CLDN5 Antibody

What is CLDN5 and why is it a significant research target?

Claudin 5 (CLDN5) is a 23 kDa transmembrane protein consisting of 218 amino acids that serves as a critical component of tight junctions. It plays a fundamental role in establishing the paracellular barrier that controls molecular flow between cells of an epithelium . CLDN5 is particularly crucial for blood-brain barrier integrity, evidenced by studies showing that CLDN5 knockout mice are not viable and die within 10 hours after birth .

The protein contains four transmembrane domains with both N-terminus and C-terminus located in the cytoplasm. Its extracellular regions form two loops that are essential for tight junction formation and have become important targets for antibody development.

How does CLDN5 expression vary across different tissues and vascular beds?

CLDN5 expression demonstrates significant tissue-specific and vessel type-specific patterns:

As demonstrated in scRNAseq and RNA in situ hybridization studies, CLDN5 exhibits a large gradual decrease in expression from arterioles through capillaries to venules in all tissues analyzed, including human dermis . This expression pattern inversely correlates with vascular permeability in some tissues.

What should researchers consider when selecting a CLDN5 antibody?

When selecting a CLDN5 antibody, researchers should consider:

• Target species: Verify reactivity with your experimental model (human, mouse, rat, etc.)

• Application compatibility: Ensure validation for your specific application (WB, IF, IHC, ELISA, Flow Cytometry)

• Epitope recognition: Consider whether extracellular region (ECR) binding is required

• Clone type: Monoclonal vs. polyclonal depending on experimental needs

• Validation data: Review published applications and validation images

For instance, the anti-CLDN5 antibody RP1043 has been validated for Western blot applications with human and rat samples, while other antibodies like clone 2B12 have been tested for ELISA, Flow Cytometry, Inhibition/Function Analysis, and Western Blotting .

What are the optimal protocols for using CLDN5 antibodies in immunohistochemistry?

For successful immunohistochemistry with CLDN5 antibodies:

• Tissue fixation: Paraformaldehyde fixation is recommended for optimal antigen preservation

• Antigen retrieval: Heat-mediated antigen retrieval in acetic acid improves detection

• Blocking: Use 1.5% serum for 1 hour at 37°C to reduce non-specific binding

• Primary antibody dilution: Typical working dilutions range from 1:200 to 1:500

• Incubation conditions: Overnight incubation at 4°C yields optimal results

• Detection system: Biotin-conjugated secondary antibodies (1:200 dilution) work effectively

This protocol has been validated for detecting CLDN5 in mouse lung and human lung tissue sections .

How can researchers effectively use CLDN5 antibodies to study blood-brain barrier integrity?

For blood-brain barrier (BBB) studies using CLDN5 antibodies:

• Permeability assessment: After CLDN5 antibody administration, measure extravasation of different molecular weight tracers (e.g., fluorescent dextrans)

• Dose-response evaluation: In non-human primate studies, 3.0 mg/kg of anti-CLDN5 mAb increased cerebrospinal fluid (CSF) concentrations of fluorescein dye (376 Da)

• Safety monitoring: Monitor behavioral changes and plasma biomarkers for inflammation, liver, and kidney injury

• Functional studies: Anti-CLDN5 monoclonal antibodies can temporarily modulate BBB permeability for potential drug delivery applications

• Therapeutic window assessment: Balance between enhanced permeability and maintenance of barrier function must be carefully determined

What techniques are recommended for validating CLDN5 antibody specificity?

To validate CLDN5 antibody specificity:

• Cross-reactivity testing: Prepare cultured cells expressing human CLDN-1 through CLDN-7 and mouse CLDN-5

• Flow cytometry analysis: Test antibody binding to each claudin-expressing cell line

• Western blot verification: Confirm recognition of the expected 23 kDa band

• Knockout/knockdown controls: Use CLDN5 knockout or knockdown samples as negative controls

• Recombinant protein testing: Verify binding to recombinant CLDN5 protein

As demonstrated in one study, five monoclonal antibodies were validated by testing their reactivity against cells expressing different claudins, confirming their specific binding to human CLDN5-expressing cells .

How does CLDN5 deficiency affect vascular permeability in different tissue types?

CLDN5 deficiency impacts vascular permeability in a tissue-specific and vessel type-specific manner:

In Cldn5 inducible endothelial cell-specific knockout (iECKO) mice, loss of CLDN5 led to vessel-specific effects. αSMA-negative capillaries showed a fourfold increase in leakage, while αSMA-positive venules demonstrated a 2.5-fold increase following histamine stimulation . Leakage sites in knockout mice appeared closer to arterioles than in controls.

What methods have been developed for engineering CLDN5 antibodies against extracellular regions?

Researchers have developed innovative approaches to generate antibodies against the extracellular regions (ECR) of CLDN5:

• Engineered liposomal immunogens: Using wheat cell-free protein synthesis systems to improve production

• GC content normalization: Suppressing and normalizing mRNA GC content dramatically improved CLDN5 production in cell-free systems

• Chimeric antigen design: Human/mouse chimeric CLDN5 (Antigen1) where mouse CLDN5 ECR was substituted with human CLDN5 sequences

• Artificial membrane protein constructs: CLDN5-based artificial membrane protein (Antigen2) consisting of symmetrically arranged ECRs

• Liposomal presentation: Both engineered antigens were synthesized as proteoliposomes containing lipid-type adjuvant

This approach successfully induced ECR-binding antibodies in mice with high success rates, yielding five monoclonal antibodies that specifically recognized CLDN5 ECR, including clone 2B12 with high affinity (<10 nM) .

What compensatory mechanisms occur following CLDN5 downregulation?

When CLDN5 is downregulated or knocked out, several compensatory changes occur in other junction proteins:

• Adherens junction proteins: Enhanced expression of VE-Cadherin (Cdh5) at both mRNA and protein levels

• Other tight junction components: Increased expression of Occludin (Ocln) and JAM-A (F11r)

• Scaffolding proteins: Decreased expression of ZO-1 (Tjp1) at both mRNA and protein levels

• Correlation patterns: CLDN5 expression levels inversely correlate with VE-Cadherin and Occludin and positively correlate with ZO-1

• Tissue-specific effects: Immunohistochemistry of ear dermis confirmed downregulation of ZO-1 and upregulation of VE-Cadherin

These compensatory changes likely represent cellular attempts to maintain barrier function despite CLDN5 loss .

What are common challenges in generating and using CLDN5 antibodies?

Researchers face several challenges when working with CLDN5 antibodies:

• Production difficulties: Membrane proteins like CLDN5 are notoriously difficult to produce due to their complex structure

• Low immunogenicity: CLDN5 has highly conserved sequences among species, resulting in poor immune responses

• Epitope accessibility: The extracellular regions of CLDN5 form small loops that may be difficult to target

• Specificity concerns: Cross-reactivity with other claudin family members due to sequence similarity

• Functional validation: Confirming that antibodies recognize native conformations in tight junctions

To overcome these challenges, researchers have developed engineered antigens and liposomal presentations that drastically improve both production and immunogenicity .

How should researchers interpret unexpected CLDN5 expression patterns?

When encountering unexpected CLDN5 staining patterns:

• Verify antibody specificity: Confirm antibody recognizes only CLDN5 and not other claudin family members

• Consider tissue heterogeneity: CLDN5 expression varies significantly across different vascular beds

• Evaluate detection sensitivity: Some vessels may express CLDN5 at levels below detection by standard immunohistochemistry

• Use multiple detection methods: Combine protein detection (immunohistochemistry) with mRNA analysis (RNA in situ hybridization)

• Consult literature: Reference tissue-specific expression data from publications

For example, researchers observed positive staining in right lung tight junctions using anti-CLDN5 antibody and questioned whether this was expected. According to literature, right lung does express CLDN5, generally in cell junctions and tight junctions, as confirmed by multiple studies (Pubmed IDs: 15489334, 14702039) .

What experimental controls are essential for CLDN5 antibody experiments?

Essential controls for CLDN5 antibody experiments include:

• Negative controls:

  • Isotype-matched non-specific antibodies

  • CLDN5 knockout or knockdown samples

  • Tissues known to lack CLDN5 expression

• Positive controls:

  • Brain endothelial cells (high CLDN5 expression)

  • Validated CLDN5-expressing cell lines

  • Recombinant CLDN5 protein

• Specificity controls:

  • Cells expressing other claudin family members

  • Pre-absorption with CLDN5 antigen

  • Multiple antibodies targeting different CLDN5 epitopes

• Technical controls:

  • RNA in situ hybridization to correlate with protein expression

  • Secondary antibody-only controls

  • Multiple validation techniques (WB, IF, IHC)

Proper controls are particularly important given the heterogeneous expression of CLDN5 across different vessel types and tissues .

How might CLDN5 antibodies advance drug delivery to the central nervous system?

CLDN5 antibodies show promise for enhancing CNS drug delivery:

• Targeted BBB modulation: Anti-CLDN5 mAbs can temporarily increase BBB permeability in a controlled manner

• Safety profile: Studies in non-human primates show that at 3.0 mg/kg, anti-CLDN5 mAb increased CSF concentrations of fluorescein dye without behavioral changes or inflammatory markers

• Size-selective permeability: Different molecular weight tracers can be used to assess the size range of molecules that can cross the modulated barrier

• Therapeutic window definition: Balancing enhanced permeability with maintenance of essential barrier function

• Combination approaches: CLDN5 antibodies could be combined with drug carriers for improved CNS delivery

This approach may enable delivery of therapeutics that normally cannot cross the BBB, potentially addressing a major challenge in treating CNS disorders .

What is the relationship between CLDN5 expression and vascular pathologies?

Understanding CLDN5's role in vascular pathologies represents an important research frontier:

• Barrier dysfunction: CLDN5 downregulation correlates with increased vascular permeability in various disease states

• Tissue-specific effects: Different vascular beds show varying dependency on CLDN5 for barrier maintenance

• Inflammatory responses: Histamine-induced vascular leakage is enhanced in CLDN5-deficient animals in specific tissues

• Compensatory mechanisms: Other junction proteins may partially compensate for CLDN5 loss in certain contexts

• Therapeutic targeting: Modulating CLDN5 function could potentially address both excessive and insufficient barrier function

Researchers should explore how CLDN5 expression changes in disease models and how these changes contribute to pathology progression or resolution.