AQP5 Antibody

What is AQP5 and why is it significant in research?

Aquaporin 5 (AQP5) is a membrane protein functioning as a water channel involved in the bidirectional transfer of water and small solutes across cell membranes. It has significant research importance due to its wide expression in tissues including digestive, renal, respiratory, and reproductive systems . AQP5 is a 28 kDa protein (observed molecular weight 27 kDa) with 265 amino acids that plays crucial roles in secretory processes, particularly in salivary and lacrimal glands . Research interest in AQP5 has intensified due to its involvement in autoimmune conditions like Sjögren's syndrome and its potential role in carcinogenesis, making AQP5 antibodies essential tools for investigating these biological processes.

What applications are AQP5 antibodies typically used for?

AQP5 antibodies are utilized across multiple experimental techniques with varying optimal dilutions:

Researchers should note that optimal dilution is sample-dependent and should be determined empirically for each experimental system .

What tissues show positive reactivity with AQP5 antibodies?

AQP5 antibodies have demonstrated positive reactivity in multiple tissue types:

• Western Blot positive detection: Mouse lung tissue, rat lung tissue

• IHC positive detection: Mouse lung tissue, mouse kidney tissue, rat kidney tissue

• IF positive detection: Rat lung tissue

For optimal IHC results, antigen retrieval with TE buffer (pH 9.0) is suggested, although citrate buffer (pH 6.0) may also be used as an alternative . AQP5 is particularly abundant in the apical membrane of type I alveolar epithelial cells, acinar epithelial cells in submucosal glands, and large airway epithelia .

How should AQP5 antibodies be stored and handled to maintain activity?

For optimal preservation of AQP5 antibody activity:

• Store at -20°C in storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• The antibody remains stable for one year after shipment when properly stored

• Aliquoting is unnecessary for -20°C storage (for most formulations)

• Some preparations (20μl sizes) contain 0.1% BSA as a stabilizer

• Avoid repeated freeze-thaw cycles that can compromise antibody functionality

Additionally, when planning experiments, researchers should account for the antibody form (liquid), purification method (typically antigen affinity purification), and any modifications such as conjugations that might affect stability or application parameters.

What are the recommended protocols for detecting AQP5 in immunohistochemistry?

For optimal AQP5 detection in tissue samples:

• Antigen retrieval: Use TE buffer (pH 9.0) as primary option; citrate buffer (pH 6.0) can serve as an alternative

• Blocking: Implement standard blocking procedures with appropriate blocking reagents (typically 5% BSA or serum matching the secondary antibody host)

• Primary antibody incubation: Apply at dilutions of 1:2500-1:10000, optimized for your specific tissue and protocol

• Visualization: Use appropriately matched secondary antibody detection systems

• Controls: Include positive controls (mouse/rat lung tissue) and negative controls (primary antibody omission)

For immunofluorescence applications, dilutions of 1:50-1:500 are typically recommended, with cell-based immunofluorescence cytochemistry (CB-IFC) assays being particularly valuable for detecting anti-AQP5 autoantibodies in clinical samples .

What are the critical considerations when using anti-AQP5 antibodies for Western blotting?

When using anti-AQP5 antibodies for Western blotting:

• Expected molecular weight: Look for bands at approximately 27-28 kDa, which is the observed molecular weight of AQP5

• Recommended dilution range: Use 1:500-1:2000 dilution, titrating to determine optimal concentration for your specific sample

• Sample preparation: Lung tissue from mouse or rat models has been validated as positive controls

• Loading controls: Include appropriate loading controls to normalize expression levels

• Membrane selection: PVDF membranes are generally preferred for optimal protein transfer and antibody binding

For detecting AQP5 phosphorylation states (important in cancer research), immunoprecipitation followed by Western blotting with phosphorylation-specific antibodies may be required .

How are AQP5 antibodies used in Sjögren's syndrome research?

AQP5 antibodies have become significant tools in Sjögren's syndrome research:

• Diagnostic biomarker potential: Anti-AQP5 autoantibodies show promise as diagnostic biomarkers for primary Sjögren's syndrome (pSS) with higher sensitivity compared to traditional anti-SSA antibodies

• Differential diagnosis: Anti-AQP5 antibody levels help distinguish pSS from other connective tissue diseases (CTD) and healthy controls

• Clinical significance: A Chinese study found anti-AQP5 levels were significantly higher in pSS patients (26.42 ng/ml) compared to CTD patients (9.10 ng/ml) and healthy controls (5.93 ng/ml)

• Functional relevance: Anti-AQP5 autoantibodies may contribute to glandular hypofunction by interfering with water transport

• Disease activity correlation: Some studies correlate anti-AQP5 antibody levels with disease activity indices

Recent research demonstrated that anti-AQP5 antibodies achieved an AUC of 0.86 (95% CI 0.80–0.93) with sensitivity of 0.95 and specificity of 0.70 when differentiating pSS from other connective tissue diseases .

What role does AQP5 play in cancer research, and how are antibodies employed in this field?

AQP5 has emerged as a subject of interest in cancer research:

• Putative oncogenic properties: Ectopic expression of human AQP5 induces phenotypic changes characteristic of transformation both in vitro and in vivo

• Phosphorylation significance: The cell proliferative ability of AQP5 depends on phosphorylation of a cAMP-protein kinase (PKA) consensus site in AQP5's cytoplasmic loop

• Differential phosphorylation: AQP5 is phosphorylated in NSCLC cell lines and primary tumor samples but not in normal lung tissues

• Functional studies: siRNA knockdown of AQP5 in cancer cell lines demonstrating AQP5 overexpression results in decreased cell proliferation

• Therapeutic targeting: AQP5 presents a potential therapeutic target in certain cancers

Anti-AQP5 antibodies are essential tools for investigating these phenomena through immunoblotting, immunohistochemistry, and immunoprecipitation techniques to detect expression levels and phosphorylation states of AQP5 in normal versus cancer tissues.

How are AQP5 antibodies utilized in respiratory disease research?

In respiratory disease research, particularly asthma research, AQP5 antibodies have revealed important insights:

• Expression patterns: Immunohistochemical analysis with AQP5 antibodies has demonstrated overexpression of AQP5 in airway epithelium and submucosal glands of asthmatics

• Knock-out studies: AQP5 knockout mice show reduced responses to house dust mite (HDM) exposure, with significantly less inflammation at the airway compared to wild-type mice

• Cytokine modulation: Anti-AQP5 antibodies help reveal that AQP5 expression influences levels of IL-4, IL-10, IL-2, and IFN-γ in bronchoalveolar lavage following allergen exposure

• Mucin regulation: Immunoblotting with AQP5 antibodies shows AQP5's involvement in the regulation of MUC5AC and MUC5B production, key components in mucous hyperproduction

These findings implicate AQP5 in the development of airway inflammation and mucous hyperproduction during chronic asthma, positioning AQP5 as a potential therapeutic target in respiratory diseases.

How can epitope mapping of AQP5 antibodies improve their application in autoimmune disease research?

Epitope mapping of AQP5 antibodies has revealed critical insights for autoimmune disease research:

• Functional epitope identification: Studies have identified multiple functional epitopes on AQP5, particularly in the extracellular loops (A, C, and E) and the second transmembrane helix

• Differential recognition patterns: Anti-AQP5 autoantibodies from Sjögren's syndrome patients and control subjects show differences in fine specificity to these functional epitopes

• E1 epitope significance: The cyclized peptide (E1) mimicking loop E is most frequently recognized and best differentiates between Sjögren's syndrome and control samples

• Diagnostic improvement: Anti-AQP5_E1 IgG demonstrated the greatest power to differentiate Sjögren's syndrome from non-Sjögren's controls based on AUC analysis

• Therapeutic implications: Understanding the exact epitopes recognized by pathological autoantibodies enables development of targeted blocking therapies

This knowledge allows researchers to design more specific ELISA assays, with anti-AQP5_E1 IgG showing a sensitivity of 0.61 and specificity of 0.77 in discriminating Sjögren's syndrome from controls .

What methodological approaches can overcome cross-reactivity challenges when using AQP5 antibodies?

Researchers can employ several strategies to address cross-reactivity issues:

• Peptide competition assays: Conducting immunofluorescence in the presence/absence of specific epitope peptides can confirm antibody specificity

• Cell-based validation: Using MDCK cells transfected with AQP5 alongside untransfected controls provides a cellular system for specificity testing

• Knockout validation: Comparing staining patterns between wild-type and AQP5 knockout tissues confirms specificity in complex tissue environments

• Epitope-specific antibodies: Utilizing antibodies targeting specific epitopes (e.g., E1 loop) reduces cross-reactivity with other aquaporins

• Multiple detection methods: Combining different techniques (CB-IFC and ELISA) provides complementary confirmation of specific binding

Research has shown that cell-based immunofluorescence cytochemistry (CB-IFC) and ELISA using specific epitope peptides as antigens demonstrate comparable performance in diagnostic accuracy (0.690 vs. 0.707) while minimizing cross-reactivity concerns .

How do phosphorylation states of AQP5 influence antibody recognition and functional studies?

The phosphorylation state of AQP5 has significant implications for antibody recognition and functional analyses:

• Key phosphorylation site: The PKA-mediated phosphorylation site at serine 156 (S156) in AQP5 is critical for its function in cell proliferation

• Antibody selection considerations: When studying AQP5 functions, researchers must consider whether their antibodies recognize phosphorylated epitopes or are phosphorylation-state independent

• Phospho-specific detection: For studies focused on AQP5 activation states, phospho-specific antibodies or (Ser/Thr) PKA substrate antibodies following immunoprecipitation are essential

• Functional mutations: Research employing site-directed mutants (N185D and S156A) demonstrates the importance of phosphorylation sites in AQP5's biological activity

• Clinical correlations: Differential phosphorylation patterns between normal and disease tissues (e.g., NSCLC) highlight the need for phosphorylation-aware antibody approaches

Studies have revealed that AQP5 is preferentially phosphorylated in tumor tissues compared to normal counterparts, indicating a potential role in carcinogenesis that requires specific antibody detection strategies .

What are the experimental design considerations for detecting anti-AQP5 autoantibodies in patient samples?

When designing experiments to detect anti-AQP5 autoantibodies in clinical samples:

• ELISA optimization: While no commercial ELISA kits are widely available, custom ELISAs can be prepared using specific proteins (Human AQP5, e.g., Ag14514) coated at 2 μg/ml in carbonate buffer

• Cutoff determination: Establish appropriate cutoff values through ROC curve analysis; studies have used 14.10 ng/ml for healthy controls and 18.79 ng/ml for differentiating from other connective tissue diseases

• Control selection: Include multiple control groups (healthy controls and disease controls) to establish specificity

• Sample preparation: Standardize serum dilution protocols (typically 1:10 for IgA and 1:100 for IgG detection)

• Antibody subclass analysis: Consider evaluating both IgG and IgA anti-AQP5 antibodies, as they may have different clinical associations

Research has demonstrated that anti-AQP5 autoantibodies can achieve high diagnostic accuracy with AUC values of 0.98 (95% CI 0.96–1.00) versus healthy controls and 0.86 (95% CI 0.80–0.93) versus other autoimmune conditions .

How can animal models be utilized to study anti-AQP5 autoantibody production and pathogenicity?

Animal models provide valuable insights into anti-AQP5 autoantibody mechanisms:

• Molecular mimicry models: Immunization with PmE-L peptide (derived from the AQP5-homologous aquaporin of Prevotella melaninogenica) can induce anti-AQP5 autoantibody production in C57BL/6 mice

• Functional consequences: Mice with anti-AQP5 antibodies demonstrate decreased salivary flow rates, mimicking Sjögren's syndrome symptoms

• B-cell receptor analysis: Characterization of AQP5-specific autoantibodies and mapping of B-cell receptor repertoires reveals that AQP5-specific B cells acquire antigen-binding ability through cumulative somatic hypermutation

• Cross-reactivity assessment: Sera containing anti-AQP5 IgG stain mouse Aqp5 in submandibular glands and detect bacterial aquaporin homologs by immunoblotting

• Phenotypic correlation: Animal models allow correlation between autoantibody titers and physiological changes in secretory function

These models provide critical platforms for testing potential therapeutic interventions targeting anti-AQP5 autoantibodies in Sjögren's syndrome and related conditions.

What role might anti-AQP5 antibodies play in the development of targeted therapies for autoimmune diseases?

Anti-AQP5 antibodies have potential therapeutic applications:

• Diagnostic stratification: Anti-AQP5 autoantibody screening may help form subgroups of Sjögren's syndrome patients for targeted therapy approaches

• Therapeutic blocking: Development of molecules that block the interaction between pathogenic autoantibodies and functional epitopes on AQP5

• Epitope-specific interventions: With knowledge that the E1 epitope is most frequently recognized in disease, targeted epitope-specific therapies can be developed

• Combination biomarkers: Anti-AQP5 antibodies could be combined with other markers (e.g., anti-PUF60) to enhance diagnostic accuracy and treatment selection

• Monitoring treatment response: Anti-AQP5 antibody levels might serve as biomarkers for monitoring response to therapy

Studies indicate that combining anti-AQP5 testing with traditional markers could enhance the diagnostic rate of Sjögren's disease, potentially leading to earlier intervention and more personalized treatment approaches .

How do technological advances in antibody development impact AQP5 research?

Emerging technologies are enhancing AQP5 antibody research:

• Phage display antibody libraries: This technology enables selection and characterization of AQP5-specific autoantibodies, providing insights into B cell receptor sequences and somatic hypermutation patterns

• Conjugated antibodies: Development of fluorescently conjugated anti-AQP5 antibodies (phycoerythrin, Cy3, Dylight488) expands applications in flow cytometry and imaging

• Multiplex detection systems: Integration of anti-AQP5 antibody detection into multiplexed autoantibody panels improves diagnostic efficiency

• Cell-based assay improvements: Refinements in cell-based immunofluorescence cytochemistry enhance detection sensitivity and specificity

• Recombinant antibody fragments: Development of smaller antibody formats may improve tissue penetration and epitope accessibility