FLT3 Antibody, Biotin conjugated

What is FLT3 and why is it an important target for antibody-based detection in hematological research? [BASIC]

FLT3 (FMS-like tyrosine kinase 3), also known as CD135, is a receptor tyrosine kinase that plays a critical role in hematopoietic progenitor cell differentiation, proliferation, and survival. It acts as a cell-surface receptor for the cytokine FLT3LG and regulates multiple signaling pathways including:

• Phosphorylation of SHC1 and AKT1

• Activation of RAS signaling

• Phosphorylation of downstream kinases (MAPK1/ERK2, MAPK3/ERK1)

• Activation of STAT5A/B signaling (though wild-type FLT3 causes only marginal activation)

FLT3 is particularly significant in leukemia research as it is frequently mutated in acute myeloid leukemia (AML), with mutations causing constitutive kinase activity that promotes cell proliferation and resistance to apoptosis . Detection of FLT3 expression patterns is therefore essential for understanding normal hematopoiesis and leukemic transformation.

What are the primary applications of biotin-conjugated FLT3 antibodies in experimental settings? [BASIC]

Biotin-conjugated FLT3 antibodies serve multiple research applications:

When selecting applications, researchers should note that different commercial antibodies have validated specific applications - for example, certain clones are only validated for ELISA while others may be validated for flow cytometry .

How should FLT3 antibody validation be performed for acute myeloid leukemia (AML) research applications? [ADVANCED]

Comprehensive validation for FLT3 antibody applications in AML research requires:

• Positive and negative cell line controls:

  • Positive: MV4-11 and EOL-1 (high FLT3 expression)

  • Negative: Cell lines known to lack FLT3 expression

• Expression verification using orthogonal methods:

  • RT-PCR to confirm mRNA expression

  • Western blotting for protein expression correlation

  • Flow cytometry using alternative FLT3 antibody clones

• Mutation-specific validation:

  • Testing against cells with known FLT3-ITD mutations versus wild-type FLT3

  • Validation against cells with FLT3 D835Y mutations

• Knockout/knockdown controls:

  • CRISPR/Cas9-mediated FLT3 knockout cells as true negative controls

  • siRNA knockdown for reduced expression controls

• Cross-reactivity assessment:

  • Testing against related tyrosine kinase receptors (c-KIT, PDGFR)

When validating for patient sample applications, include primary AML samples with known FLT3 mutation status to establish detection thresholds relevant to clinical samples .

What is the optimal protocol for flow cytometric detection of FLT3 using biotin-conjugated antibodies in primary AML samples? [ADVANCED]

The following optimized protocol enhances detection sensitivity in primary AML samples:

• Sample preparation:

  • Process fresh bone marrow or peripheral blood within 24 hours of collection

  • Isolate mononuclear cells using density gradient centrifugation

  • Resuspend cells at 1×10^6 cells/100μL in PBS with 2% FBS

• Blocking step (critical):

  • Incubate cells with 10% normal serum (matching secondary reagent species) and 1% BSA for 20 minutes at 4°C

  • Include Fc receptor blocking reagent to reduce non-specific binding

• Antibody staining:

  • Use titrated biotin-conjugated FLT3 antibody (typically 0.25-1.0 μg/mL)

  • Incubate for 30 minutes at 4°C in the dark

  • Wash twice with cold PBS containing 2% FBS

• Secondary detection:

  • Apply streptavidin-fluorochrome conjugate (APC or PE recommended for sensitivity)

  • Incubate for 20 minutes at 4°C in the dark

  • Wash twice with cold PBS containing 2% FBS

• Multi-parameter considerations:

  • Include CD34, CD38, and CD45 for blast identification

  • Add CD3, CD19, and CD33 for lineage determination

  • Include viability dye (7-AAD or DAPI) to exclude dead cells

• Analysis considerations:

  • Establish gating strategy using FMO (fluorescence minus one) controls

  • Use isotype-biotin control to set background threshold

  • Calculate mean fluorescence intensity (MFI) and percentage of positive cells

This protocol shows optimal results when compared to direct conjugate approaches, with superior signal-to-noise ratio for detecting variable FLT3 expression levels in AML blast populations .

How can I compare the detection sensitivity between FLT3 antibody-based assays and genetic testing for FLT3 mutations in research settings? [ADVANCED]

The complementary nature of antibody-based and genetic detection approaches requires systematic comparison:

For comprehensive characterization, employ both approaches in parallel:

• Flow cytometry with biotin-conjugated FLT3 antibody to quantify receptor expression levels

• Molecular testing for FLT3-ITD and TKD mutations (D835Y being particularly important)

Research demonstrates that FLT3 protein overexpression doesn't always correlate with mutation status, highlighting the need for both methodologies in research settings. For example, wild-type FLT3 overexpression can drive leukemic phenotypes in certain AML subtypes even without mutations .

What factors affect the storage stability of biotin-conjugated FLT3 antibodies, and how can researchers maximize shelf-life? [BASIC]

Several factors impact stability of biotin-conjugated FLT3 antibodies:

• Temperature effects:

  • Optimal storage is at -20°C or -80°C for long-term preservation

  • Avoid repeated freeze-thaw cycles which accelerate degradation

  • Aliquot upon receipt to minimize freeze-thaw events

• Buffer composition impact:

  • Glycerol (typically 50%) acts as cryoprotectant

  • Preservatives like Proclin 300 (0.03%) prevent microbial growth

  • pH maintenance (typically pH 7.4) prevents denaturation

• Light exposure:

  • Protect from light to prevent photobleaching of biotin

  • Store in amber vials or wrapped in aluminum foil

• Protein concentration:

  • Higher concentration antibodies (>0.5 mg/mL) typically show better stability

  • Carrier proteins (BSA) may improve stability at lower concentrations

• Solution phase stability:

  • Working dilutions are less stable than stock solutions

  • Prepare fresh working solutions when possible

Stability testing shows that properly stored biotin-conjugated FLT3 antibodies maintain >90% activity for approximately 12 months at -80°C, compared to 6-8 months at -20°C. For optimized preservation, use cryopreservation buffers containing 50% glycerol and 0.01M PBS (pH 7.4) .

What are the primary sources of non-specific binding when using FLT3 antibodies, and how can they be mitigated? [ADVANCED]

Non-specific binding represents a significant challenge when working with FLT3 antibodies, particularly in complex clinical samples:

• Fc receptor interactions:

  • Primary issue: FcγR-expressing cells (monocytes, macrophages, B cells) bind antibody Fc regions

  • Mitigation: Use Fc receptor blocking reagents (10-20 μg/mL human IgG or commercial Fc block)

  • Validation: Include blocking controls to quantify improvement

• Biotin-streptavidin system complications:

  • Primary issue: Endogenous biotin in samples, particularly after fixation

  • Mitigation: Pre-block with avidin/biotin blocking kit

  • Validation: Compare staining in avidin-blocked vs. non-blocked samples

• Cross-reactivity with related receptors:

  • Primary issue: FLT3 shares structural homology with other Class III receptor tyrosine kinases

  • Mitigation: Use monoclonal antibodies targeting unique epitopes of FLT3

  • Validation: Test on cells expressing related receptors (c-KIT, PDGFR-α, PDGFR-β)

• Non-specific protein interactions:

  • Primary issue: Hydrophobic interactions with dead/damaged cells

  • Mitigation: Include viability dye; use higher protein concentrations in blocking buffer (5-10% BSA)

  • Validation: Compare viable vs. non-viable cell staining patterns

• Epitope masking:

  • Primary issue: Ligand binding or receptor internalization reducing antibody access

  • Mitigation: Use multiple antibody clones targeting different epitopes

  • Validation: Compare staining after acid washing to remove bound ligand

Empirical testing with multiple controls is essential for distinguishing specific from non-specific signals. Include isotype-biotin controls matched for species, isotype, and biotin:protein ratio to establish background thresholds accurately .

How can researchers interpret variability in FLT3 expression levels across different AML patient samples? [ADVANCED]

Interpreting heterogeneous FLT3 expression patterns across AML samples requires multi-dimensional analysis:

• Establish reference ranges:

  • Measure FLT3 expression in normal hematopoietic stem cells (CD34+/CD38-)

  • Determine expression in mature myeloid populations as comparative baselines

  • Calculate fold-change relative to these controls rather than using absolute values

• Correlation with genetic characteristics:

  • Stratify samples by FLT3 mutation status (wild-type, ITD, TKD mutations)

  • Assess FLT3 expression relative to allelic ratio in ITD+ cases

  • Research shows FLT3 is genetically essential for ITD-mutated leukemic stem cells but dispensable for normal hematopoietic stem cells

• Multi-parameter analysis:

  • Combine FLT3 detection with stem cell markers (CD34, CD38)

  • Correlate with differentiation markers (CD33, CD117)

  • Compare expression in blast vs. non-blast populations

• Functional interpretation:

  • High FLT3 expression without mutation suggests ligand-dependent activation

  • Variable expression within a patient sample indicates subclonal heterogeneity

  • Low expression may indicate internalization due to constitutive activation

• Clinical correlation:

  • Monitor expression changes during disease progression

  • Evaluate changes following FLT3 inhibitor therapy

  • Compare primary diagnosis vs. relapse samples

Research demonstrates that FLT3 expression analysis provides complementary information to mutational analysis. Studies show that FLT3-ITD AML with high allelic ratio responds differently to targeted therapies compared to those with low allelic ratio, emphasizing the importance of quantitative expression assessment .

How should researchers design experiments to evaluate the efficacy of FLT3-targeted therapies using biotin-conjugated FLT3 antibodies? [ADVANCED]

Robust experimental design for FLT3-targeted therapy evaluation requires:

• Baseline expression profiling:

  • Quantify pre-treatment FLT3 surface expression using biotin-conjugated antibodies

  • Measure receptor density (antibody binding capacity) rather than just percent positive

  • Correlate with FLT3 mutation status (ITD, D835Y, or wild-type)

• Post-treatment monitoring protocol:

  • Design time-course experiments (6h, 24h, 72h, 7d)

  • Evaluate both receptor internalization and expression changes

  • Include parallel assessment of downstream signaling molecules (phospho-STAT5, phospho-ERK)

• Comparative analysis of different therapeutic modalities:

  • FLT3 tyrosine kinase inhibitors (quizartinib, gilteritinib)

  • Bispecific FLT3×CD3 antibodies (in IgG or Fabsc format)

  • FLT3-targeted immunotherapy approaches

• Microenvironmental considerations:

  • Compare efficacy in suspension vs. stromal co-culture conditions

  • Evaluate under normoxic vs. hypoxic conditions

  • Research shows gilteritinib maintains stronger proapoptotic effects in hypoxia and co-culture with bone marrow stromal cells compared to quizartinib

• Resistance monitoring:

  • Track changes in FLT3 expression during treatment

  • Identify emergence of FLT3-negative subpopulations

  • Correlate with functional assays (proliferation, apoptosis)

For optimal experimental design, implement a factorial approach that systematically tests multiple variables:

This comprehensive approach enables identification of resistant populations and optimization of therapeutic strategies for different AML subtypes .

How can biotin-conjugated FLT3 antibodies be utilized in developing bispecific antibody constructs for therapeutic applications? [ADVANCED]

Biotin-conjugated FLT3 antibodies serve as critical tools in the development pipeline for bispecific therapeutic antibodies:

• Epitope mapping and selection:

  • Use biotinylated FLT3 antibodies to identify optimal binding domains

  • Research shows antibodies targeting domain 4 (like 4G8) versus domain 2 (like BV10) of FLT3 have different therapeutic efficacy

  • Screen panels of biotinylated antibodies to identify those that don't block ligand binding or impede T-cell engagement

• Multiparametric screening methodology:

  • Develop competitive binding assays using biotinylated reference antibodies

  • Quantify epitope occupancy during therapeutic antibody development

  • Create screening cascades to identify candidates with desired binding characteristics

• Bispecific format evaluation:

  • Compare different bispecific formats (bssc, Fabsc, IgG-scFv fusion)

  • Research demonstrates that Fabsc format shows superior properties compared to bssc format including:

    • Better production yield from transfected cells

    • Reduced aggregation tendency

    • Improved affinity to FLT3

• Target validation strategies:

  • Use biotinylated antibodies to confirm accessibility of FLT3 epitopes in patient samples

  • Quantify receptor density on different AML subtypes to predict response

  • Evaluate dual targeting of FLT3 with other leukemia antigens

• Quality control applications:

  • Develop competitive binding assays for lot-to-lot consistency testing

  • Establish reference standards for therapeutic antibody characterization

  • Create bioanalytical methods for pharmacokinetic studies

Studies have demonstrated that bispecific FLT3×CD3 antibodies in different formats can effectively redirect T cells to eliminate FLT3-expressing leukemic cells. The latest generation IgG-based bispecific antibodies (like CLN-049) show promising preclinical characteristics, including high-affinity FLT3 binding and potent T-cell activation at low E:T ratios .

How are FLT3 antibodies being utilized in research examining the differences between leukemic stem cells (LSCs) and normal hematopoietic stem cells (HSCs)? [ADVANCED]

FLT3 antibodies are enabling critical discoveries about the differential biology of LSCs versus HSCs:

• Differential dependency studies:

  • Recent CRISPR/Cas9 knockout studies demonstrate FLT3 is genetically essential for ITD-mutated LSCs but dispensable for normal HSCs

  • FLT3 antibodies enable precise quantification of expression differences between these populations

  • These findings establish FLT3 as an ideal therapeutic target where ITD+ LSCs are eradicated upon FLT3 deletion, while HSCs are spared

• Stem cell niche interactions:

  • Multi-parameter analysis with FLT3 antibodies reveals differential responses to microenvironmental factors

  • LSCs show distinct FLT3 signaling patterns in hypoxic conditions compared to HSCs

  • FLT3×CD3 bispecific antibodies demonstrate greater efficacy against leukemic cells in bone marrow compared to peripheral blood

• Therapeutic window identification:

  • Quantitative flow cytometry using biotin-conjugated FLT3 antibodies helps establish the therapeutic window

  • Enables direct comparison of receptor density between normal and leukemic stem cells

  • Supports selection of antibody affinities that preferentially target LSCs over HSCs

• Resistance mechanism exploration:

  • FLT3 antibodies track changes in expression during therapy

  • Research with FLT3 inhibitors reveals LSCs can develop resistance through receptor downregulation

  • Biotinylated antibodies enable sensitive detection of residual FLT3+ cells during treatment

• Single-cell analysis applications:

  • Integration with mass cytometry (CyTOF) or spectral cytometry

  • Compatible with single-cell sorting for downstream genomic analysis

  • Enables correlation of FLT3 protein expression with transcriptomic profiles

Recent research has demonstrated that FLT3-ITD with high allelic ratio creates a genetic dependency in LSCs that can be therapeutically exploited. Bispecific FLT3×CD3 antibodies show particular promise for targeting these cells while sparing normal HSCs, representing a paradigm shift in leukemia therapy .

What methodological approaches can researchers use to study FLT3 receptor internalization dynamics following ligand binding or antibody engagement? [ADVANCED]

Studying FLT3 receptor trafficking and internalization requires sophisticated methodological approaches:

• Time-resolved flow cytometry protocol:

  • Surface staining: Use biotin-conjugated FLT3 antibodies targeting non-overlapping epitopes

  • Total receptor: Perform intracellular staining after fixation/permeabilization

  • Internalization index: Calculate ratio of surface:total FLT3 expression

  • Kinetic analysis: Measure at multiple timepoints (5, 15, 30, 60, 120 min)

• Imaging-based approaches:

  • Confocal microscopy: Trace biotin-FLT3 antibody with fluorescent streptavidin

  • Live cell imaging: Use quantum dot-conjugated streptavidin for extended visualization

  • Colocalization analysis: Combine with endosomal markers (Rab5, Rab7, LAMP1)

  • Quantitative analysis: Measure nuclear translocation of receptor fragments

• Biochemical fractionation techniques:

  • Surface biotinylation: Label all surface proteins before internalization

  • Membrane fractionation: Separate plasma membrane from endosomal compartments

  • Western blot analysis: Quantify FLT3 in different cellular compartments

  • Phosphorylation status: Track activation state during trafficking

• Advanced receptor dynamics methodologies:

  • FRET-based approaches: Measure conformational changes during internalization

  • pH-sensitive fluorophores: Track endosomal acidification during trafficking

  • Pulse-chase experiments: Follow receptor fate after initial labeling

  • Recycling assays: Distinguish recycling from degradation pathways

• Therapeutic implications assessment:

  • Compare natural ligand vs. antibody-induced internalization

  • Evaluate differential internalization with various antibody formats (IgG vs. bispecific)

  • Correlate internalization rates with therapeutic efficacy

  • Test combinatorial approaches targeting different internalization mechanisms

This methodological toolkit has revealed that FLT3 receptor internalization dynamics differ significantly between wild-type and mutated receptors. FLT3-ITD shows constitutive internalization and recycling that contributes to abnormal signaling patterns. These differences can be exploited therapeutically, as certain bispecific antibody formats induce more efficient internalization and degradation of FLT3 compared to others .