ADAM33 Antibody

What is ADAM33 and what is its significance in disease pathology?

ADAM33 is a transmembrane protein containing multiple domains including a metalloprotease-like domain, disintegrin cell adhesion domain, and cysteine-rich domain. It plays a critical role in airway function maintenance, with significant involvement in asthma pathogenesis. ADAM33 functions as a local tissue susceptibility gene that promotes allergic asthma, particularly through its soluble form (sADAM33). The soluble enzymatically active form is increased in asthmatic airways and contributes to airway remodeling independent of inflammation processes . Beyond respiratory diseases, ADAM33 also shows altered expression patterns in certain cancers, including breast cancer, where its downregulation has been explored as a potential biomarker .

How does ADAM33 structure influence antibody design and selection?

ADAM33's complex structure consists of several distinct domains that affect antibody design strategies. The mature ectodomain of human ADAM33 shares approximately 79% amino acid sequence identity with mouse ADAM33, which provides important considerations for cross-reactivity in experimental design . Commercial antibodies like MAB5565 typically target specific epitopes within the Val30-Lys501 region of the protein . For custom antibody development, regions encompassing parts of the disintegrin and cysteine-rich domains have proven effective targets, as demonstrated by the successful GMGK06 monoclonal antibody that recognizes these domains . When selecting antibodies, researchers should consider which domain they need to target based on their specific research question, particularly if distinguishing between membrane-bound and soluble forms.

What validation steps are essential before using an ADAM33 antibody in research?

Rigorous validation is critical when working with ADAM33 antibodies. A comprehensive validation protocol should include:

• Western blot analysis using known positive and negative control cell lines. For instance, breast cancer cell lines PMC42, MCF7, and SKBR3 have been established as ADAM33-positive, while MDA-MB-231 and MDA-MB-436 serve as negative controls .

• Cross-reactivity testing against other ADAM family members, particularly ADAM9 and ADAM12 which share structural similarities. The specificity of your antibody should be confirmed as demonstrated with GMGK06, which shows no reactivity with other ADAM proteins .

• Immunocytochemistry verification in appropriate cell lines to confirm cellular localization patterns.

• Flow cytometry validation if the antibody will be used for this application, as demonstrated with the MAB5565 antibody in A549 human lung carcinoma cells .

• Positive control tissue testing, such as human lung tissue which strongly expresses ADAM33 .

How can I differentiate between various forms of ADAM33 in my experiments?

ADAM33 exists in multiple forms including the full-length membrane-bound protein and several soluble forms. In bronchoalveolar lavage fluid (BALF), immunoreactive sADAM33 can be detected using antibodies against the metalloprotease (MP) domain, showing distinct bands: approximately 25 kDa (MP domain), 52 kDa (unprocessed Pro-MP domain), and higher molecular weight bands representing ectodomain fragments containing the MP domain .

To confirm the identity of specific bands, researchers can use multiple antibodies targeting different domains, such as an antibody against the Pro domain to verify the 52-kDa band as the unprocessed Pro-MP domain . Additionally, enzymatic activity assays using FRET-based substrates can complement protein detection methods to distinguish active from inactive forms of ADAM33, as demonstrated in studies comparing wild-type and Adam33-null mice .

What are the optimal protocols for detecting ADAM33 in different sample types?

Different sample types require optimized protocols for ADAM33 detection:

For protein lysates and Western blot analysis:

• Standard SDS-PAGE protocols are effective, with special attention to sample preparation to preserve protein integrity

• For detecting soluble forms in biological fluids, concentration steps may be necessary before SDS-PAGE

For tissue sections (IHC protocols):

• Immunohistochemical scoring systems have been established based on intensity and extension parameters

• A validated scoring system assigns: Intensity (0=none, 1=weak, 2=strong) and Extension (0=none, 1=<10% positive cells, 2=>10% positive cells)

• The final score (0-4) is calculated as the sum of intensity and extension scores

For flow cytometry applications:

• Cell fixation with paraformaldehyde and permeabilization with saponin facilitates intracellular staining

• Secondary antibody selection is critical; Phycoerythrin-conjugated Anti-Mouse IgG F(ab')2 has been successfully used with primary ADAM33 antibodies

How can I assess ADAM33 enzymatic activity rather than just protein expression?

Measuring ADAM33 enzymatic activity provides crucial functional information beyond mere protein presence. Researchers have employed FRET (Fluorescence Resonance Energy Transfer) assays to quantify metalloprotease activity of ADAM33. This approach involves:

• Collection of bronchoalveolar lavage fluid (BALF) or other biological samples

• Incubation with specific FRET substrates designed for metalloprotease activity

• Measurement of fluorescence intensity as an indicator of enzymatic activity

The specificity of such assays can be validated using samples from Adam33-null mice as negative controls. After HDM (house dust mite) allergen challenge, wild-type mice show increased sADAM33 enzyme activity in BALF, while Adam33-null mice show no significant effect, confirming the specificity of the ADAM33 FRET assay . This methodological approach allows researchers to distinguish between the presence of inactive ADAM33 protein and functionally active enzyme.

How do ADAM33 polymorphisms affect experimental outcomes and antibody selection?

ADAM33 polymorphisms have been significantly associated with asthma susceptibility and bronchial hyperresponsiveness . These genetic variations may affect:

• Protein conformation and epitope accessibility, potentially altering antibody binding efficacy

• Expression levels of different protein isoforms

• Enzymatic activity of the metalloprotease domain

• Cellular localization and trafficking of the protein

When designing experiments, researchers should consider the specific ADAM33 polymorphisms present in their study population or cell models. Antibodies targeting conserved regions might be less affected by polymorphisms than those targeting variable regions. For population studies, documenting known ADAM33 polymorphisms in your subjects can help explain variable antibody reactivity or phenotypic differences. Publications have demonstrated that ADAM33 polymorphism is associated with progression from preschool wheeze into childhood asthma, highlighting the clinical relevance of these variations .

What approaches can detect the interaction between ADAM33 and environmental factors in disease models?

The interaction between ADAM33 and environmental factors represents a critical gene-environment interface in disease pathogenesis. Research approaches to study these interactions include:

• In utero expression models: Studies have shown that when sADAM33 is induced in utero or added ex vivo, it causes structural remodeling of airways, which enhances postnatal airway eosinophilia and bronchial hyperresponsiveness following subthreshold challenge with aeroallergens . This experimental approach helps explain the end-organ expression of allergic asthma in genetically susceptible individuals.

• Allergen challenge models: HDM (house dust mite) allergen challenge demonstrates how ADAM33 mediates responses to environmental triggers. In wild-type mice, allergen challenge increases sADAM33 enzyme activity, while Adam33-null mice show suppression of both remodeling and inflammation after allergen challenge .

• Controlled exposure studies: Measuring changes in sADAM33 levels and activity before and after specific environmental exposures can reveal mechanisms of disease exacerbation.

• Longitudinal studies: These can track how ADAM33 expression/activity changes over time with environmental exposures, particularly in developmental contexts where maternal allergy has been shown to induce sADAM33 in utero .

How can ADAM33 antibodies be used to study airway remodeling mechanisms?

ADAM33 antibodies offer valuable tools for investigating airway remodeling mechanisms, particularly in asthma research. Methodological approaches include:

• Temporal analysis of ADAM33 expression: Using antibodies to track changes in ADAM33 protein levels before, during, and after remodeling processes can reveal its temporal role in disease progression.

• Cellular localization studies: Immunohistochemistry with ADAM33 antibodies can identify which cell types express ADAM33 during remodeling events. This helps determine whether ADAM33 expression is altered in fibroblasts, smooth muscle cells, or other structural cells involved in remodeling.

• Intervention studies: ADAM33 antibodies can monitor protein changes during therapeutic interventions aimed at reversing remodeling. Research has shown that sADAM33-induced airway remodeling is reversible, highlighting the therapeutic potential of targeting ADAM33 in asthma .

• Co-localization experiments: Combining ADAM33 antibodies with markers for extracellular matrix proteins (collagens, fibronectin) can reveal relationships between ADAM33 and structural changes. Studies have shown that after HDM challenge, mRNAs for Acta2, Col1a1, Col3a1, and Fn1 were significantly increased in wild-type mice but suppressed in Adam33-deleted mice in a gene dosage-dependent manner .

What is the significance of ADAM33 methylation in cancer research and how can antibodies complement methylation studies?

ADAM33 methylation has emerged as an important area of investigation in cancer research, particularly in breast cancer. The relationship between methylation status and protein expression offers insights into epigenetic regulation mechanisms:

• Integrated analysis approach: ADAM33 antibodies provide crucial protein-level validation for methylation studies. In breast cancer research, tissue sections from 44 cases with methylated ADAM33 (as determined by methylation-specific PCR) were evaluated using ADAM33 antibodies to establish the relationship between methylation and protein expression .

• Cell line models: Breast cancer cell lines with known ADAM33 methylation status serve as experimental models. The monoclonal antibody GMGK06 revealed strong immunoreactivity in PMC42, MCF7, and SKBR3 cells but no signal in MDA-MB-231 and MDA-MB-436 cells, correlating with gene expression patterns .

• Scoring systems for clinical samples: A standardized scoring system based on intensity (0=none, 1=weak, 2=strong) and extension (0=none, 1=<10%, 2=>10% positive cells) provides quantitative assessment of ADAM33 protein expression in clinical specimens, allowing correlation with methylation data .

• Biomarker development: The correlation between ADAM33 methylation and protein expression is being explored as a potential biomarker in breast cancer, with researchers investigating whether antibody-based detection methods could offer clinically applicable diagnostic or prognostic tools .