CLDN5 Recombinant Monoclonal Antibody

What is CLDN5 and why is it challenging to produce antibodies against it?

CLDN5 (Claudin-5) is a multispanning membrane protein with two highly conserved extracellular loops that forms tight junctions between cells. Production of antibodies against CLDN5 is notoriously difficult due to its complex structure, highly conserved sequences among species, and low immunogenicity . Unlike soluble proteins, membrane proteins like CLDN5 are challenging to purify in their native conformation, which further complicates antibody production . Additionally, the extracellular regions (ECR) of CLDN5 are particularly small, presenting limited epitope availability for antibody recognition .

What innovations have improved the generation of anti-CLDN5 antibodies?

Several breakthrough approaches have enhanced CLDN5 antibody development. Researchers have successfully increased CLDN5 productivity in cell-free systems by suppressing and normalizing mRNA GC content to approximately 50.8%, eliminating high local GC content peaks that inhibit translation . Additionally, engineered immunogens have been designed as proteoliposomes using wheat cell-free protein synthesis systems . Two particularly effective engineered antigens include human/mouse chimeric CLDN5 (Antigen1) and a CLDN5-based artificial membrane protein with symmetrically arranged ECRs (Antigen2) . These innovations have dramatically improved the success rate of generating high-quality antibodies against CLDN5.

What is the difference between conventional CLDN5 antibody production and engineered approaches?

Conventional approaches to CLDN5 antibody production often yield poor results. When researchers attempted immunization with wild-type C-terminal-truncated CLDN5 via subcutaneous tail base injection, none of the 20 immunized mice produced antibodies recognizing human CLDN5 ECR . In contrast, engineered approaches using redesigned antigens delivered intraperitoneally achieved remarkable success rates of 84.2% (16/19) for Antigen1 and 82.4% (14/17) for Antigen2 . The table below compares these approaches:

What experimental applications are suitable for anti-CLDN5 monoclonal antibodies?

Anti-CLDN5 monoclonal antibodies have been validated for multiple research applications. Based on available data, these antibodies work effectively in Western blotting (WB) and immunohistochemistry (IHC) . For Western blot applications, dilutions typically range from 1:500-1:2000, while IHC applications generally require dilutions between 1:50-1:200 . While not explicitly confirmed in the data provided, researchers also commonly use these antibodies in immunocytochemistry, flow cytometry, and immunoprecipitation experiments. When selecting antibodies for specific applications, researchers should verify that validation has been performed for their target species and tissue type .

How can researchers validate CLDN5 antibody specificity?

Thorough validation of CLDN5 antibody specificity involves multiple complementary approaches. First, researchers should test antibody reactivity against cells expressing different claudin family members (CLDN-1 through CLDN-7) using flow cytometry to confirm specificity for CLDN5 . A comprehensive approach involves testing against all 27 human claudins using proteoliposome ELISA . Additionally, mutational analysis using alanine scanning (substituting individual amino acids with alanine) helps identify specific epitopes recognized by the antibody . For antibodies suspected to be conformation-sensitive, comparing binding to native versus denatured CLDN5 through SDS-PAGE under reducing conditions is essential. The research shows that some antibodies (like 1B3 and 4F1) completely lose binding to denatured CLDN5, while others (like 2B12) maintain binding capability .

What controls should be incorporated when using CLDN5 antibodies?

Robust experimental design requires appropriate controls when using CLDN5 antibodies. Negative controls should include tissues or cells known to lack CLDN5 expression, while positive controls should include samples with confirmed CLDN5 expression (such as brain endothelial cells) . For specificity validation, blocking peptides corresponding to the immunogen can be used to confirm selective binding . In cross-reactivity studies, researchers should include orthologous CLDN5 from different species, as demonstrated by testing antibodies against both human and mouse CLDN5 . For functional assays such as Trans-Epithelial/Endothelial Electrical Resistance (TEER), isotype controls and antibodies targeting different epitopes serve as important controls to distinguish specific from non-specific effects .

How can epitope mapping inform the selection of CLDN5 antibodies for specific applications?

Epitope mapping provides critical insights for selecting the most appropriate CLDN5 antibody for specific research questions. Through alanine scanning mutagenesis, researchers have identified that most anti-CLDN5 ECR monoclonal antibodies (except clone 4F1) recognize epitopes containing serine 151 in the second extracellular loop . More specifically, antibodies like 1B3 show impaired binding when mutations occur at positions Y148A, D149A, V152A, and P153A, with partial effects at E146A and S151A . Different antibodies exhibited unique binding patterns - for instance, clones 1D1 and 2B1 failed to bind to E146A, D149A, P150A, S151A, and P153A mutants . Understanding these epitope specificities allows researchers to select antibodies that target accessible regions in their experimental system and avoid those that might be masked by protein interactions or conformational changes.

What explains the discrepancy between observed and calculated molecular weights of CLDN5?

Researchers often observe CLDN5 at approximately 130 kDa by Western blot, despite its calculated molecular weight of 23,147 Da . This substantial discrepancy stems from several factors that researchers must consider when interpreting results. Post-translational modifications, particularly oligomerization of CLDN5 in tight junction complexes, significantly affect migration patterns. Additionally, the hydrophobic nature of this membrane protein causes unusual migration in SDS-PAGE. CLDN5 often forms detergent-resistant complexes even under denaturing conditions, resulting in higher apparent molecular weights. When conducting Western blot analysis, researchers should carefully optimize sample preparation conditions, including detergent selection, reducing agent concentration, and heating duration, to ensure consistent results across experiments .

How can CLDN5 antibodies be used to assess blood-brain barrier function?

CLDN5 antibodies enable sophisticated assessment of blood-brain barrier (BBB) integrity through multiple experimental approaches. The trans-epithelial/endothelial electrical resistance (TEER) assay using CLDN5-expressing MDCKII cells provides a quantitative measure of barrier function . Treatment with specific antibodies like clone 2B12 significantly decreases TEER in human CLDN5-expressing MDCKII cell monolayers, indicating functional disruption of CLDN5-based tight junctions . Importantly, this effect showed species specificity, as 2B12 did not disrupt mouse CLDN5 barriers despite binding to human CLDN5 . By contrast, clone 4F1, which binds both human and mouse CLDN5, did not modulate barrier function in either species . These differential effects highlight the importance of epitope specificity in functional studies. Researchers can employ these antibodies to study how various pathological conditions or therapeutic interventions affect BBB integrity by monitoring CLDN5 expression, localization, and function.

How can researchers determine if their anti-CLDN5 antibody recognizes conformational or linear epitopes?

Distinguishing between antibodies recognizing conformational versus linear epitopes is crucial for experimental design. Western blot analysis under reducing conditions provides valuable insights—antibodies detecting denatured CLDN5 likely recognize linear epitopes, while those failing to bind under these conditions target conformational epitopes . In the reported study, clone 2B12 effectively bound denatured human CLDN5 in cell lysates separated by SDS-PAGE under reducing conditions at 10 μg/mL, suggesting recognition of a linear epitope . In contrast, clones 1B3 and 4F1 showed no binding to denatured CLDN5 even at 50 μg/mL, indicating strict conformational dependence . Clones 1D1 and 2B1 exhibited minimal binding at 50 μg/mL, suggesting partial conformational dependence . These characteristics significantly impact application suitability—conformational antibodies excel in applications maintaining native protein structure (flow cytometry, immunoprecipitation) but may perform poorly in applications involving denaturation (western blotting, certain IHC protocols).

What approaches can improve detection of CLDN5 in difficult tissue samples?

Detecting CLDN5 in challenging tissue samples requires optimized protocols. For tissue samples with high autofluorescence or background, researchers should consider antigen retrieval methods specifically optimized for membrane proteins. Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) often improves signal-to-noise ratio . Given that some anti-CLDN5 antibodies (like clones 1B3 and 4F1) recognize conformational epitopes, researchers should carefully select fixation methods that preserve protein structure—mild fixatives like 2% paraformaldehyde may be preferable to harsh denaturants . For frozen tissues, optimization of section thickness (8-10 μm typically works well) and proper fixation prior to antibody incubation is critical . When working with barrier tissues like brain endothelium, gentle permeabilization techniques using low concentrations of detergents (0.1% Triton X-100 or 0.05% Saponin) help maintain claudin architecture while allowing antibody access.

What factors influence cross-reactivity of CLDN5 antibodies between species?

Cross-species reactivity of CLDN5 antibodies depends on epitope conservation and can be predicted through sequence analysis. Among the five monoclonal antibodies characterized in the research, only clone 4F1 bound to mouse CLDN5, while all five recognized human CLDN5 . Sequence analysis revealed that in the extracellular region of human CLDN5, amino acids D68, T75, and S151 differ from mouse CLDN5 . Through mutational studies using chimeric constructs (D68E, T75A, and S151T), researchers determined that four antibodies (except 4F1) bound to D68E and T75A mutants but failed to bind the S151T mutant . This indicates that serine 151, located in the second extracellular loop, is critical for antibody recognition. When considering antibody application to unstudied species, researchers should analyze sequence conservation at key epitope regions, particularly around position 151 for most anti-CLDN5 antibodies. For pig tissues, which were specifically queried, detailed sequence alignment focusing on these critical residues would help predict cross-reactivity .

How are function-blocking CLDN5 antibodies being used in neurovascular research?

Function-blocking antibodies like clone 2B12 are powerful tools for studying the dynamic regulation of the blood-brain barrier in neurovascular research. These antibodies can temporarily disrupt tight junctions without genetic manipulation, allowing researchers to examine the consequences of controlled, transient barrier disruption . This approach offers significant advantages over genetic knockout models, which are often lethal—CLDN5 knockout mice die within 10 hours after birth . By measuring TEER in cellular models, researchers can quantitatively assess the degree of barrier disruption caused by specific antibodies under various experimental conditions . This methodology enables investigation of size-selective BBB permeability changes, as CLDN5 regulates the passage of molecules smaller than 800 Da. Ongoing research applications include studying drug delivery across the BBB, modeling neurodegenerative diseases with BBB disruption, examining pathogen neuroinvasion mechanisms, and investigating the contribution of BBB dysfunction to neuroinflammatory conditions.

What considerations are important when using CLDN5 antibodies in multiplex immunostaining?

Multiplex immunostaining with CLDN5 antibodies requires careful optimization to ensure specific detection without cross-reactivity. When co-staining with other tight junction proteins (occludin, ZO-1), researchers should select primary antibodies raised in different host species to allow for species-specific secondary antibodies . For instance, rabbit monoclonal anti-CLDN5 antibodies can be paired with mouse antibodies against other targets . Given the variable affinity of different CLDN5 antibody clones, titration experiments are essential to determine optimal concentrations that provide specific signals without background or bleed-through. Sequential staining protocols may be necessary when using multiple rabbit antibodies, employing complete blocking steps between rounds of staining. When designing multiplex panels, researchers should consider that some CLDN5 antibodies (like 1B3 and 4F1) recognize conformational epitopes that may be affected by aggressive antigen retrieval methods required for other targets . Finally, proper controls including single-stained samples and isotype controls are critical for accurate interpretation of multiplex results.

What are the latest advances in engineering CLDN5 antibodies for therapeutic applications?

Engineering CLDN5 antibodies for therapeutic applications represents a frontier in BBB drug delivery research. While standard monoclonal antibodies like clone 2B12 can modulate CLDN5 function in vitro, their large size (~150 kDa) limits BBB penetration in vivo . Research is advancing toward creating smaller antibody fragments such as single-chain variable fragments (scFv, ~25 kDa) or nanobodies (~15 kDa) derived from effective CLDN5 binders like 2B12 . These smaller formats may achieve better tissue penetration while maintaining binding specificity. Another approach involves engineering bispecific antibodies that target both CLDN5 and disease-relevant targets, potentially enabling simultaneous BBB modulation and therapeutic action. Given that complete CLDN5 knockout is lethal, researchers are focusing on antibodies that cause partial or temporary modulation of barrier function without complete disruption . For any therapeutic development, species cross-reactivity is crucial for translational research—understanding epitope differences between human and animal CLDN5 (like the S151 residue) informs the development of antibodies suitable for both preclinical models and human applications .