AOP2 Antibody

What is AOP2 and why is it relevant to immunological research?

AOP2 (Antioxidant Protein 2) is a heavily O-glycosylated protein that forms important immunological complexes in human plasma. It has a molecular weight of approximately 98 kDa and contains about 51% carbohydrate content . AOP2 is significant in immunological research because it forms triplet immune complexes with anti-α-galactoside (anti-Gal) and anti-β-glucoside (ABG) antibodies in plasma, potentially modulating immune responses . These complexes consist of antibody-AOP2-albumin triplets that have important biological functions in recognizing serine- and threonine-rich peptide sequences (STPS) as surrogate antigens .
Methodologically, researchers investigating AOP2 should employ affinity chromatography using appropriate matrices such as guar galactomannan gel for anti-Gal and cellulose for ABG to isolate these triplet complexes .

How do AOP2 antibodies differ from other common research antibodies?

AOP2 antibodies differ from conventional research antibodies in that they recognize a protein involved in natural immune complex formation. Unlike antibodies against standard protein targets that typically recognize folded epitopes, anti-AOP2 antibodies must be validated against highly glycosylated forms of the protein to ensure specificity .
When working with AOP2 antibodies, researchers should implement the following methodological considerations:

• Confirm specificity against both glycosylated and deglycosylated forms of AOP2

• Use parallel validation with jacalin (O-glycan-specific lectin) to verify glycoprotein identity

• Employ alkaline PAGE separation techniques for analyzing triplet complexes containing AOP2

What are the recommended storage and handling conditions for AOP2 antibodies?

Based on standard antibody practices and the glycoprotein nature of AOP2, optimal storage and handling conditions include:

What are the validated applications for AOP2 antibodies in research?

AOP2 antibodies have been validated for several experimental applications, primarily focusing on its role in immune complex formation and antioxidant functions:

How should researchers optimize immunohistochemical protocols for AOP2 detection?

For optimal immunohistochemical detection of AOP2:

• Fixation: Use 4% paraformaldehyde; avoid methanol fixation which can disrupt glycoprotein epitopes

• Antigen retrieval: Implement gentle heat-mediated retrieval (80-90°C) in citrate buffer (pH 6.0)

• Blocking: Use 5-10% serum with 0.1% Triton X-100 to reduce background

• Primary antibody incubation: Optimize concentrations between 1:100-1:1000; incubate overnight at 4°C

• Detection system: Use fluorophore-conjugated secondary antibodies for co-localization studies

• Controls: Include both positive controls (tissues known to express AOP2) and negative controls (omitting primary antibody)
  When examining eye tissues, where AOP2 has demonstrated importance in lens epithelial cell protection, researchers should be particularly careful with fixation protocols as inappropriate fixation can affect glycoprotein antigenicity .

What is the role of AOP2 in lens epithelial cell protection, and how can this be studied experimentally?

AOP2 has been implicated in protecting lens epithelial cells (LECs) through its antioxidant properties. Studies have shown that LEDGF (Lens Epithelium-Derived Growth Factor) regulates AOP2 expression, and this pathway may prevent the progression of age-related cataracts .
To experimentally investigate this protective role:

• Cell culture models: Use lens epithelial cell lines exposed to oxidative stressors (H₂O₂, UV radiation)

• Gene expression analysis: Employ RT-PCR to measure AOP2 mRNA levels under stress conditions

• Protein quantification: Implement Western blotting with anti-AOP2 antibodies to assess protein expression

• Functional assays: Measure cell viability and ROS levels in cells with normal vs. knocked-down AOP2 expression

• Animal models: Examine AOP2 expression in cataract models such as the Shumiya cataract rat (SCR)
  Research findings indicate that cataractous lenses from 11 and 13-week-old SCR rats showed significant downregulation of AOP2 protein and mRNA levels, supporting its role in cataract prevention .

How do AOP2-containing immune complexes interact with platelet surface glycoproteins?

Recent research has revealed complex interactions between AOP2-containing immune complexes and platelet surface glycoproteins, with significant implications for platelet function:
Upon treatment with α-galactosides and β-glucosides, normal platelets release triplet immune complexes identical to those found in plasma. These complexes contain anti-Gal/ABG antibodies, AOP2, and albumin . The interaction occurs through:

• Recognition of serine- and threonine-rich peptide sequences (STPS) on platelet surface O-glycoproteins by anti-Gal/ABG antibodies

• Binding particularly to GPIIb/IIIa (fibrinogen receptor), with the IIb subunit being heavily O-glycosylated

• Formation of a protective "shield" that prevents aberrant platelet activation
  Experimentally, researchers can study these interactions by:

• Treating platelets with specific sugars (methyl-α-galactoside, cellobiose) to release triplet complexes

• Analyzing released complexes via ELISA using antibodies against component parts

• Examining platelet aggregation behavior before and after complex removal

• Using jacalin (O-glycan-binding lectin) to block STPS recognition sites
  Of particular clinical relevance, high glucose concentrations (as seen in diabetes) can remove these protective complexes from platelets, potentially explaining increased platelet aggregation in diabetic patients .

What is the relationship between AOP2, LEDGF/p75, and autoantibodies in systemic autoimmune diseases?

The relationship between AOP2, LEDGF/p75 (Lens Epithelium-Derived Growth Factor p75), and autoantibodies presents a complex area of immunological research:
LEDGF/p75 (also known as DFS70) is targeted by autoantibodies that are commonly observed in clinical laboratory referrals but have a uniquely low frequency in patients with systemic autoimmune rheumatic diseases (SARD) . This protein regulates expression of stress survival genes, including AOP2 .
Key research findings include:

• Anti-DFS70/LEDGF autoantibodies predominantly occur in apparently healthy individuals or non-SARD conditions

• They target the C-terminal autoepitope (aa 347-429) that overlaps with the integrase binding domain (IBD)

• These autoantibodies might serve as biomarkers to exclude SARD diagnosis

• LEDGF/p75 regulates AOP2 expression as part of cellular stress response
  For researchers investigating this relationship, methodological approaches should include:

• Testing for co-expression of LEDGF/p75 and AOP2 in stressed cells

• Analyzing autoantibody binding to both proteins in patient samples

• Examining downstream effects of anti-LEDGF/p75 autoantibodies on AOP2 expression

• Comparing autoantibody profiles between healthy individuals, SARD, and non-SARD inflammatory conditions

How can researchers distinguish between specific and non-specific binding when using AOP2 antibodies?

Distinguishing between specific and non-specific binding is crucial when working with AOP2 antibodies due to the complex nature of AOP2-containing immune complexes. Researchers should implement the following validation approaches:

What are common sources of experimental variability when working with AOP2 antibodies?

When working with AOP2 antibodies, researchers frequently encounter several sources of experimental variability:

• Glycosylation heterogeneity: AOP2 is heavily O-glycosylated (51% carbohydrate), leading to variations in antibody recognition

• Complex formation dynamics: AOP2 exists in triplet complexes that may partially dissociate during experimental procedures

• Donor-dependent variations: Natural antibody levels against triplet components vary between individuals

• Sample handling effects: Freeze-thaw cycles can affect complex integrity

• Buffer composition impacts: Ionic strength and pH significantly affect antibody-antigen interactions
  To minimize these variations, researchers should standardize protocols by:

• Using pooled samples when possible

• Maintaining consistent buffer compositions across experiments

• Implementing stringent quality control for antibody lots

• Including internal controls in each experiment

• Documenting detailed methods to facilitate reproducibility

How should researchers interpret conflicting results between different antibody-based detection methods for AOP2?

When faced with conflicting results between different detection methods:

• Assess epitope accessibility:

  • Western blotting detects denatured epitopes

  • ELISA may detect conformational epitopes

  • Immunohistochemistry is affected by fixation method

• Consider complex integrity:

  • Some methods may disrupt AOP2-containing triplet complexes

  • Alkaline electrophoresis separates triplet components

  • Gentle methods like native PAGE may preserve complexes

• Evaluate detection sensitivity:

  • Different techniques have varying detection thresholds

  • Amplification steps in each method vary in efficiency

  • Signal-to-noise ratios differ between methods

• Implement resolution strategy:

  • Prioritize results from methods with appropriate controls

  • Consider orthogonal validation with non-antibody methods

  • Use knockout/knockdown validation when possible

  • Consult literature for similar discrepancies

What is the significance of AOP2 in the context of amyloid β binding, and how can this interaction be studied?

Recent research has uncovered an important relationship between AOP2 and amyloid β (Aβ-42) binding, with potential implications for Alzheimer's disease research:
Aβ-42 has been shown to bind specifically to the serine- and threonine-rich peptide sequences (STPS) of AOP2 in triplet complexes, but not to albumin or the antibodies themselves . This interaction provides a potential mechanistic link between diabetes and increased Alzheimer's disease susceptibility.
To study this interaction, researchers can employ the following methodological approaches:

• Co-immunoprecipitation assays:

  • Use anti-AOP2 antibodies to pull down complexes

  • Probe for Aβ-42 using specific antibodies

  • Confirm specificity with appropriate controls

• Surface Plasmon Resonance (SPR):

  • Immobilize purified AOP2 on sensor chips

  • Measure binding kinetics with different Aβ-42 preparations

  • Determine affinity constants and binding dynamics

• Platelet-based assays:

  • Compare Aβ-42 binding to normal vs. denuded platelets

  • Measure effects of high glucose on Aβ-42 sequestration

  • Quantify Aβ-42 binding through fluorescence microscopy or flow cytometry

• Competitive binding assays:

  • Determine if other STPS-binding molecules compete with Aβ-42

  • Evaluate if Aβ-42 displaces anti-Gal or ABG from AOP2

  • Assess effects of glycosylation on binding affinity
    Research findings indicate that platelets are major carriers of Aβ-42 , and the presence of protective triplet complexes on normal platelets may be part of a defense system against diabetes-driven platelet malfunctions and associated vascular and neurodegenerative complications.

How is AOP2 involved in oxidative stress response pathways in different tissues?

AOP2 has been implicated in oxidative stress response across multiple tissue types, with tissue-specific functions:
In lens epithelial cells, AOP2 plays a critical role in protection against oxidative damage. Research shows that:

• AOP2 expression is regulated by LEDGF (Lens Epithelium-Derived Growth Factor)

• Downregulation of AOP2 correlates with cataract progression in animal models

• AOP2 functions as part of an antioxidant defense mechanism in the eye
  In photoreceptors, AOP2 gene expression is activated during degeneration, suggesting a response to oxidative stress in retinal tissue . Study approaches should include:

• Tissue-specific expression analysis using qRT-PCR and Western blotting

• Functional assays measuring ROS levels in cells with modified AOP2 expression

• ChIP assays to identify transcription factors regulating AOP2 in different tissues

• Comparative analysis of oxidative stress markers and AOP2 levels across tissues
  Research findings suggest that AOP2, along with other genes involved in oxidative stress (Gstm1, Ogg1), may represent a common response mechanism activated during cellular stress in multiple tissues .

What are the structural determinants of AOP2 that enable its diverse binding interactions?

The structural features of AOP2 that facilitate its diverse binding interactions include:

• Heavy O-glycosylation: AOP2 contains approximately 51% carbohydrate content , creating a unique surface topology

• Serine- and threonine-rich peptide sequences (STPS): These regions serve as recognition sites for anti-Gal and ABG antibodies

• Albumin-binding domain: Allows formation of stable complexes with albumin in circulation
  Researchers investigating AOP2 structure should consider:

• X-ray crystallography or cryo-EM studies of purified AOP2

• Mapping of glycosylation sites using mass spectrometry

• Mutagenesis studies to identify critical binding residues

• Computational modeling of AOP2-antibody-albumin triplet complexes
  Understanding the structural basis of these interactions has significant implications for developing targeted interventions for conditions associated with AOP2 dysfunction.

How might understanding AOP2 immune complexes contribute to novel therapeutic approaches for diabetes-related complications?

The discovery that high glucose removes protective AOP2-containing immune complexes from platelets provides important insights for potential therapeutic strategies:

• Platelet dysfunction in diabetes:

  • High glucose (at concentrations typical in diabetes) removes anti-Gal/ABG-AOP2-albumin triplets from platelets

  • Denuded platelets show increased spontaneous and ADP-mediated aggregation

  • This may contribute to increased thrombotic risk in diabetic patients

• Potential therapeutic approaches:

  • Development of synthetic triplet mimetics that resist glucose displacement

  • Engineering modified antibodies that maintain platelet binding in hyperglycemic conditions

  • Targeting the pathway by which glucose disrupts these protective complexes

• Broader implications:

  • Connection to Alzheimer's disease risk through Aβ-42 binding to triplet complexes

  • Potential explanation for vascular complications in diabetes

  • Novel biomarkers for monitoring diabetic complications risk
    Experimental approaches should include:

• In vitro platelet aggregation studies under various glucose conditions

• Animal models of diabetes with modified AOP2 expression

• Clinical studies correlating natural antibody levels with diabetes complications

• Screening of small molecules that stabilize triplet complexes on platelets
  Research findings suggest this could represent a novel defensive system against diabetes-driven platelet malfunctions, which when compromised by high glucose, may contribute to the development of vascular diseases and potentially increased risk of neurodegenerative conditions .