ompF Antibody, FITC conjugated is a polyclonal or monoclonal antibody specifically targeting the outer membrane porin F (OmpF) protein, chemically linked to fluorescein isothiocyanate (FITC). OmpF is a β-barrel porin protein critical for regulating permeability in Gram-negative bacterial membranes, enabling nutrient uptake and antibiotic resistance . FITC conjugation allows visualization of OmpF in techniques like flow cytometry (FC), immunofluorescence (IF), and immunohistochemistry (IHC) .
FITC-conjugated ompF antibodies are pivotal for:
Bacterial Surface Labeling: Detecting OmpF expression in E. coli, Pseudomonas aeruginosa, and Yersinia species .
Flow Cytometry: Quantifying OmpF surface display in genetically engineered bacterial strains (e.g., ER0808) .
Immunofluorescence Microscopy: Localizing OmpF in paraffin-embedded tissues or cultured cells .
Antibiotic Resistance Studies: Investigating OmpF’s role in membrane permeability and drug efflux .
Constitutive OmpF expression in E. coli ER0808 resulted in 99.1% FC-positive cells when labeled with FITC-conjugated antibodies, outperforming plasmid-induced systems (96.3%) .
Immunoelectron microscopy confirmed gold-labeled OmpF epitopes on bacterial surfaces, validating antibody specificity .
Recombinant OmpF (rtOmpF) conjugated with adjuvant ISA763 increased survival rates in fish infected with Yersinia ruckeri by 75.86%, highlighting its immunogenic potential .
OmpF-targeting antibodies enhanced rifamycin efficacy against E. coli, reducing MIC values from 12.5 µg/mL to 0.78 µg/mL .
FITC conjugation involves:
FITC:Antibody Ratio: Titrate between 10–400 µg FITC per mg antibody to balance brightness and solubility .
Reaction: Incubate with FITC-DMSO solution (1 hr, room temperature, dark) .
Purification: Remove unbound FITC via dialysis or gel filtration .
Quality Control: Measure F/P ratio (A495/A280 absorbance; ideal range: 3–6 FITC per IgG) .
Photobleaching: FITC degrades under prolonged light exposure; store in dark .
Cross-Reactivity: Validate specificity using knockout strains (e.g., ompF⁻ E. coli) .
Buffer Compatibility: Use PBS with BSA or glycerol to prevent aggregation .
KEGG: ecj:JW0912
STRING: 316385.ECDH10B_0999
OmpF is a major outer membrane porin protein found predominantly in gram-negative bacteria that forms water-filled channels allowing passive diffusion of small hydrophilic molecules across the bacterial outer membrane. The protein plays crucial structural roles in determining cell shape and membrane integrity while functioning as a selective filter for molecules entering the periplasmic space. The significance of ompF in bacterial research stems from its role in antibiotic resistance, environmental adaptation, and bacterial pathogenesis. OmpF expression levels are regulated in response to environmental conditions, making it an important marker for bacterial stress responses . Research utilizing ompF has expanded to include novel applications in bacterial surface display systems, where it serves as a carrier protein for presenting viral epitopes or other molecules of interest .
FITC conjugation to antibodies occurs through a chemical reaction between the isothiocyanate group (-N=C=S) of FITC and primary amine groups (particularly lysine residues) on the antibody molecule, forming stable covalent thiourea bonds . The conjugation process requires specific buffer conditions: typically, antibodies must be dialyzed against an alkaline buffer (pH 9.2 for FITC labeling) to remove interfering NH4+ ions and optimize reaction conditions . The reaction involves adding 20 μl of 5 mg/ml FITC in anhydrous DMSO for each milligram of antibody, followed by a 2-hour incubation at room temperature . Unbound FITC molecules must be removed through dialysis against an appropriate buffer. This process results in antibodies labeled with multiple FITC molecules, typically at a fluorophore-to-protein (F:P) ratio of 3:1 to 8:1 for optimal performance without compromising antibody binding capacity.
OmpF antibody, FITC conjugated reagents are valuable in multiple research applications:
Flow cytometry analysis of bacterial populations for quantifying ompF expression levels and heterogeneity
Immunofluorescence microscopy for visualizing ompF distribution on bacterial membranes
Validation of bacterial surface display systems utilizing ompF as a carrier protein
Monitoring changes in porin expression under various environmental conditions or antibiotic treatments
Detection of gram-negative bacteria in mixed microbial communities
Study of bacterial outer membrane vesicles (OMVs) containing ompF
Flow cytometry data from constitutive expression-based ompF display systems have demonstrated high positive cell ratios (>90%) compared to plasmid-induced expression systems, indicating efficient surface display . This method allows researchers to quantitatively assess bacterial population responses to environmental changes through shifts in fluorescence intensity distributions.
When designing experiments with FITC-conjugated ompF antibodies, researchers should incorporate several controls to ensure valid interpretation of results:
Control Type | Purpose | Implementation |
---|---|---|
Negative control | Assess background fluorescence | Use bacterial strains lacking ompF expression (ompF knockout) |
Isotype control | Evaluate non-specific binding | Use FITC-conjugated antibody of same isotype but irrelevant specificity |
Secondary antibody control | Measure background from secondary reagents | Omit primary antibody in immunostaining protocol |
Autofluorescence control | Distinguish endogenous fluorescence | Examine unstained bacteria at FITC emission wavelengths |
Positive control | Confirm staining protocol efficacy | Use bacterial strains with known high ompF expression levels |
Blocking validation | Verify specificity | Pre-incubate antibody with recombinant ompF protein |
Flow cytometry analysis should establish appropriate gating strategies based on these controls to differentiate genuine ompF-positive bacterial populations from background signals. In constitutive expression systems, control strains like ER2566 and ER0808 have been used to establish baseline fluorescence before analyzing ompF-display constructs .
Optimizing FITC-conjugated ompF antibody performance requires attention to several experimental parameters:
Experimental validation using both Western blot and immunohistochemistry techniques should be performed to confirm optimized conditions work across multiple detection platforms . For flow cytometry, signal-to-noise ratio can be enhanced by adjusting detector voltage based on unstained control samples.
Cross-reactivity assessment is critical when applying ompF antibodies across different bacterial species due to porin homology. The following approaches help address this concern:
Sequence homology analysis: Conduct bioinformatic analysis of ompF protein sequences across target species to predict potential cross-reactivity. Higher homology (>70%) increases likelihood of antibody cross-reactivity.
Western blot validation: Perform Western blots on membrane fractions from multiple bacterial species to identify potential cross-reactive proteins based on molecular weight differences.
Absorption controls: Pre-absorb antibodies with lysates from related bacterial species to remove cross-reactive antibodies before experimental use.
Epitope mapping: Determine the specific epitope recognized by the antibody and assess its conservation across species.
Competitive binding assays: Use recombinant ompF proteins from different species to compete for antibody binding in ELISA or flow cytometry assays.
Antibodies designed against conserved regions like transmembrane domains may show broader cross-reactivity than those targeting loop regions, which typically exhibit greater sequence variation. Published data indicate that polyclonal ompF antibodies tend to show specific reactivity with their target bacterial species but may cross-react with related Enterobacteriaceae family members .
OmpF expression is highly responsive to environmental conditions through complex regulatory mechanisms. FITC-conjugated antibodies provide valuable tools for quantifying these changes:
Environmental Factor | Effect on ompF Expression | Quantification Method |
---|---|---|
Osmotic pressure | Decreases at high osmolarity | Flow cytometry mean fluorescence intensity (MFI) |
Temperature | Downregulated at high temperatures | Quantitative immunofluorescence microscopy |
pH | Reduced expression at low pH | Flow cytometry with calibration beads |
Nutrient limitation | Generally upregulated | Comparative MFI across growth conditions |
Antibiotic exposure | Variable depending on antibiotic class | Time-course flow cytometry analysis |
Research has revealed that promoter mutations can significantly enhance ompF expression levels, as demonstrated in the engineered bacterial strain ER0808, which showed substantially higher ompF expression compared to the standard ER2566 strain . The mutation rendered ompF an ideal carrier protein for enriched display of target epitopes on the bacterial surface.
For quantification, standardized flow cytometry protocols using FITC-conjugated ompF antibodies should include calibration beads to normalize fluorescence intensity values across experiments. This approach enables reliable comparison of expression levels under different environmental conditions or between different bacterial strains.
Bacterial surface display systems utilizing ompF as a carrier protein represent an innovative application that can be optimized and validated using FITC-conjugated antibodies:
Insertion site selection: Loop 8 of ompF has been identified as an effective insertion locus for peptide fusion based on structural analysis and genomic PAM (protospacer adjacent motif) assessment . FITC-conjugated antibodies targeting the inserted epitopes can confirm surface accessibility.
Expression system comparison: Flow cytometry analysis of FITC-antibody binding has demonstrated that constitutive expression of ompF fusion proteins yields significantly higher surface display efficiency (99.1% and 91.6% positive cells for different epitopes) compared to plasmid-induced expression systems (96.3% and 62.9% positive cells) .
Validation through multiple techniques: Combine flow cytometry with immunoelectron microscopy using gold particle-conjugated antibodies to visually confirm surface localization of ompF fusion proteins .
Functional assessment: Evaluate whether the displayed peptides/proteins maintain their functional activity through binding assays with appropriate ligands or receptors, using FITC-conjugated antibodies as reference markers.
Stability testing: Monitor the stability of the display system over time through periodic sampling and flow cytometry analysis with FITC-conjugated antibodies.
Researchers have successfully used genome editing techniques to introduce viral epitope sequences (such as hepatitis B virus surface antigen and human papillomavirus L2 protein) into loop 8 of ompF, creating effective surface display systems that can be readily detected using epitope-specific antibodies .
The standard FITC conjugation protocol can be modified in several ways to improve efficiency specifically for ompF antibodies:
Optimal F:P ratio determination: The fluorophore-to-protein ratio significantly impacts antibody performance. For ompF antibodies, aim for a 4:1 to 6:1 ratio, which balances fluorescence intensity with antibody binding capacity. This can be calculated by measuring absorbance at 280 nm (protein) and 495 nm (FITC).
Buffer optimization: The standard protocol recommends dialyzing antibodies against FITC labeling buffer at pH 9.2 . For ompF antibodies, extending dialysis time to 3 days with 4-5 buffer changes can remove interfering substances more effectively.
Reaction conditions: While the standard protocol suggests 2 hours at room temperature , extending incubation to 3-4 hours at 4°C can improve conjugation while preserving antibody activity, particularly important for antibodies targeting conformational epitopes like those in ompF.
Purification enhancement: After conjugation, purify FITC-antibody conjugates using size-exclusion chromatography rather than dialysis alone to achieve more complete removal of unconjugated FITC, resulting in lower background fluorescence.
Stabilization additions: Add 1% BSA and 0.02% sodium azide to the final conjugate solution to enhance stability. For long-term storage, aliquot and store at -20°C in the dark to prevent photobleaching.
The protocol can be monitored by measuring the absorbance spectrum of the conjugate after each step, with successful conjugation showing characteristic peaks at both 280 nm (protein) and 495 nm (FITC).
Researchers commonly encounter several issues when working with FITC-conjugated ompF antibodies. Here are methodological approaches to address them:
Issue | Possible Causes | Troubleshooting Approaches |
---|---|---|
Low signal intensity | Insufficient antibody concentration, epitope masking, photobleaching | Increase antibody concentration, optimize fixation protocol, add antifade reagents, adjust detector settings |
High background | Incomplete blocking, non-specific binding, excessive antibody concentration | Extend blocking time (2-3 hours), use combination blockers (BSA+serum), titrate antibody concentration, include 0.1% Tween-20 in wash buffers |
Variable staining patterns | Heterogeneous ompF expression, membrane disruption during preparation | Standardize culture conditions, optimize fixation protocols, use gentle permeabilization methods |
Photobleaching | FITC sensitivity to light exposure | Minimize exposure time, use antifade mounting media, consider alternative fluorophores (Alexa Fluor 488) |
Poor correlation with protein expression | Post-translational modifications, conformational changes | Validate with alternative methods (Western blot), use multiple antibody clones targeting different epitopes |
Researchers have reported successful immunohistochemical analysis using ompF antibodies at 1/200 dilution for both fluorescent (with Cy3-conjugated secondary antibody) and DAB staining approaches . When optimizing for bacterial samples, testing fixation in 2-4% paraformaldehyde for varying times (10-30 minutes) can help identify conditions that preserve both membrane integrity and epitope accessibility.
Standardization is essential for comparing data across different experiments using FITC-conjugated ompF antibodies. Implement these methodological approaches:
Fluorescence calibration: Use calibration beads with defined fluorescence intensities to normalize flow cytometry data across experiments. Convert raw fluorescence values to molecules of equivalent soluble fluorochrome (MESF) units for quantitative comparisons.
Internal reference samples: Include a standard bacterial strain with stable ompF expression in each experiment as a reference point. Express experimental sample values relative to this standard.
Consistent acquisition parameters: Maintain identical instrument settings (voltage, gain, threshold) across experiments. If settings must change, use calibration beads to establish conversion factors.
Antibody lot validation: Test each new antibody lot against a reference lot using standard samples to determine correction factors for lot-to-lot variations.
Data normalization approaches: For flow cytometry data, normalize using:
Ratio of sample median fluorescence intensity (MFI) to negative control MFI
Z-score normalization within each experimental batch
Percentile rank within distribution of reference samples
Statistical validation: Implement statistical tests appropriate for non-parametric distributions typical of flow cytometry data, such as Mann-Whitney U test or Kolmogorov-Smirnov test.
Research comparing constitutive expression-based ompF display systems with plasmid-induced systems demonstrated that data normalization is critical for accurate comparison, with the constitutive expression system consistently showing higher positive cell ratios than plasmid-induced systems .
Quantitative analysis of fluorescence microscopy data requires rigorous methodological approaches:
Image acquisition standardization: Collect all images using identical exposure settings, gain, and offset. Include internal controls on each slide to monitor staining consistency.
Background correction methods: Apply appropriate background subtraction techniques:
Local background subtraction: Measure intensity near but outside the cell
Rolling ball algorithm: Removes smooth continuous backgrounds
Negative control subtraction: Use images from negative control samples
Signal intensity quantification:
Mean fluorescence intensity (MFI) per cell
Integrated density (area × mean intensity)
Intensity distribution analysis within individual cells
Membrane localization assessment:
Line profile analysis across cellular membranes
Ratio of membrane-to-cytoplasmic fluorescence
Co-localization analysis with membrane-specific markers
Population analysis approaches:
Single-cell analysis for population heterogeneity
Classification of expression patterns (uniform vs. patchy distribution)
Statistical analysis comparing experimental conditions
Immunoelectron microscopy has been used to validate fluorescence microscopy findings, showing gold particles specifically visualized on the surfaces of bacteria expressing ompF fusion proteins . This complementary approach provides nanometer-scale resolution of protein localization that supports fluorescence data interpretation.
Several pitfalls can complicate interpretation of results from experiments using FITC-conjugated ompF antibodies:
Bacterial autofluorescence interference: Many bacteria produce endogenous fluorophores that overlap with FITC's emission spectrum. Control for this by measuring unstained samples and implementing appropriate spectral compensation.
Fixation artifacts: Overfixation can create artificial fluorescence patterns or mask epitopes. Validate findings using multiple fixation protocols and complementary techniques like immunoelectron microscopy .
Expression level vs. accessibility confusion: Surface accessibility of ompF can change without changes in total expression level. Distinguish between these possibilities by comparing flow cytometry data (surface accessibility) with Western blot results (total protein).
Strain-specific differences: OmpF expression regulation varies between bacterial strains. For example, the ER0808 strain shows enhanced ompF expression due to a promoter mutation compared to the ER2566 strain . Always include appropriate strain controls.
Media and growth conditions effects: OmpF expression is highly regulated by environmental conditions. Standardize culture conditions or explicitly test their effects as experimental variables.
Photobleaching misinterpretation: FITC is susceptible to photobleaching, which can be mistaken for biological differences. Include time-matched controls and consider using more photostable fluorophores for extended imaging.
Research has shown that constitutive expression-based ompF display systems generate higher positive cell ratios than plasmid-induced expression systems , but this difference could be misinterpreted without proper controls and awareness of the technical limitations of each approach.
FITC-conjugated ompF antibodies present valuable opportunities for application in several emerging research areas:
Single-cell bacterial analysis: Correlating ompF expression patterns with individual bacterial phenotypes, particularly in heterogeneous populations responding to environmental stresses or antibiotic treatments.
Bacterial biofilm studies: Investigating the distribution and accessibility of ompF proteins within complex biofilm structures, potentially revealing novel mechanisms of antibiotic resistance or environmental adaptation.
Vaccine development research: Building on successful bacterial surface display systems , FITC-conjugated antibodies can validate the expression and accessibility of vaccine antigens displayed via ompF carrier proteins.
Bacterial diagnostic development: Creating rapid detection systems for pathogenic gram-negative bacteria based on ompF recognition combined with FITC fluorescence detection.
Nanoparticle-bacteria interactions: Studying how engineered nanoparticles interact with bacterial outer membrane proteins, with potential applications in targeted antimicrobial delivery.
Research has demonstrated that ompF can serve as an effective carrier protein for displaying viral epitopes on bacterial surfaces , suggesting that FITC-conjugated antibodies against these epitopes could facilitate the development and validation of novel bacterial vaccine platforms and diagnostic tools.
Several methodological advances could significantly enhance the utility of FITC-conjugated ompF antibodies:
Development of site-specific conjugation: Current conjugation protocols lead to random FITC attachment to lysine residues . Site-specific conjugation technologies would preserve antibody binding capacity while ensuring consistent fluorophore placement.
Super-resolution microscopy optimization: Adapting protocols for techniques like STORM or PALM imaging would enable nanoscale visualization of ompF distribution patterns in bacterial membranes.
Multiplexed detection systems: Developing compatible protocols for simultaneous detection of ompF along with other bacterial proteins using spectrally distinct fluorophores would enhance experimental efficiency.
In vivo imaging applications: Adapting FITC-conjugated ompF antibodies for in vivo bacterial tracking during infection models would connect molecular details to pathogenesis.
Machine learning analysis integration: Implementing automated image analysis algorithms to quantify complex ompF distribution patterns would improve objectivity and throughput in data analysis.
These advances would build upon existing platforms where immunoelectron microscopy has already been successfully used to visualize ompF fusion proteins on bacterial surfaces , extending these capabilities to more dynamic, higher-resolution, and multiplexed applications.