjugated

What is CD164 and what are its primary functions in hematopoiesis?

CD164 is a novel sialomucin that plays dual roles in hematopoiesis. It functions as an adhesion receptor on human CD34+ cell subsets in the bone marrow and simultaneously acts as a potent negative regulator of CD34+ hemopoietic progenitor cell proliferation . These diverse biological effects are mediated by at least two functional epitopes that have been defined by specific monoclonal antibodies. The molecule is expressed on subsets of CD34+ cells and plays a critical role in the regulation of hematopoietic stem cell development and proliferation .

Which CD164 epitopes are commonly targeted by research antibodies?

Research antibodies against CD164 typically target one of several well-characterized epitopes. The most extensively studied epitopes are those recognized by the monoclonal antibodies 103B2/9E10, 105A5, N6B6, and 67D2 . These epitopes have been precisely mapped and categorized into three distinct classes based on their sensitivity to various enzyme treatments:

• Class I epitopes (e.g., 105A5): Sensitive to sialidase, O-glycosidase, and O-sialoglycoprotease treatments

• Class II epitopes (e.g., 103B2/9E10): Sensitive to N-glycanase, O-glycosidase, and O-sialoglycoprotease treatments

• Class III epitopes (e.g., N6B6 and 67D2): Resistant to these enzyme treatments

What are the optimal conditions for using FITC-conjugated CD164 antibodies in flow cytometry?

When using FITC-conjugated CD164 antibodies for flow cytometry analysis of hematopoietic cells, several methodological considerations should be addressed:

• Sample preparation: Fresh or properly cryopreserved cells should be used, with viability exceeding 90%

• Titration: Perform antibody titration to determine optimal concentration (typically 0.5-10 μg/mL depending on the specific clone)

• Blocking: Pre-block cells with 2-5% serum matching the secondary antibody species if using indirect methods

• Incubation: Stain cells at 4°C for 30 minutes in the dark to preserve FITC fluorescence

• Washing: Use PBS containing 0.1-0.5% BSA for washing steps to minimize background

• Controls: Include appropriate isotype controls (matching the same isotype as your CD164 antibody - IgG1, IgG2a, IgG3, or IgM as appropriate)

Compensation controls are essential when performing multicolor analysis to correct for spectral overlap between FITC and other fluorochromes such as PE.

How can CD164 epitope expression be validated across different cell types?

Validating CD164 epitope expression across different cell types requires a systematic approach:

• Select multiple CD164 monoclonal antibodies recognizing different epitopes (e.g., 103B2/9E10, 105A5, N6B6, and 67D2)

• Perform parallel staining of target cell populations and analyze by flow cytometry

• Compare expression patterns to determine if all epitopes are equally accessible

• For comprehensive validation, conduct enzyme digestion studies to characterize the post-translational modifications present on CD164 in different cell types

Research has shown that the differential expression of CD164 epitopes in adult tissues is linked with cell type-specific post-translational modifications . This suggests that researchers should test multiple CD164 antibodies recognizing different epitopes when characterizing a new cell type or tissue.

What approaches can be used to distinguish different CD164 splice variants in experimental systems?

Distinguishing CD164 splice variants requires specialized techniques beyond standard antibody-based detection:

• RT-PCR using splice variant-specific primers targeting unique exon junctions

• Western blotting using antibodies that recognize conserved regions to identify size differences

• Custom antibodies raised against splice variant-specific sequences

• Recombinant expression systems using the CD164-Fc* chimeric proteins corresponding to different splice variants

It's worth noting that most commercially available CD164 antibodies, including FITC-conjugated versions, do not distinguish between the different CD164 splice variants as demonstrated in comprehensive epitope mapping studies . For definitive splice variant identification, researchers should consider genetic approaches such as variant-specific PCR or sequencing.

How can FITC-conjugated CD164 antibodies be employed to investigate CD164's role in hematopoietic stem cell development?

FITC-conjugated CD164 antibodies provide powerful tools for investigating CD164's role in hematopoietic stem cell development through several advanced approaches:

• Multi-parameter flow cytometry: Combining CD164-FITC with antibodies against other stem cell markers (e.g., CD34-PE, CD38-APC) to isolate specific progenitor populations

• Cell sorting: Using FITC-conjugated CD164 antibodies for FACS isolation of CD164+ cell subsets for functional assays

• Clonogenic assays: Adding CD164 antibodies to cultures to study their effects on colony formation by primitive granulocyte-monocyte and erythroid precursors

• Cell cycle analysis: Combining CD164-FITC staining with DNA content analysis to examine the relationship between CD164 expression and cell cycle status

• Adhesion assays: Using FITC-labeled cells to quantify the effect of CD164 antibodies on progenitor cell adhesion to bone marrow stroma

Experimental data have shown that specific CD164 monoclonal antibodies (103B2/9E10 and 105A5) can inhibit nucleated cell production in liquid cultures and colony formation by primitive hematopoietic precursors . The 103B2/9E10 antibody specifically prevents recruitment of CD34+CD38low/− cells into cell cycle in the presence of cytokines IL-3, IL-6, G-CSF, and SCF .

What methods can be used to characterize the post-translational modifications of CD164 epitopes in different cellular contexts?

Characterizing post-translational modifications of CD164 epitopes requires sophisticated biochemical approaches:

• Sequential enzymatic digestion: Treating cells or purified CD164 with specific enzymes:

  • Sialidase to remove sialic acid moieties

  • O-glycosidase to remove O-linked glycans

  • O-sialoglycoprotease to cleave sialomucins

  • N-glycanase to remove N-linked glycans

• Epitope accessibility assays: Monitoring changes in antibody binding after enzyme treatments using flow cytometry or ELISA

• Mass spectrometry: Analyzing glycopeptides to precisely identify glycosylation patterns

• Lectin binding assays: Using plant lectins with known glycan specificities as complementary tools

This systematic approach allows researchers to classify CD164 epitopes into three distinct categories, similar to the established CD34 epitope classification system . The table below summarizes the sensitivity of different CD164 epitopes to enzymatic treatments:

How can epitope mapping data be used to select the optimal CD164 antibody for specific research applications?

Epitope mapping data provides crucial guidance for selecting the optimal CD164 antibody:

• For adhesion studies: The 103B2/9E10 antibody partially inhibits adhesion of CD34+ cells to bone marrow stroma, making it suitable for investigating adhesion processes

• For proliferation studies: Both 103B2/9E10 and 105A5 antibodies inhibit nucleated cell production and colony formation, making them valuable tools for studying CD164's role in proliferation regulation

• For structural studies: The N6B6 and 67D2 antibodies recognize complex conformational epitopes requiring the integrity of cysteine-rich regions encoded by exons 2 and 3, making them useful for monitoring protein structure

• For glycobiology research: Class I (105A5) and Class II (103B2/9E10) epitopes are differentially sensitive to glycosidase treatments, providing tools to study glycosylation patterns

• For splice variant analysis: None of the standard CD164 antibodies distinguish between splice variants, so researchers must use alternative methods for this purpose

The detailed epitope mapping studies show that 105A5 and 103B2/9E10 functional epitopes map to distinct glycosylated regions within the first mucin domain of CD164, while N6B6 and 67D2 recognize complex epitopes encompassing cysteine-rich regions encoded by exons 2 and 3 .

What are the common causes of variable or weak staining with FITC-conjugated CD164 antibodies?

Several factors can contribute to variable or weak staining with FITC-conjugated CD164 antibodies:

• Post-translational modifications: Cell type-specific glycosylation patterns can mask certain CD164 epitopes, particularly those recognized by class I and II antibodies

• Fluorochrome degradation: FITC is sensitive to photobleaching and pH changes; store antibodies protected from light at appropriate temperature (typically 4°C)

• Fixation effects: Some fixation protocols can alter CD164 epitope accessibility; optimize fixation conditions or perform live cell staining when possible

• Epitope accessibility: The conformational epitopes recognized by N6B6 and 67D2 require intact protein structure that may be disrupted during sample preparation

• Alternative splice variants: The presence of CD164 splice variants lacking specific exons can result in loss of certain epitopes

To address these issues, researchers should test multiple CD164 antibody clones, optimize sample preparation protocols, and validate their specific experimental system with appropriate controls.

How can researchers distinguish between specific and non-specific binding when using CD164-FITC antibodies?

Distinguishing specific from non-specific binding requires rigorous controls:

• Isotype controls: Include matched isotype controls (IgG1, IgG2a, IgG3, or IgM) conjugated to FITC to establish background fluorescence levels

• Blocking experiments: Pre-incubate cells with unconjugated CD164 antibody before adding CD164-FITC to demonstrate binding competition

• Epitope competition assays: In competitive binding assays, the 103B2/9E10 and 105A5 mAbs do not significantly compete with one another or with N6B6 or 67D2 mAbs, confirming the distinctness of these epitopes

• Enzyme treatments: Selectively remove specific post-translational modifications to verify epitope-specific binding

• Negative control cells: Include cell types known to lack CD164 expression to establish true negative staining parameters

These approaches collectively provide robust validation of antibody specificity in experimental systems.

What strategies can be employed when encountering conflicting results between different CD164 antibody clones?

When facing conflicting results between different CD164 antibody clones, consider these research-based strategies:

• Epitope mapping: Determine which domains of CD164 are recognized by each antibody using the exon-specific recombinant constructs approach

• Domain-specific expression analysis: Use soluble recombinant chimeric proteins encoded by specific exons to characterize antibody binding, as demonstrated with the CD164-Fc* fusion protein series

• Functional validation: Test the effects of different antibodies in functional assays such as adhesion and proliferation inhibition assays

• Post-translational modification assessment: Evaluate the sensitivity of epitopes to glycosidase treatments to determine if differential glycosylation is responsible for conflicting results

• Cross-validation with non-antibody methods: Confirm expression patterns using alternative techniques such as RT-PCR, RNA-seq, or CRISPR-based approaches

The research demonstrates that the previously observed differential expression of CD164 epitopes in adult tissues is linked with cell type-specific post-translational modifications and suggests that epitope-associated carbohydrate structures play important roles in CD164 function .

How might genomic and proteomic approaches complement antibody-based studies of CD164?

Genomic and proteomic approaches offer powerful complementary methods to antibody-based CD164 studies:

• Single-cell RNA sequencing: Enabling precise characterization of CD164 splice variant expression across heterogeneous cell populations

• CRISPR-Cas9 genome editing: Creating specific CD164 domain deletions or mutations to study structure-function relationships

• Glycoproteomics: Providing comprehensive mapping of site-specific glycosylation patterns that influence antibody binding

• Spatial transcriptomics: Revealing the tissue-specific expression patterns of CD164 with spatial resolution

• Protein interactome analysis: Identifying CD164 binding partners under different physiological and pathological conditions

These approaches can overcome some limitations of antibody-based detection methods, particularly in distinguishing splice variants and characterizing post-translational modifications with high precision.

What are the emerging applications of CD164 antibodies in studying hematological malignancies?

CD164 antibodies are finding increasing applications in hematological malignancy research:

• Diagnostic marker identification: Evaluating CD164 expression patterns across leukemia subtypes using standardized flow cytometry panels

• Therapeutic targeting: Developing antibody-drug conjugates targeting CD164-expressing malignant cells

• Minimal residual disease detection: Using high-sensitivity flow cytometry with CD164-FITC to identify rare leukemic stem cells

• Functional studies: Investigating how CD164-mediated adhesion and proliferation pathways may be dysregulated in malignancy

• Cancer stem cell characterization: Examining the relationship between CD164 expression and cancer stem cell properties

The functional studies showing that CD164 antibodies can inhibit hematopoietic progenitor cell proliferation suggest potential therapeutic applications in targeting leukemic stem cells that may rely on similar pathways .

How can structure-function relationship studies of CD164 inform the development of next-generation antibodies?

Structure-function studies of CD164 provide valuable insights for next-generation antibody development:

• Epitope-specific functional effects: The observation that different epitopes mediate distinct biological effects (adhesion vs. proliferation) suggests that highly targeted antibodies could selectively modulate specific CD164 functions

• Domain-specific targeting: The mapping of functional epitopes to distinct glycosylated regions within the first mucin domain provides a rationale for developing domain-specific antibodies

• Conformation-sensitive antibodies: The complex epitopes recognized by N6B6 and 67D2, which rely on conformational integrity of the CD164 molecule, represent a model for developing antibodies that detect specific protein conformations

• Glycoform-specific antibodies: The differential sensitivity of epitopes to glycosidase treatments suggests the possibility of developing antibodies that specifically recognize particular glycoforms of CD164

• Bispecific antibody design: Understanding the spatial relationship between different CD164 epitopes enables rational design of bispecific antibodies that could have novel functional properties

The comprehensive epitope mapping studies using recombinant CD164 constructs provide a foundation for these advanced antibody development approaches .