DPEP Antibody

What are the different types of DPEP proteins and their associated antibodies?

DPEP antibodies target members of the dipeptidase family, primarily:

• DPEP1 (Dipeptidase 1): Also known as MDP or RDP, is a 45.7 kDa glycoprotein with 411 amino acid residues that functions in inflammatory response pathways . DPEP1 is primarily expressed in the small intestine, pancreas, kidney, duodenum, and colon, and is localized to the cell membrane .

• DPPX (Dipeptidyl-peptidase-like protein 6): An extracellular subunit of voltage-gated potassium channel 4.2, targeted by antibodies associated with autoimmune encephalitis .

• DPEP3: A glycosylphosphatidylinositol-anchored metallopeptidase that shares structural similarities with DPEP1 (49% sequence identity) but contains key variations in active site residues that affect its enzymatic function .

What are the optimal applications and protocols for DPEP1 antibodies?

Different DPEP1 antibodies show varying efficacy across applications. Based on validation data:

Recommended Applications and Dilutions:

• Western Blot (WB): Most DPEP1 antibodies perform well, with dilution ranges from 1:1000-1:50000 depending on the antibody .

• Immunohistochemistry (IHC): Optimal for detecting DPEP1 in kidney, colon, and pancreatic tissue sections at 1:500-1:2000 dilution .

• Immunofluorescence (IF): Effective for visualizing DPEP1 in tissue sections, particularly in kidney tissue, at 1:50-1:800 dilution .

Protocol Optimization Tips:

• For IHC, antigen retrieval with TE buffer (pH 9.0) often yields better results than citrate buffer for DPEP1 detection .

• For IF, CoraLite® Plus 488 conjugated antibodies provide excellent visualization of DPEP1 in kidney tissue with excitation/emission at 493/522 nm .

• When assessing peritubular capillary expression, DPEP1-binding peptides like LSALT can be used as additional visualization tools .

Sample Preparation:

• For serum samples: Separate serum from cells within 2 hours of collection and transfer to standard transport tubes (minimum 0.2 mL) .

• For tissue samples: DPEP1 is highly expressed in kidney proximal tubules and can be detected in both human and rodent samples .

What is the mechanism of action of anti-DPPX antibodies in autoimmune encephalitis?

Anti-DPPX antibodies cause encephalitis through a well-characterized immunological mechanism:

• Target interaction: Anti-DPPX antibodies (predominantly IgG1 and IgG4 subclasses) target the extracellular domain of DPPX, which is an auxiliary subunit of Kv4.2 potassium channels .

• Internalization effect: When cultured hippocampal neurons are exposed to purified patient IgG containing DPPX antibodies, there is a significant decrease in the density of surface DPPX clusters compared to controls .

• Secondary effects on Kv4.2: Quantitative immunoblot analysis shows that anti-DPPX antibodies also reduce surface expression of Kv4.2 channels, despite not directly binding to Kv4.2 itself .

• Functional consequences: In vitro experiments demonstrate that patient sera containing anti-DPPX antibodies increase the excitability and action potential frequency in enteric nervous system neurons, explaining gastrointestinal symptoms often seen in these patients .

• Reversibility: When antibodies are removed, surface levels of both DPPX and Kv4.2 progressively recover, reaching normal baseline levels within 7 days .

This mechanism explains the characteristic clinical triad of anti-DPPX encephalitis:

• Gastrointestinal symptoms (predominantly diarrhea)

• Cognitive-psychiatric dysfunction

• CNS hyperexcitability (with hyperekplexia being a specific feature)

How can I use DPEP1 antibodies to study its role in inflammation and neutrophil recruitment?

DPEP1 functions as an adhesion receptor for neutrophil recruitment, particularly in lungs and liver, independent of its enzymatic activity . To study this role:

Experimental Approaches:

• Endothelial expression studies: Use immunohistochemistry with DPEP1 antibodies to monitor expression changes in peritubular capillaries following injury or inflammation .

• Flow cytometry analysis: DPEP1 expression on endothelium, epithelium, and infiltrating leukocytes can be quantified using flow cytometry with specific antibodies .

• Blocking studies: Utilize DPEP1-binding peptides like LSALT as functional blockers to abrogate neutrophil recruitment experimentally .

• Confocal intravital imaging: This approach can visualize DPEP1-mediated neutrophil adhesion in real-time in living tissue .

Key Research Findings:

• DPEP1 expression increases in peritubular capillaries following ischemia-reperfusion injury (IRI) or LPS administration .

• DPEP1 upregulation is accompanied by increased molecular mass, suggesting post-translational modifications that may enhance its adhesion properties .

• Genetic ablation or peptide blocking of DPEP1 significantly reduces neutrophil recruitment to lungs and liver and improves survival in endotoxemia models .

What are the current approaches for rational design of antibodies targeting specific DPEP epitopes?

Recent advances have improved our ability to design antibodies against specific epitopes within DPEP proteins:

Rational Design Methodology:

• Complementary peptide identification: This approach identifies peptides complementary to target regions within disordered proteins or domains .

• CDR grafting: The complementary peptide is grafted onto the Complementarity-Determining Region (CDR) of an antibody scaffold .

• Energy-based preference optimization: For antibody-antigen interaction optimization, direct energy-based preference optimization techniques can guide the generation of antibodies with both rational structures and high binding affinities .

Advanced Design Techniques:

• Residue-level decomposed energy preference: This fine-tuned approach enhances binding specificity by optimizing energy at individual amino acid levels .

• Energy decomposition strategies: Various energy types (attraction, repulsion) can be independently optimized to enhance binding parameters .

• Gradient surgery: This technique addresses conflicts between different types of energy during optimization .

Experimental Results:

Models using these approaches have demonstrated effectiveness in generating antibodies with energies resembling natural antibodies, achieving state-of-the-art performance in designing high-quality antibodies with low total energy and high binding affinity simultaneously .

How are anti-DPPX antibodies detected and quantified in clinical samples?

Anti-DPPX antibodies are detected using specialized laboratory techniques:

Detection Methods:

• Cell-Based Indirect Fluorescent Antibody (CBA-IFA): This semi-quantitative assay utilizes DPPX-transfected cells for detection and titer determination of DPPX IgG antibodies .

• Tissue-Based Assay (TBA): This complementary method examines fluorescence patterns in the stratum molecular of the hippocampus (neuropil staining) and cerebellum .

• IgG Subclass Determination: Specific testing for IgG1, IgG2, IgG3, and IgG4 subclasses can provide additional diagnostic information. Studies show anti-DPPX antibodies are predominantly IgG1 and IgG4 .

Specimen Requirements and Handling:

• Preferred specimen: Serum in serum separator tube, with 1 mL transferred to standard transport tube (minimum 0.2 mL) .

• Processing: Separate serum from cells within 2 hours of collection .

• Storage stability: After separation from cells: Ambient: 48 hours; Refrigerated: 2 weeks; Frozen: 30 days (avoid repeated freeze/thaw cycles) .

• CSF testing: Can also be performed but should be paired with serum testing when possible .

Interpretation Considerations:

• A positive result indicates the presence of DPPX antibodies, which may be associated with hyperexcitability, pleocytosis, and frequent diarrhea .

• DPPX encephalitis may be paraneoplastic with hematologic malignancies reported in a subset of patients .

• Decreasing antibody levels may correlate with therapeutic response .

• A negative test result does not rule out autoimmune neurologic disease and should be interpreted alongside clinical history and other laboratory findings .

What are the structural and functional differences between DPEP family members that affect antibody development?

Understanding structural differences between DPEP family members is crucial for specific antibody development:

Structural Comparisons:

• DPEP1 and DPEP3: Share a conserved, dimeric TIM (β/α)8-barrel fold consistent with 49% sequence identity .

• Active Site Variations: DPEP3 differs from DPEP1 in key positions:

  • Histidine to tyrosine variation at position 269 reduces affinity for β zinc and may cause substrate steric hindrance

  • Aspartate to asparagine change at position 359 abolishes activation of the nucleophilic water/hydroxide

Functional Implications:

• DPEP1 actively hydrolyzes a wide range of dipeptides, including leukotriene D4 conversion to leukotriene E4, and possesses β-lactamase activity .

• DPEP3 shows no in vitro activity against dipeptides and biological substrates (imipenem, leukotriene D4, cystinyl-bis-glycine) due to the structural variations .

• DPPX (DPP6) is not enzymatically active but serves as an auxiliary subunit that modulates Kv4.2 potassium channel function .

Epitope Considerations:

• For DPEP3, the SC-003 antibody targets an eighteen-residue epitope across the dimerization interface, distinct from the enzymatic active site .

• For DPEP1, antibodies targeting the brush border of proximal tubules versus those targeting endothelial-expressed DPEP1 may have different binding characteristics .

• Anti-DPPX antibodies target extracellular domains that affect Kv4.2 channel clustering .

These structural differences must be considered when selecting or designing antibodies for specific research applications.

What are the best practices for studying DPEP1 expression changes in disease models?

To effectively study DPEP1 expression changes in disease models:

Methodological Approaches:

• Multiple detection techniques: Combine conventional immunohistochemistry, fluorescent peptide binding (e.g., LSALT), and flow cytometry for comprehensive analysis .

• Temporal analysis: Collect samples at multiple timepoints (baseline, early response, late response) to track expression dynamics. DPEP1 protein expression increases in kidney homogenates within 8 hours of ischemia-reperfusion injury (IRI) or LPS administration .

• Compartmental analysis: Differentiate DPEP1 expression between different cellular compartments (e.g., epithelium vs. endothelium) as expression patterns may vary by cell type .

• Post-translational modifications: Monitor changes in DPEP1 molecular mass, as injury can induce slight increases consistent with post-translational modifications that may affect function .

Experimental Controls:

• Use DPEP1 knockout models as negative controls for antibody specificity .

• Include multiple tissue types to assess differential expression (DPEP1 is expressed in kidney, liver, intestine, and other tissues) .

• For visualization studies, have blinded radiologists or specialists interpret imaging results to avoid bias .

Expression Assessment Example:

In renal injury models, DPEP1 expression increases substantially in both proximal tubular cells and peritubular capillaries following injury, with particularly strong enhancement in peritubular capillaries after IRI or LPS administration .

What are the emerging therapeutic applications of DPEP-targeted antibodies?

DPEP-targeted antibodies show promising therapeutic potential:

Anti-DPEP3 Antibody Drug Conjugates:

• SC-003, a pyrrolobenzodiazepine conjugate targeting DPEP3, has shown efficacy in preclinical models and entered phase I clinical trials (NCT02539719) .

• DPEP3 expression is normally limited to testis but can be elevated in ovarian cancer, making it a potentially selective target .

DPEP1 as an Anti-inflammatory Target:

• DPEP1 functions as a major adhesion receptor for neutrophil recruitment in lungs and liver .

• Peptide blockers of DPEP1 significantly reduce neutrophil recruitment and improve survival in endotoxemia models .

• DPEP1-targeted therapeutics could benefit neutrophil-driven inflammatory diseases of the lungs and acute kidney injury .

Anti-DPPX Immunotherapy:

• Anti-DPPX encephalitis responds well to immunotherapy, with most patients ultimately presenting with a good prognosis .

• Monitoring DPPX antibody titers may help assess treatment response, as decreasing levels have been associated with clinical improvement .

• One study described significant decreases in anti-DPPX antibody titer after tumor resection in paraneoplastic cases .

Novel Approaches:

Cell-penetrating peptides like Dpep (targeting transcription factors ATF5, CEBPB, and CEBPD) selectively promote apoptotic death of multiple tumor cell types while sparing normal cells, representing a potential therapeutic strategy .

How can I troubleshoot common issues with DPEP1 antibody experiments?

When encountering challenges with DPEP1 antibody experiments:

Western Blot Troubleshooting:

• Multiple bands: DPEP1 undergoes post-translational modifications; additional bands at slightly higher molecular weight may represent glycosylated forms . Validate with positive controls from tissues known to express DPEP1 (kidney, intestine).

• Weak signal: DPEP1 antibodies show wide variation in optimal dilutions (1:1000-1:50000); titrate antibody concentrations carefully .

• Background issues: PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) is the recommended storage buffer for many DPEP1 antibodies; improper buffer conditions may affect performance .

Immunohistochemistry Issues:

• Poor tissue staining: For kidney samples, antigen retrieval with TE buffer (pH 9.0) often yields better results than citrate buffer .

• Background staining: Mouse monoclonal antibodies may require special blocking when used on mouse tissues to minimize background.

• Inconsistent results: Store antibody at -20°C in aliquots to maintain stability. Repeated freeze-thaw cycles can reduce antibody performance .

Flow Cytometry Considerations:

• Low detection: When analyzing endothelial DPEP1 expression, note that baseline expression may be minimal and only increase substantially after activation or injury .

• Autofluorescence: In tissues with high autofluorescence (like kidney), using conjugated antibodies with appropriate fluorophores (e.g., CoraLite 488 with excitation/emission at 493/522 nm) can help distinguish true signal .

Sample-Specific Recommendations:

For kidney tissue, DPEP1 is strongly expressed in proximal tubular cells; for lung and liver inflammation studies, focus on endothelial expression which increases after injury or inflammation .

How do energy-based optimization methods improve antibody design for DPEP targets?

Energy-based optimization has revolutionized DPEP-targeted antibody design:

Technical Foundations:

• Pre-trained diffusion models: These serve as the foundation for antibody design, jointly modeling sequences and structures with equivariant neural networks .

• Direct energy-based preference optimization (AbDPO): This approach fine-tunes pre-trained models using residue-level decomposed energy preferences .

• Energy decomposition: Multiple energy types are identified and optimized separately:

  • Rationality energy: Evaluates structural feasibility

  • Functionality energy: Measures binding affinity to target antigens

  • Attraction and repulsion energies: Address different aspects of molecular interaction

Experimental Results:

Energy-based optimization methods significantly outperform traditional approaches:

• AbDPO achieves superior performance in designing antibodies with low total energy and high binding affinity simultaneously .

• When evaluated on the RAbD benchmark, AbDPO was the only method to achieve CDR-Ag lower than 0, indicating favorable binding energy .

• These methods effectively optimize the energy of generated antibodies to resemble natural antibodies while maintaining appropriate structural parameters .