PLA2G7 Antibody Pair

What are the optimal combinations of PLA2G7/Lp-PLA2 antibodies for developing a sensitive sandwich ELISA?

Several validated antibody pairs have demonstrated high sensitivity and specificity for PLA2G7/Lp-PLA2 detection in sandwich ELISA formats. The most effective combinations include:

• MAb combination A: DMAB-SP01 (capture) and DMAB-SP02 (detection)

• MAb combination C: DMAB-SP02 (capture) and DMAB-SP01 (detection)

R&D Systems offers validated antibody pairs such as:

• Mouse Anti-Human PLA2G7/PAF-AH/Lp-PLA2 Monoclonal Antibody (Catalog # MAB51061) as capture antibody

• Mouse Anti-Human PLA2G7/PAF-AH/Lp-PLA2 Monoclonal Antibody (Catalog # MAB5106) as detection antibody

These combinations have demonstrated linear detection ranges from 0.13 to 1000 ng/mL in ELISA assays , with intra-assay and inter-assay precision coefficients of variation typically between 4.7-6.7% .

How does the expression and localization of PLA2G7/Lp-PLA2 vary across tissues and cell types?

PLA2G7/Lp-PLA2 demonstrates distinct expression patterns across various tissues and cell types:

• Cellular sources: Predominantly produced by mature macrophages and activated platelets

• Cancer cells: Expression observed in renal cancer cell lines (ACHN and 786-O), with little or no expression in other renal cancer lines (e.g., 769-P)

• Subcellular localization: Immunofluorescence experiments show that PLA2G7/Lp-PLA2 is distributed throughout renal cancer cells, with predominant localization in the cytoplasm

In plasma, PLA2G7/Lp-PLA2 is found primarily bound to lipoproteins in the following proportions:

• 80% bound to LDL

• 15-20% bound to HDL

• Remainder bound to VLDL

This distribution profile is important when developing detection methods for circulating PLA2G7/Lp-PLA2, as it may influence antibody accessibility to the target protein.

What are the critical factors for validating PLA2G7/Lp-PLA2 antibody specificity?

Thorough validation of PLA2G7/Lp-PLA2 antibodies is essential for reliable experimental results. Key considerations include:

• Cross-reactivity assessment: Verify species-specific reactivity against recombinant PLA2G7/Lp-PLA2. Many commercial antibodies show reactivity to human, mouse, rat, and pig PLA2G7/Lp-PLA2

• Epitope mapping: Confirm the specific protein region recognized by the antibody. Commercial antibodies target distinct regions:

  • AA 22-441 (full-length mature protein)

  • AA 49-441

  • AA 200-228

  • AA 301-441

• Multiple detection methods: Validate specificity using complementary techniques:

  • Western blotting to confirm single-band specificity at approximately 45 kDa

  • Immunofluorescence to verify subcellular localization

  • Knockdown validation to demonstrate signal reduction

• Functional validation: For antibodies targeting functional domains, confirm impact on enzymatic activity using PLA2G7 inhibitors like darapladib for comparison

How do PLA2G7 gene polymorphisms affect antibody binding and assay performance?

PLA2G7 gene polymorphisms can significantly impact antibody binding efficiency through several mechanisms:

Several common polymorphisms in the PLA2G7 gene cause amino acid substitutions that may alter protein conformation:

• rs1051931 (c.1136T>C p.Val379Ala) - highly prevalent in Han Chinese CHD patients (94.62%)

• rs1805017 (c.275G>A p.Arg92His)

• rs1805018 (c.593T>C p.Ile198Thr)

• rs76863441 (c.835G>T p.Val279Phe)

Different polymorphisms show varying correlations with Lp-PLA2 activity:

• rs1805017 (Arg92His) shows positive correlation with serum Lp-PLA2 activity

• rs1051931 (Val379Ala) shows negative correlation with serum Lp-PLA2 activity

• Strong linkage disequilibrium exists between rs1805018 (Ile198Thr) and rs76863441 (Val279Phe), both related to lower Lp-PLA2 activity

When developing immunoassays for populations with known polymorphism prevalence, researchers should consider using antibodies targeting conserved regions unaffected by common variants or develop detection methods that account for these genetic variations.

What methodological approaches can resolve discrepancies between PLA2G7/Lp-PLA2 protein levels and enzymatic activity?

Discrepancies between immunologically detected PLA2G7/Lp-PLA2 levels and enzymatic activity require systematic investigation using multiple approaches:

Parallel assessment methods:

• Implement both antibody-based detection (ELISA, Western blot) and activity-based assays simultaneously

• Use recombinant Lp-PLA2 standards with known activity levels as calibrators

Genetic analysis:

• Screen for PLA2G7 polymorphisms known to affect activity but not protein expression

• Common variants to consider include rs1051931 (Val379Ala) and rs1805017 (Arg92His)

Oxidative modification assessment:

• Recent research has shown that oxidation of Met117 and nitration of Tyr307/Tyr335 can reduce enzymatic activity without affecting antibody detection

• Oxidation of Met117 induces enhanced flexibility and decreased compactness in the oxidized state, potentially affecting catalytic function but not antibody recognition

• Nitration of Tyr307 and Tyr335 leads to disorientation of the catalytic triad, impacting activity

By implementing these approaches, researchers can better understand the biological significance of discrepancies between protein levels and activity in experimental and clinical samples.

What experimental strategies can evaluate the role of PLA2G7/Lp-PLA2 in ferroptosis and oxidative stress?

Recent research has identified PLA2G7/Lp-PLA2 as a key player in ferroptosis and oxidative stress regulation. The following strategies can effectively evaluate its role:

Genetic manipulation approaches:

• Overexpression systems: Transfect cells with PLA2G7 expression vectors to assess protective effects against ferroptosis inducers

• RNA interference: Use siRNA or shRNA to knockdown PLA2G7 expression

• CRISPR-Cas9: Generate PLA2G7 knockout cell lines for complete gene elimination

Ferroptosis induction models:

• Punicic acid (PunA) treatment: Demonstrated to induce ferroptosis in prostate cancer cells

• Combined inhibition of GPX4 and PLA2G7 strongly increases lipid peroxidation

Functional readouts:

• Lipid peroxidation assessment: PLA2G7 overexpression has been shown to decrease lipid peroxidation levels, suggesting it hydrolyzes hydroperoxide-containing phospholipids to prevent ferroptosis

• Cell viability assays: Treatment with PLA2G7 inhibitors like darapladib significantly decreases viability of PLA2G7-expressing cancer cells like 786-O and ACHN renal cancer cells

Research has demonstrated that PLA2G7 acts complementary to GPX4 to protect cells from ferroptosis by eliminating oxidized phospholipids from cell membranes . This functional relationship provides insights into potential therapeutic approaches targeting these pathways.

How can researchers distinguish between PLA2G7/Lp-PLA2 associated with different lipoprotein fractions?

Differentiating PLA2G7/Lp-PLA2 associated with different lipoprotein fractions requires specialized techniques that separate and analyze these distinct pools:

Lipoprotein fractionation methods:

• Ultracentrifugation: Sequential density gradient ultracentrifugation to isolate LDL (containing ~80% of Lp-PLA2), HDL (containing 15-20%), and VLDL (containing the remainder)

• Size exclusion chromatography: Separation based on particle size differences

• Immunoprecipitation: Using antibodies against specific lipoproteins (anti-ApoB for LDL, anti-ApoA-I for HDL)

Post-fractionation analysis:

• ELISA: Quantify Lp-PLA2 mass in each lipoprotein fraction using validated antibody pairs

• Activity assays: Measure enzymatic activity in each fraction using PAF or oxidized phospholipid substrates

• Western blotting: Detect Lp-PLA2 in each fraction while simultaneously confirming lipoprotein identity

Understanding the lipoprotein-specific distribution of PLA2G7/Lp-PLA2 is crucial for cardiovascular research, as variations in this distribution have been associated with coronary heart disease risk .

What protocols can enhance detection sensitivity for PLA2G7/Lp-PLA2 in samples with low expression?

Enhancing detection sensitivity for PLA2G7/Lp-PLA2 in low-abundance samples requires optimized protocols:

Sample preparation optimization:

• Lipoprotein isolation: Enrich for LDL fractions where approximately 80% of PLA2G7/Lp-PLA2 is found

• Protease inhibitor cocktails: Prevent degradation during processing

• Standardized freeze-thaw protocols: Minimize activity loss during sample handling

ELISA sensitivity enhancement:

• Signal amplification systems: Employ polymer-HRP conjugates or tyramide signal amplification

• Extended substrate incubation: Optimize development time to maximize signal-to-noise ratio

• Optimized antibody pairs: Select validated high-affinity combinations

Quality control measures:

• Standard curve optimization: Use the appropriate range for low-abundance samples (0.13 to 1000 ng/mL)

• Inter-assay calibration: Include consistent control samples across multiple assays

• Batch-to-batch consistency: Assess reproducibility across different antibody lots

Implementing these strategies can significantly improve detection limits, enabling more reliable quantification in samples with low PLA2G7/Lp-PLA2 expression.

How do post-translational modifications affect PLA2G7/Lp-PLA2 recognition by antibodies?

Post-translational modifications (PTMs) of PLA2G7/Lp-PLA2 can significantly impact antibody recognition, affecting research and clinical assay results:

Oxidative modifications:

• Methionine oxidation: Met117 oxidation increases protein flexibility and decreases compactness, potentially altering conformational epitopes

• Tyrosine nitration: Nitration of Tyr307 and Tyr335 leads to disorientation of the catalytic triad and reduced molecular interactions

Glycosylation:

• PLA2G7/Lp-PLA2 is N-glycosylated, which can mask epitopes or create steric hindrance

• Glycosylation profiles may vary between different biological sources

Strategies to address PTM-related antibody binding issues:

• Epitope mapping: Characterize antibody binding sites in relation to known PTM sites

• Deglycosylation experiments: Compare antibody binding to native and enzymatically deglycosylated protein

• Multiple antibody approach: Use antibodies targeting different epitopes to ensure detection regardless of PTM status

Recent molecular dynamics simulation studies have elucidated the effect of oxidative modifications on PLA2G7/Lp-PLA2 structure, showing that these modifications can affect substrate binding and catalytic activity without necessarily impacting antibody recognition . This understanding is crucial for developing robust detection methods and interpreting experimental results accurately.

How can PLA2G7/Lp-PLA2 antibody pairs be utilized in cancer biomarker research?

Recent studies have highlighted PLA2G7/Lp-PLA2's potential as a cancer biomarker and therapeutic target:

Diagnostic applications:

• PLA2G7 has demonstrated excellent diagnostic accuracy in various cancers, with notable AUC values for hepatocellular carcinoma, renal cancer, and other malignancies

• Antibody pairs can be used to develop sensitive immunoassays for detecting PLA2G7/Lp-PLA2 in patient samples

Prognostic significance:

Therapeutic targeting potential:

• Inhibition of PLA2G7 gene expression significantly decreases the viability of renal cancer cell lines (786-O and ACHN)

• PLA2G7 has been identified as promoting hepatocellular carcinoma through the STAT1/PD-L1 axis

Immunohistochemical analysis using validated PLA2G7 antibodies can help stratify patients and identify those who might benefit from targeted therapies, highlighting the translational potential of PLA2G7 antibody research beyond basic laboratory applications.