dlk-1 Antibody
Description
What Is DLK1 Antibody?
DLK1 antibodies are monoclonal or polyclonal immunoglobulins designed to bind DLK1, a protein expressed in stem cells and overexpressed in cancers such as neuroblastoma, adrenocortical carcinoma (ACC), and hepatocellular carcinoma (HCC). DLK1 exists in membrane-bound and soluble forms, regulating cell differentiation and tumorigenesis through Notch-dependent and independent pathways . Antibodies targeting DLK1 are engineered for:
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Diagnostic use: Detecting DLK1 expression in tissues via immunohistochemistry (IHC) or flow cytometry .
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Therapeutic use: Delivering cytotoxic payloads via antibody-drug conjugates (ADCs) or inhibiting DLK1 signaling to suppress tumor growth .
Development and Characterization of DLK1 Antibodies
Key steps in DLK1 antibody development include:
Antigen Preparation
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Recombinant DLK1-Fc fusion proteins are generated for immunization and screening .
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Phage display libraries are used to isolate single-chain variable fragments (scFv) with high specificity for DLK1 .
Validation
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Binding affinity: Surface plasmon resonance (SPR) and ELISA confirm nanomolar-range affinity (e.g., CBA-1205: IC₅₀ = 3.433 ng/mL) .
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Specificity: Antibodies like 10636-1-AP (Proteintech) react with human, mouse, and rat DLK1 but not homologous proteins like DLL1 or JAG1 .
Cancer Diagnostics and Prognostics
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Lung cancer: DLK1 is expressed in 20.5% of small-cell lung cancer (SCLC) and 16.8% of non-small-cell lung cancer (NSCLC) cases. In NSCLC, DLK1 positivity correlates with reduced recurrence-free survival (P < 0.01) .
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Adrenocortical carcinoma (ACC): DLK1 is expressed in 97% of ACC tumors, with higher levels in metastatic disease. DLK1 overexpression doubles recurrence risk .
Therapeutic Mechanisms
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Antibody-drug conjugates (ADCs):
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Neutralizing antibodies: Anti-DLK1 monoclonal antibodies inhibit bone resorption in estrogen-deficient mice, offering protection against osteoporosis .
Clinical Applications and Trials
Challenges and Future Directions
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Mitochondrial localization to the axon after injury is regulated in part by dual-leucine zipper kinase 1 (DLK-1), a conserved regulator of axon regeneration. PMID: 28009276
- DLK-1, SEK-3 and PMK-3 have roles in C. elegans adaptation to disruption of the mitochondrial electron transport chain, and in extended lifespan. PMID: 27420916
- These findings explain how DLK specifically mediates nerve injury responses and reveal a novel cellular mechanism that ensures the specificity of neuronal kinase signaling. PMID: 26719418
- Independent of myosin light chain activation, RHGF-1 acts via Rho-dependent kinase LET-502/ROCK and activates a conserved, retrograde DLK-1 MAPK pathway, triggering synaptic branch retraction and overgrowth of the PLM neurite in a dose-dependent manner. PMID: 25359212
- Genetic, transgenic, and biochemical studies indicated that PPM-2 functions coordinately with the ubiquitin ligase activity of RPM-1 and the F-box protein FSN-1 to negatively regulate DLK-1. PMID: 24810406
- DLK-1 is activated by calcium-mediated dissociation from an inhibitory isoform. PMID: 23141066
- These findings reveal the relationship between Ca(2+)/cAMP signaling and the DLK-1 MAPK (mitogen-activated protein kinase) cascade in regeneration. PMID: 20203177
- Findings demonstrate that the DLK-1 mitogen-activated protein (MAP) kinase pathway is essential for regeneration in Caenorhabditis elegans motor neurons. PMID: 19164707
- Understanding how the DLK-1 pathway acts in development, plasticity, and regeneration may shed light on the evolution of mechanisms regulating axon regeneration. PMID: 19417215
- Research shows that in C. elegans DLK-1 regulates not only proper synapse formation and axon morphology but also axon regeneration by influencing mRNA stability. PMID: 19737525
Q&A
What is DLK-1 and what is its molecular structure?
DLK-1 (Delta-like 1 homolog), also known as PREF1, FA1, or pG2, is a transmembrane protein belonging to the epidermal growth factor (EGF)-like superfamily. Its structure includes six tandem EGF-like motifs in the extracellular region. The protein has a calculated molecular weight of 41 kDa, but is typically observed at 45-60 kDa in experimental conditions due to post-translational modifications. DLK-1 functions as a non-canonical Notch ligand and plays significant roles in various biological processes, including regulation of adipocyte differentiation, where it acts as a negative regulator .
Which tissues and cell types express DLK-1 under normal conditions?
DLK-1 shows distinct expression patterns across various tissues and cell types:
| Tissue Type | DLK-1 Expression |
|---|---|
| Mouse placenta tissue | Positive |
| Mouse ovary tissue | Positive |
| Mouse brain tissue | Positive |
| Rat brain tissue | Positive |
| Human placenta tissue | Positive |
| 3T3-L1 cells | Positive |
| MCF-7 cells | Positive |
| A549 cells | Positive |
| Preadipocytes | Abundant expression |
DLK-1 is particularly abundant in preadipocytes where it regulates differentiation. It's also frequently found in tissues with neuroendocrine features, suggesting involvement in neuroendocrine differentiation processes .
What are the recommended protocols for using DLK-1 antibodies in Western Blot analysis?
For optimal Western Blot results with DLK-1 antibodies, researchers should consider the following parameters:
| Antibody Clone | Recommended Dilution | Expected MW | Sample Types |
|---|---|---|---|
| 10636-1-AP | 1:500-1:1000 | 45-60 kDa | Mouse placenta, ovary, brain; 3T3-L1, MCF-7, A549 cells |
| 29288-1-AP | 1:500-1:3000 | 60 kDa | Mouse and rat brain tissue |
The molecular weight discrepancy between calculated (41 kDa) and observed (45-60 kDa) is likely due to post-translational modifications such as glycosylation. Researchers should prepare samples in standard denaturing conditions and include appropriate positive controls from the list of validated tissues or cell lines .
What are the optimal conditions for DLK-1 immunohistochemical staining?
For successful immunohistochemistry applications targeting DLK-1:
| Parameter | Recommendation |
|---|---|
| Antibody Dilution (10636-1-AP) | 1:500-1:2000 |
| Antibody Dilution (29288-1-AP) | 1:50-1:500 |
| Primary Antigen Retrieval | TE buffer pH 9.0 |
| Alternative Antigen Retrieval | Citrate buffer pH 6.0 |
| Positive Control Tissues | Human placenta, pancreatic cancer, breast cancer |
The antigen retrieval step is critical for exposing the DLK-1 epitope in formalin-fixed paraffin-embedded (FFPE) tissues. Researchers should optimize dilutions for each specific tissue type and include appropriate positive controls to validate staining patterns .
How prevalent is DLK-1 expression in different cancer types?
DLK-1 expression varies significantly across cancer types. In lung cancer specifically:
| Cancer Type | DLK-1 Positive Cases | Percentage |
|---|---|---|
| Small-cell lung cancer (SCLC) | 23/112 patients | 20.5% |
| Non-small-cell lung cancer (NSCLC) | 17/101 patients | 16.8% |
Additionally, DLK-1 overexpression has been reported in hepatocellular carcinoma (HCC) and other cancers with neuroendocrine features. Immunohistochemical studies have also identified DLK-1 expression in human pancreatic cancer and breast cancer tissues .
What is the prognostic significance of DLK-1 expression in cancer?
The prognostic value of DLK-1 varies by cancer type:
These findings suggest that DLK-1 could serve as a promising prognostic factor specifically for recurrence in patients with resected NSCLC, while its prognostic utility in other cancer types requires further investigation .
How is DLK-1 being targeted in cancer therapeutics?
Current therapeutic approaches targeting DLK-1 include:
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Radioimmunotherapy (RIT): Research has demonstrated that iodine-125 (¹²⁵I)-labeled anti-DLK1 antibody specifically targets DLK1 on human SCLC tumor cell lines and localizes to tumor tissue in mouse models. This approach shows promise for targeted therapy, with potential for using alternative radionuclides like the alpha-particle-emitter astatine-211 (²¹¹At) .
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Novel Antibody Therapeutics: CBA-1205, a glycoengineered humanized anti-DLK-1 antibody developed using GlymaxX® technology, enhances antibody-dependent cellular cytotoxicity. Early clinical trials show promising safety profiles and disease control in some patients with advanced solid tumors .
What is the current status of clinical trials targeting DLK-1?
A phase I, first-in-human study of CBA-1205 (anti-DLK-1 antibody) has been conducted with the following key findings:
| Study Design | Results |
|---|---|
| Multicenter, open-label Phase I | 22 patients with advanced solid tumors enrolled |
| Seven dose cohorts (0.1-30 mg/kg) | Well tolerated at all dose levels |
| 2-week interval administration in 28-day cycles | No dose-limiting toxicity observed |
| Primary endpoint: safety and tolerability | No ≥ Grade 3 treatment-related adverse events |
| Secondary endpoints: pharmacokinetics, efficacy | Disease control in 8 patients (38.1%, all with stable disease) |
The study demonstrated dose-proportional increases in serum concentration of CBA-1205, with exposure exceeding effective concentrations observed in mouse xenograft models. Importantly, no anti-CBA-1205 antibodies were detected in patient serum, suggesting low immunogenicity .
How do researchers assess DLK-1 expression in clinical samples?
Clinical assessment of DLK-1 expression employs multiple complementary approaches:
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Immunohistochemistry (IHC): FFPE tissue sections are analyzed using anti-DLK-1 antibodies (such as clone DI-2-20) with appropriate antigen retrieval methods. This technique allows visualization of DLK-1 protein expression and localization within tissue architecture.
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Serum ELISA: Circulating DLK-1 levels can be quantified using the Human Pref-1/DLK1/FA1 DuoSet ELISA (R&D Systems), providing a non-invasive method to monitor DLK-1 levels.
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Flow Cytometry: Cell surface DLK-1 expression can be assessed on fresh cell suspensions, as demonstrated in studies with HepG2 human hepatocellular carcinoma cell lines .
Why might I observe variable molecular weights for DLK-1 in Western blot analysis?
Researchers frequently observe DLK-1 at molecular weights ranging from 45-60 kDa despite its calculated weight of 41 kDa. This variability stems from:
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Post-translational modifications: Glycosylation patterns can vary by tissue type and physiological condition.
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Protein isoforms: Alternative splicing may generate different isoforms.
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Sample preparation: Different lysis buffers or denaturation conditions may affect apparent molecular weight.
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Tissue-specific processing: The extent of protein processing may differ between tissues.
To address this variability, researchers should include appropriate positive controls from tissues known to express DLK-1 and consider using multiple antibody clones targeting different epitopes to confirm specificity .
How can I optimize DLK-1 detection in challenging sample types?
For improved DLK-1 detection in difficult samples:
| Challenge | Optimization Strategy |
|---|---|
| Low expression levels | Use more sensitive detection methods (chemiluminescence-based Western blot or tyramide signal amplification for IHC) |
| High background in IHC | Optimize blocking (5% BSA or 10% normal serum) and increase washing steps |
| Variable antigen retrieval efficacy | Compare TE buffer (pH 9.0) with citrate buffer (pH 6.0) for each tissue type |
| Weak Western blot signal | Transfer smaller proteins at lower voltage for longer duration; consider PVDF membranes for better protein retention |
| Cross-reactivity concerns | Validate findings with multiple antibody clones targeting different epitopes |
Each sample type may require specific optimization, and researchers should titrate antibodies in each testing system to obtain optimal results .
Beyond cancer, what other research areas involve DLK-1?
DLK-1 functions extend beyond oncology into several important biological processes:
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Adipogenesis regulation: DLK-1 is abundant in preadipocytes and negatively regulates adipocyte differentiation. Studies with DLK-1 deficient mouse models demonstrate growth retardation and accelerated adiposity, highlighting its role in adipose tissue development.
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Neuroendocrine differentiation: DLK-1 expression in tumors with neuroendocrine features suggests involvement in neuroendocrine differentiation pathways.
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Developmental biology: As a non-canonical Notch ligand, DLK-1 likely plays important roles in developmental processes that rely on Notch signaling .
What novel antibody-based approaches are being developed for DLK-1?
Recent advances in antibody engineering are expanding the therapeutic potential of anti-DLK-1 approaches:
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Glycoengineered antibodies: CBA-1205 represents a novel approach using GlymaxX® technology to enhance antibody-dependent cellular cytotoxicity, potentially improving efficacy against DLK-1 expressing tumors.
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Radioimmunotherapy conjugates: Beyond the studied iodine-125 labeled antibodies, research is exploring alternative radionuclides like astatine-211 that offer enhanced therapeutic potential through alpha-particle emission.
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Combinatorial approaches: Future directions may include combining DLK-1 targeting with immune checkpoint inhibitors or other targeted therapies to enhance efficacy .
