AAD14 Antibody

What is ADAMTS4 antibody and what is its target protein?

ADAMTS4 antibody is a monoclonal antibody (clone #416608) that targets human ADAMTS4 protein, also known as aggrecanase-1. ADAMTS4 belongs to a family of secreted zinc proteases with a multi-domain structure. The protein consists of a signal peptide (amino acids 1-51), a pro domain (amino acids 52-212), and a mature chain (amino acids 213-837) containing several functional domains including catalytic, disintegrin, TSP type-1, cysteine-rich, and spacer domains . ADAMTS4 is the only ADAMTS protein identified that has one thrombospondin (TS) type I motif, making it structurally unique within its family .

What is mAb AA4 and what cellular targets does it bind to?

Monoclonal antibody AA4 (mAb AA4) binds specifically to novel derivatives of ganglioside GD1b found exclusively on mast cells in rat tissues . These gangliosides are located in close proximity to the high-affinity IgE receptor (FcεRI), and some research indicates that binding of mAb AA4 can inhibit FcεRI-mediated histamine release . The binding specificity of mAb AA4 makes it a valuable tool for studying mast cell biology and function in rat models.

What are the primary research applications for ADAMTS4 antibody?

ADAMTS4 antibody is primarily intended for assay development on various platforms requiring antibody pairs . Its main application is in the development of sandwich ELISAs for detecting and quantifying ADAMTS4 protein. The antibody can be used to coat microplates for capturing ADAMTS4 protein, and when paired with a biotinylated detection antibody, it creates a sensitive detection system that can be visualized using streptavidin-HRP and appropriate substrates . This system allows researchers to generate standard curves for quantitative analysis of ADAMTS4 in experimental samples.

How does mAb AA4 affect mast cell morphology and what are the methodological implications?

When mAb AA4 binds to mast cells, it induces dramatic morphological changes within 1 minute of binding. Cells lose their normal spindle-shaped appearance, increase membrane ruffling, and spread over the culture surface . These changes are accompanied by a redistribution of cytoskeletal elements, including actin, tubulin, and vimentin, though only actin associates with the membrane ruffles . When designing experiments, researchers should be aware that these morphological changes could influence other cellular processes being studied simultaneously. Control experiments should be performed at 4°C or in the absence of extracellular calcium, conditions under which these morphological changes do not occur .

What are the implications of ADAMTS4 in osteoarthritis research and how can the antibody contribute to this field?

ADAMTS4 has been implicated in osteoarthritis due to its ability to cleave aggrecan, a major structural component of cartilage. Interestingly, studies with ADAMTS4 knockout mice did not show significant protective effects in osteoarthritis models, suggesting complex mechanisms at play . When designing osteoarthritis research using ADAMTS4 antibody, investigators should consider:

• Comparing ADAMTS4 expression levels between normal and osteoarthritic tissue

• Evaluating the effects of inflammatory mediators like Interleukin-1 on ADAMTS4 expression

• Studying the interaction between ADAMTS4 and its natural inhibitor TIMP-3

• Developing combinatorial approaches that target multiple aggrecanases simultaneously

Such approaches may provide more complete insights than focusing on ADAMTS4 alone, given the complex nature of osteoarthritis pathogenesis.

How can mAb AA4 be used to investigate the relationship between gangliosides and FcεRI function?

Given that mAb AA4 binds to gangliosides located in proximity to FcεRI and inhibits FcεRI-mediated histamine release, it serves as an excellent tool for studying the modulatory role of gangliosides in receptor function. Researchers can use mAb AA4 to:

• Investigate the spatial organization of gangliosides and FcεRI in the plasma membrane

• Examine how ganglioside-antibody interactions affect receptor clustering and activation

• Study the role of gangliosides in regulating signal transduction downstream of FcεRI

• Explore the ganglioside-cytoskeleton interactions that influence receptor mobility and function

While mAb AA4 does not directly trigger histamine release, it enhances calcium ionophore A23187-mediated release, suggesting a modulatory rather than direct role in secretory processes .

What are the optimal conditions for using ADAMTS4 antibody in ELISA development?

When developing a sandwich ELISA using ADAMTS4 antibody, researchers should optimize several parameters:

• Coating concentration: Determine the optimal concentration of capture antibody (MAB43071) for coating microplates

• Sample dilution: Establish appropriate dilution series for recombinant ADAMTS4 standards

• Detection antibody concentration: Optimize the concentration of biotinylated detection antibody (MAB4307)

• Incubation conditions: Determine optimal temperatures and duration for each step

• Washing protocol: Establish effective washing procedures to minimize background

• Substrate development: Optimize the timing for enzymatic reaction before adding stop solution

According to the available data, recombinant Human ADAMTS4 protein can be effectively detected using a system where Mouse Anti-Human ADAMTS4 Monoclonal Antibody (MAB43071) is used for capture and biotinylated Mouse Anti-Human ADAMTS4 Monoclonal Antibody (MAB4307) is used for detection .

What controls should be included when using mAb AA4 in experimental studies?

When conducting experiments with mAb AA4, researchers should include several controls to ensure valid interpretations:

• Isotype controls: Include nonspecific IgG of the same isotype to confirm specificity

• Temperature controls: Compare results at physiological temperature (37°C) versus 4°C

• Calcium dependency controls: Perform parallel experiments in the presence and absence of extracellular calcium

• Alternative antibody controls: Include other anti-cell surface antibodies to confirm that observed effects are specific to mAb AA4

• Positive controls: Include established mast cell activators like antigen-IgE complexes for comparison

The search results indicate that nonspecific IgG, IgE, or four other anti-cell surface antibodies did not induce the same changes associated with mAb AA4 binding, emphasizing the importance of proper controls .

How should ADAMTS4 antibody be stored and handled to maintain its activity?

Proper storage and handling of ADAMTS4 antibody is critical for maintaining its activity:

• Long-term storage: Store at -20°C to -70°C for up to 12 months from date of receipt

• After reconstitution: Store at 2-8°C under sterile conditions for up to 1 month

• Extended storage after reconstitution: Store at -20°C to -70°C under sterile conditions for up to 6 months

• Avoid repeated freeze-thaw cycles by aliquoting the antibody before freezing

• Use a manual defrost freezer to prevent damage from automatic defrost cycles

These recommendations ensure maximum antibody stability and performance in experimental applications.

How do the effects of mAb AA4 compare to those observed with FcεRI activation?

The morphological and biochemical effects produced by mAb AA4 binding are similar to those seen following activation of the cell through the IgE receptor (FcεRI), but there are important differences:

• Signal intensity: mAb AA4 induces calcium flux, phosphatidylinositol breakdown, and PKC activation, but the extent of these changes is less than that observed with FcεRI stimulation

• Histamine release: Unlike FcεRI activation, mAb AA4 binding does not directly stimulate histamine release, though it enhances calcium ionophore-mediated release

• Cytoskeletal reorganization: Both mAb AA4 and FcεRI activation induce cytoskeletal changes, but there may be differences in the specific patterns and extent of reorganization

• Temporal dynamics: Researchers should carefully compare the timing of events triggered by mAb AA4 versus FcεRI activation

These similarities and differences suggest that the gangliosides which bind mAb AA4 may function in modulating secretory events, possibly through interactions with the cytoskeleton and calcium signaling machinery .

What factors might affect the reliability of ADAMTS4 detection in experimental samples?

Several factors can influence the reliability of ADAMTS4 detection in experimental samples:

• Post-translational modifications: ADAMTS4 undergoes processing from its precursor form, which can affect antibody recognition

• Protein interactions: Binding to substrates or inhibitors might mask antibody epitopes

• Sample preparation: Improper handling can lead to protein degradation or aggregation

• Cross-reactivity: The amino acid sequence of human ADAMTS4 is 100% identical to chimpanzee, 97% to dog, and 94% to mouse/rat/bovine ADAMTS4, which may affect species-specific detection

• Matrix effects: Components in complex biological samples might interfere with antibody binding

Researchers should validate antibody performance in their specific experimental context and include appropriate positive and negative controls to account for these factors.