petA Antibody

What is PETA-3/CD151 and what role does it play in cellular processes?

PETA-3/CD151 is a member of the tetraspanin family of proteins that has been identified as a positive effector of metastasis. This cell surface protein plays a critical role in mediating tumor cell migration but does not appear to significantly affect cell adhesion to various purified matrix proteins. Research has demonstrated that PETA-3/CD151 is specifically involved in early steps of metastatic foci formation, making it a valuable target for cancer research .

The protein has a molecular weight of approximately 29,000 daltons in its glycosylated form, with a nonglycosylated form appearing at approximately 25,000 daltons. This is slightly smaller than its predicted mass of 27.8 kDa, likely due to the hydrophobic nature of the protein and/or the presence of disulfide bonds resulting from its 15 cysteine residues .

How are antibodies against PETA-3/CD151 used in metastasis research?

Monoclonal antibodies targeting PETA-3/CD151, including mAb 50-6 and mAb 1A5, have demonstrated significant capabilities in inhibiting both spontaneous and experimental metastasis in cancer models. These antibodies do not affect primary tumor growth or in vitro cell proliferation, making them valuable tools for specifically studying metastatic processes .

In experimental models using HEp-3 human epidermoid carcinoma cells, these antibodies have shown impressive inhibition of metastasis:

• mAb 50-6 and mAb 1A5 inhibited HEp-3 migration by approximately 45% and 44%, respectively, compared to controls (P = 0.034)

• Previous studies with mAb 1A5 demonstrated inhibition of spontaneous HEp-3 metastasis by approximately 90%

These antibodies appear to specifically block an early step in the formation of metastatic foci, providing researchers with valuable tools to study the mechanisms of metastasis.

What experimental methods are commonly used to detect and quantify PETA-3/CD151 expression?

Several established methods exist for detecting and quantifying PETA-3/CD151 expression in experimental settings:

Whole-Cell ELISA:

• Seed cells (2.0 × 10^4/0.1 ml) in 96-well culture plates and culture for 36 hours

• Wash with PBS, fix with 0.25% glutaraldehyde for 5 minutes at room temperature

• Quench with 1.0 M glycine (pH 8.0) for 2 hours

• Block overnight at 4°C

• Incubate with primary antibody (1 μg/ml) for 2 hours

• Detect with horseradish peroxidase-conjugated secondary antibody

• Develop with o-phenylene diamine substrate (0.34 mg/ml, 0.1 M sodium citrate, pH 4.5, 0.012% H2O2)

• Read absorbance at 405 nm

Western Blotting:
This technique can distinguish between the glycosylated (29 kDa) and nonglycosylated (25 kDa) forms of PETA-3/CD151. Treatment with tunicamycin or N-glycanase generates the nonglycosylated form that can be detected with specific antibodies .

What are the key differences between animal-derived and recombinant antibodies for research applications?

According to recent scientific studies, almost one-third of antibodies produced using animals don't effectively bind to their specific target molecules, potentially contributing to the high failure rate in scientific studies .

How does PETA-3/CD151 mechanistically influence cancer cell migration and metastasis?

PETA-3/CD151's role in promoting metastasis appears to be primarily mediated through its effects on cell migration rather than proliferation or adhesion. Evidence supporting this mechanism includes:

• Migration vs. Adhesion Distinction: Studies show that PETA-3/CD151 expression levels correlate with migratory capability but not with adhesion properties. HeLa cells transfected with and overexpressing PETA-3/CD151 demonstrated enhanced migration compared to control transfectants expressing minimal CD151 .

• Antibody Inhibition Specificity: Both mAb 50-6 and mAb 1A5 antibodies targeting PETA-3/CD151 specifically inhibit migration without affecting:

  • Primary tumor growth on the chorioallantoic membrane

  • Tumor cell growth in vitro

  • Cell adhesion to various purified matrix proteins

• Tetraspanin Functionality: As part of the tetraspanin family, PETA-3/CD151 likely functions as a "molecular facilitator" that influences cell migration through interactions with integrins, consistent with known tetraspanin functions .

These findings collectively suggest that PETA-3/CD151 promotes metastasis by specifically enhancing the migratory capabilities of cancer cells, making it the first tetraspanin family member identified as a positive effector of metastasis.

What are the current challenges in antibody structure prediction for research?

Accurately predicting antibody structures faces several significant challenges:

• Underrepresentation in Databases: Antibodies constitute only approximately 4% of structures in the Protein Data Bank (PDB), with only around 8,000 antibody structures out of more than 200,000 total protein structures. This underrepresentation limits the training data available for machine learning models .

• Conformational Flexibility: Antibody loops are highly flexible, making their structure prediction particularly challenging. Current tools like AlphaFold and RoseTTAFold provide rigid structure outputs that may not capture this flexibility adequately .

• Developability Parameter Inconsistency: Even when prediction tools produce structures that closely resemble ground truth structures, developability measurements often vary across different structure prediction tools, creating consistency challenges .

• Dynamic vs. Static Structures: Developability parameters measured on rigid predicted structures may not align with those measured on experimental structures. Implementing molecular dynamics (MD) simulations is often necessary to achieve higher agreement with experimental measurements .

These challenges highlight why antibody structure prediction remains more difficult than prediction for other protein types, despite recent advancements in the field.

How can researchers optimize experimental design when studying PETA-3/CD151 in metastasis models?

To maximize the validity and impact of PETA-3/CD151 metastasis research, consider these research design optimizations:

• Select Appropriate Metastasis Models:

  • Spontaneous metastasis models allow observation of the complete metastatic cascade

  • Experimental metastasis models isolate specific steps in the metastatic process

  • The chicken embryo metastasis assay has proven effective for studying PETA-3/CD151 functions

• Control for Growth vs. Metastasis Effects:

  • Always assess both primary tumor growth and metastatic behavior separately

  • Include controls to distinguish between effects on proliferation versus migration

  • Monitor in vitro growth to rule out general cytotoxicity of interventions

• Expression Level Validation:

  • Use whole-cell ELISA to quantify PETA-3/CD151 expression levels across different cell lines

  • Compare expression with functional outcomes to establish dose-response relationships

  • Consider both glycosylated and nonglycosylated forms in your analysis

• Functional Validation Through Transfection:

  • Perform gain-of-function studies by transfecting PETA-3/CD151 into low-expressing cell lines

  • Validate antibody specificity using transfected cells as positive controls

  • Establish clear functional correlations between expression levels and migratory behavior

What methodologies are being developed to replace animal-derived antibodies in research?

Several advanced methodologies are being developed and refined to replace traditional animal-derived antibodies:

• Recombinant Antibody Technology:

  • Involves determining the DNA sequence of existing animal-derived antibodies

  • Allows production in cell culture systems without further animal use

  • Provides consistent, reproducible antibodies across batches

  • Offers long-term, scalable supply for ongoing research needs

• Aptamer Development:

  • Short segments of DNA or RNA that bind to specific molecular targets

  • Can be designed to have high specificity similar to antibodies

  • Produced through entirely synthetic processes without animal use

  • Often more stable than protein-based antibodies in various experimental conditions

• Implementation Strategy Example:
  The PETA Science Consortium International project with the USDA demonstrates a practical approach:

  • Identified an existing animal-derived antibody used in leptospirosis vaccine testing

  • Funded biotech company Absolute Antibody to determine its DNA sequence

  • Produced a recombinant version with identical binding properties

  • Validated the recombinant antibody in the same in vitro potency test

  • Created a sustainable, animal-free antibody supply for ongoing testing needs

These methodologies not only address ethical concerns but also improve scientific reliability by reducing batch variation and enhancing specificity.

How can molecular dynamics simulations improve antibody developability assessment?

Molecular dynamics (MD) simulations offer significant advantages for antibody developability assessment:

• Beyond Static Structures: While tools like AlphaFold provide static antibody structures, MD simulations capture the crucial dynamic behavior of antibodies, particularly in flexible loop regions .

• Improved Measurement Accuracy: Studies have demonstrated that developability parameters measured using MD simulations show higher agreement with experimental measurements compared to those calculated from rigid structures alone .

• Implementation Methodology:

  • Begin with a predicted antibody structure from tools like AlphaFold

  • Place the structure in a solvated environment that mimics physiological conditions

  • Run simulations over nanosecond to microsecond timescales

  • Analyze the resulting conformational ensemble rather than a single structure

  • Calculate developability parameters across the ensemble for more robust predictions

• Parameter Stability Assessment: MD simulations allow researchers to assess how stable developability parameters remain across different conformational states, providing confidence metrics for predictions .

By incorporating MD simulations into antibody developability workflows, researchers can achieve more accurate predictions that better align with experimental results, potentially reducing development failures and accelerating research timelines.