AHL3 Antibody
Description
Development and Affinity Profiling
AHL3 antibodies were developed using hapten-carrier conjugates (e.g., RS3-KLH) to immunize mice. Hybridoma screening identified clones with specificity for 3-oxo-C6-AHL :
Affinity Characteristics of RS3-Derived mAbs :
| Target AHL | Median K<sub>d</sub> | Cross-Reactivity |
|---|---|---|
| 3-oxo-C6-HSL | 10–50 μM | Minimal (<5%) with long-chain AHLs |
| 3-oxo-C12-HSL | >100 μM | None |
These mAbs showed no significant binding to structurally distinct AHLs (e.g., C4-HSL), highlighting their specificity for short-chain 3-oxo variants .
Functional Efficacy in Bacterial Inhibition
AHL3 antibodies disrupt QS in Pseudomonas aeruginosa, reducing virulence factor production:
Experimental Outcomes :
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Pyocyanin suppression: 40–60% reduction in toxin levels at 50 μM AHL3 antibody concentrations.
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Biofilm inhibition: Limited efficacy compared to long-chain-targeting mAbs (e.g., RS2-1G9) .
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Cytokine modulation: Indirect anti-inflammatory effects observed via AHL neutralization .
Challenges and Limitations
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Low affinity: K<sub>d</sub> values in the micromolar range limit therapeutic utility .
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Short half-life: IgG3 subclass antibodies exhibit a 7-day serum half-life, necessitating frequent dosing .
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Narrow spectrum: Specificity for short-chain AHLs restricts applicability to pathogens using these signaling molecules .
Comparative Analysis of AHL-Targeting Antibodies
| Parameter | AHL3 Antibody | RS2-1G9 (Long-Chain) |
|---|---|---|
| Target AHL | 3-oxo-C6-HSL | 3-oxo-C12-HSL |
| Affinity (K<sub>d</sub>) | 10–50 μM | 150 nM |
| QS Inhibition | Moderate | High |
| Therapeutic Potential | Limited | High (validated in vivo) |
Future Directions
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Cell-to-cell communication mediated by mobile AHL3 and AHL4 is essential for establishing the boundary between the procambium and xylem. PMID: 23335615
Q&A
Here’s a structured FAQ collection for "AHL3 Antibody" in academic research, integrating methodological guidance and evidence from peer-reviewed studies:
Advanced Research Questions
How to resolve contradictory data arising from AHL3 cross-reactivity in multi-species studies?
Scenario: Inconsistent staining in co-culture models with Syrian hamster cells. Methodological solution:
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Pre-adsorb AHL3 with Syrian hamster serum proteins to eliminate cross-reactivity .
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Validate using dual-labeling experiments with species-specific Fab fragments .
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Confirm via mass spectrometry if off-target binding persists .
What optimization strategies improve AHL3 performance in multiplex immunoassays?
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Titration matrix: Test AHL3 at 0.1–2.0 µg/mL against dilutions of primary antibodies .
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Buffer compatibility: Use low-surfactant PBS (pH 7.4) to prevent aggregation .
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Signal enhancement: Combine with streptavidin-biotin amplification for low-abundance targets .
How to validate AHL3’s role in cytokine modulation studies (e.g., IL-10/TNF-α assays)?
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In macrophages: Treat RAW264.7 cells with 25–100 µM 3-oxo-C12:2-HSL (AHL3’s target context) and measure IL-10/TNF-α via qPCR and ELISA .
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Controls: Include LPS-stimulated cells without AHL3 to baseline cytokine levels .
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Dose-response analysis: Ensure linear regression fits (R² > 0.95) for TNF-α suppression .
Data Contradiction Analysis
How to interpret conflicting results in AHL3-mediated NF-κB pathway studies?
Case: AHL3 reduces TNF-α in PBMCs but exacerbates inflammation in alveolar macrophages . Resolution framework:
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Cell-type specificity: Test AHL3 in both PBMCs and MH-S alveolar macrophages under identical LPS conditions .
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Pathway mapping: Compare transcriptomes (RNA-seq) for NF-κB regulators like IκBα .
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Dose calibration: Re-evaluate thresholds (e.g., 50 µM vs. 100 µM AHL3) .
