NS Antibody, FITC conjugated
Description
Overview of NS Antibody, FITC Conjugated
The NS Antibody, FITC conjugated refers to a polyclonal rabbit IgG antibody targeting the NS1 (nonstructural protein 1) of the dengue virus (DENV), specifically type 2 (strain New Guinea C). This antibody is covalently labeled with fluorescein isothiocyanate (FITC), a fluorescent dye with excitation/emission spectra of 495 nm/519 nm, enabling its use in fluorescence-based assays such as ELISA, Western blot, and imaging techniques .
Conjugation Process and Optimization
The conjugation of FITC to NS1 antibodies involves:
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Primary amine reaction: FITC reacts with lysine residues on the antibody via nucleophilic substitution .
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Titration: Initial conjugations use 10–400 µg FITC/mg antibody to avoid quenching and solubility issues .
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Purification: Unbound FITC is removed via desalting columns to prevent background fluorescence .
Critical Considerations:
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Labeling index: Higher FITC-to-antibody ratios (>6) reduce binding affinity (–0.4–0.6 log units per 10-fold increase in labeling) .
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Storage: FITC is unstable post-reconstitution; conjugation must occur immediately .
Applications in Research
4.1. Binding Affinity and Specificity
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Trade-off: Higher FITC labeling enhances sensitivity but reduces specificity due to non-specific binding (e.g., 2.3-fold increase in background staining at 400 µg FITC/mg) .
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Optimal labeling: Studies recommend 40–80 µg FITC/mg antibody for maximal signal-to-noise ratio .
4.2. Role in Dengue Pathogenesis
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
What is FITC and how does it function as an antibody conjugate?
FITC (fluorescein isothiocyanate) is a derivative of fluorescein modified with an isothiocyanate reactive group (-N=C=S). This fluorescent dye functions as a marker by chemically binding to antibodies, enabling visualization of specific cellular targets. The isothiocyanate group reacts with amino-terminal and primary amine groups on antibodies, forming stable covalent thiourea bonds . FITC exhibits distinctive spectral properties with excitation and emission peak wavelengths at approximately 495nm and 525nm, respectively, resulting in bright yellow-green fluorescence when excited by blue or ultraviolet light . This specificity makes FITC-conjugated antibodies particularly valuable for numerous detection methods in biological research.
What are the primary applications of FITC-conjugated antibodies in laboratory research?
FITC-conjugated antibodies serve as versatile tools across multiple research applications:
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Flow Cytometry: Enables analysis of cells' physical and chemical characteristics, allowing researchers to detect specific cell populations and measure parameters including size, granularity, and protein expression levels with high sensitivity .
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Fluorescence Microscopy: Facilitates visualization of cellular structures and processes with exceptional specificity, making it ideal for multi-color imaging experiments .
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Immunoassays: Detects specific antigens or antibodies with high sensitivity, providing valuable diagnostic capabilities in clinical and research settings .
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Immunocytochemistry/Immunohistochemistry: Labels specific cellular components in fixed cells or tissue sections for detailed structural analysis .
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Fluorescence in situ Hybridization (FISH): When conjugated to nucleic acid probes, allows visualization of specific DNA or RNA sequences within cells .
What are the optimal storage conditions for FITC-conjugated antibodies?
To maintain functionality and fluorescence intensity, FITC-conjugated antibodies require specific storage conditions:
| Storage Phase | Temperature | Duration | Conditions |
|---|---|---|---|
| As supplied | -20 to -70°C | 12 months from receipt | Original container |
| After reconstitution | 2 to 8°C | 1 month | Sterile conditions |
| Long-term storage after reconstitution | -20 to -70°C | 6 months | Sterile conditions |
It is crucial to use a manual defrost freezer and avoid repeated freeze-thaw cycles, which can significantly degrade antibody quality and fluorescence intensity . Store antibodies in the dark to prevent photobleaching of the FITC fluorophore.
How does the pH environment affect FITC fluorescence properties?
FITC fluorescence intensity is pH-dependent, with optimal fluorescence occurring at slightly alkaline conditions:
| pH Value | Effect on Fluorescence |
|---|---|
| <6.0 | Significantly reduced fluorescence intensity |
| 7.0 | Moderate fluorescence intensity |
| 8.0-9.0 | Optimal fluorescence intensity |
| >10.0 | Potential chemical degradation of fluorophore |
This pH sensitivity must be considered when designing experiments, particularly when working with acidic cellular compartments such as lysosomes or when conducting experiments involving pH changes .
What factors determine the optimal FITC-to-antibody ratio during conjugation?
The FITC-to-antibody ratio (F/P ratio) critically influences conjugate performance. According to experimental studies, several parameters affect optimal conjugation:
| Parameter | Optimal Condition | Effect on Conjugation |
|---|---|---|
| pH | 9.5 | Maximizes reaction efficiency between isothiocyanate and primary amines |
| Temperature | Room temperature (20-25°C) | Balances reaction rate with antibody stability |
| Reaction time | 30-60 minutes | Achieves maximal labeling without over-modification |
| Protein concentration | ~25 mg/ml | Higher concentration enhances conjugation efficiency |
| Buffer composition | Carbonate/bicarbonate buffer (pH 9.5) | Provides optimal conditions for isothiocyanate reactivity |
Purification via gradient DEAE Sephadex chromatography effectively separates optimally labeled antibodies from under- and over-labeled proteins . The ideal F/P ratio typically ranges from 3:1 to 6:1 for most applications, balancing fluorescence intensity with preserved antibody functionality .
How does the conjugation strategy affect antibody orientation and targeting efficiency?
The method used to conjugate FITC to antibodies significantly impacts their orientation and consequently their targeting performance:
| Conjugation Strategy | Effect on Antibody Orientation | Impact on Targeting Efficiency |
|---|---|---|
| Random conjugation (e.g., isothiocyanate reaction with lysine residues) | Random antibody orientation | Reduced targeting efficiency due to potential modification of antigen-binding domains |
| Site-specific conjugation (e.g., thiol-maleimide coupling) | More controlled orientation | Improved target binding by preserving antigen-binding regions |
| Copper-free click chemistry | Highly oriented antibody attachment | Superior targeting efficiency with preserved binding domains |
Research demonstrates that properly oriented antibodies (via copper-free click chemistry) exhibit significantly better target recognition than randomly conjugated antibodies, even when surface antibody density is comparable . This orientation factor becomes particularly critical for applications requiring high sensitivity or specificity, such as in nanocarrier drug delivery systems .
What strategies help prevent photobleaching of FITC during imaging experiments?
FITC is susceptible to photobleaching, which can limit experimental duration and data quality. Several approaches minimize this effect:
| Anti-Photobleaching Strategy | Mechanism | Implementation |
|---|---|---|
| Anti-fade mounting media | Contains oxygen scavengers and radical quenchers | Replace standard mounting media with commercial anti-fade formulations |
| Reduced light exposure | Minimizes photochemical damage | Lower excitation intensity, shorter exposure times, neutral density filters |
| Sample temperature control | Slows photobleaching reactions | Maintain samples at 4°C during preparation and imaging |
| Imaging optimization | Reduces cumulative light exposure | Use lower laser power, faster scanning speeds, frame averaging |
| Alternative detection | Signal amplification with less exposure | Anti-FITC antibodies to enhance signal without increased excitation |
For quantitative studies particularly vulnerable to photobleaching artifacts, consider mathematical correction based on photobleaching curves or switching to more photostable alternatives like Alexa Fluor 488 with similar spectral properties .
How can researchers validate the specificity of FITC-conjugated antibodies?
Proper validation ensures experimental reliability. The scientific community recommends a multi-faceted validation approach:
| Validation Method | Technical Approach | Expected Outcome |
|---|---|---|
| Positive and negative controls | Test antibody on samples known to express or lack target | Signal in positive samples, no signal in negative samples |
| Blocking experiments | Pre-incubate with unlabeled primary antibody or specific blocking peptides | Significant reduction in signal intensity |
| Isotype controls | Use FITC-conjugated isotype-matched irrelevant antibodies | No specific binding pattern |
| Knockdown/knockout validation | Test antibody in cells with genetic depletion of target | Substantial reduction or elimination of signal |
| Western blot correlation | Compare with unconjugated antibody in Western blot | Recognition of same molecular weight band |
| Cross-platform verification | Compare results across different detection techniques | Consistent pattern of target recognition |
According to recent guidelines, responsibility for antibody validation is shared between manufacturers and investigators . While commercial antibodies often come with validation data, researchers should independently verify specificity in their specific experimental systems .
What are the best practices for multiplexing FITC with other fluorophores?
Effective multiplexing requires strategic fluorophore selection and experimental design:
| Consideration | Best Practice | Rationale |
|---|---|---|
| Spectral compatibility | Pair FITC (Ex:495nm, Em:525nm) with spectrally distinct fluorophores | Minimizes spectral overlap for clearer signal separation |
| Recommended combinations | FITC + PE/R-PE (Em:578nm) + APC (Em:660nm) | Provides well-separated emission peaks |
| FITC + Cy5 (Em:670nm) + DyLight 650 (Em:672nm) | Allows multi-color imaging with minimal bleed-through | |
| Controls | Include single-stained controls for each fluorophore | Enables proper compensation settings in flow cytometry |
| Antibody titration | Optimize concentration of each conjugate separately | Prevents over-staining and cross-interference |
| Detection strategy | Use narrow bandpass filters or sequential acquisition | Isolates specific emissions to reduce spectral overlap |
Advanced microscopy platforms may employ spectral unmixing algorithms to separate overlapping signals mathematically, enhancing multiplexing capabilities .
How can researchers troubleshoot poor signal strength when using FITC-conjugated antibodies?
Poor signal strength can result from multiple factors. A systematic troubleshooting approach includes:
| Issue Category | Potential Problem | Solution Strategy |
|---|---|---|
| Antibody factors | Degraded fluorophore | Check expiration, prepare fresh working solutions |
| Suboptimal F/P ratio | Verify F/P ratio, consider alternative conjugates | |
| Improper storage | Maintain proper temperature and light protection | |
| Sample preparation | Overfixation masking epitopes | Test different fixation methods and durations |
| Inadequate permeabilization | Optimize detergent concentration and incubation time | |
| Insufficient blocking | Improve blocking conditions to enhance signal-to-noise ratio | |
| Technical aspects | Suboptimal instrument settings | Verify detector sensitivity, gain settings, laser power |
| pH effects | Ensure buffer pH 8.0-8.5 for optimal FITC fluorescence | |
| Quenching by mounting media | Test anti-fade reagents specifically compatible with FITC |
If problems persist, consider signal amplification techniques or switch to more photostable alternatives like Alexa Fluor 488 with similar spectral properties but greater stability .
What methods can enhance detection sensitivity when using FITC-conjugated antibodies?
Several approaches can significantly improve detection sensitivity:
| Enhancement Approach | Methodology | Relative Improvement |
|---|---|---|
| Tyramide Signal Amplification (TSA) | HRP-coupled antibodies catalyze FITC-tyramide deposition | 10-50 fold signal enhancement |
| Layered detection | Primary → Biotinylated secondary → FITC-streptavidin | 2-5 fold signal enhancement |
| Anti-FITC antibodies | Secondary amplification with anti-FITC antibodies | 2-3 fold signal enhancement |
| Optical enhancement | Confocal microscopy, deconvolution, structured illumination | Improves signal-to-noise ratio |
| Sample optimization | Optimized fixation, permeabilization, antigen retrieval | Increases epitope accessibility |
| Extended incubation | Overnight primary antibody incubation at 4°C | Enhances signal without increasing background |
For extremely low abundance targets, combining multiple enhancement techniques may be necessary to achieve adequate sensitivity .
How does FITC conjugation impact antibody functionality and binding capacity?
The impact of FITC conjugation on antibody performance depends on several factors:
| Factor | Effect on Antibody Function | Optimization Strategy |
|---|---|---|
| F/P Ratio | Over-labeling (>8 FITC molecules per antibody) can impair binding | Aim for 3-6 FITC molecules per antibody |
| Conjugation site | Modification near antigen-binding region reduces affinity | Use site-specific conjugation methods |
| Purification quality | Residual free FITC increases background | Employ thorough dialysis or size exclusion chromatography |
| Antibody isotype | Different isotypes show varying sensitivity to conjugation | Validate each isotype-specific conjugate independently |
Research indicates that when properly optimized, FITC conjugation typically preserves 70-90% of antibody binding capacity compared to unconjugated counterparts . Electrophoretically distinct IgG molecules appear to have similar affinity for FITC, suggesting consistent conjugation behavior across different antibody populations .
What are the critical quality control parameters for evaluating FITC-conjugated antibodies?
Quality assessment of FITC-conjugated antibodies should evaluate multiple parameters:
| Quality Parameter | Measurement Method | Acceptable Range |
|---|---|---|
| F/P ratio | Spectrophotometric measurement (A495/A280) | 3.0-6.0 for most applications |
| Functional activity | Flow cytometry or immunofluorescence with known positive controls | ≥70% of unconjugated antibody activity |
| Specificity | Testing against positive and negative cell lines/tissues | Strong signal in positive samples, negligible in negative samples |
| Aggregation level | Size exclusion chromatography or dynamic light scattering | <10% aggregates |
| Free FITC content | TLC or gel filtration | <5% free FITC |
| Lot-to-lot consistency | Comparative analysis between different production lots | <15% variation in key parameters |
Regular quality control testing is essential, particularly for antibodies stored for extended periods, as FITC fluorescence can deteriorate over time even under optimal storage conditions .
What recent advances have improved FITC-conjugated antibody performance?
Several technological innovations have enhanced FITC-conjugated antibody applications:
| Innovation | Mechanism | Performance Improvement |
|---|---|---|
| Site-specific conjugation | Targets specific residues away from binding domains | Preserves >90% of binding activity |
| Copper-free click chemistry | Enables oriented antibody attachment to carriers | Significantly improves targeting efficiency |
| Hydrophilic linkers | Reduces aggregation and non-specific binding | Enhances signal-to-noise ratio |
| Anti-FITC secondary amplification | Uses anti-FITC antibodies for signal boosting | Increases detection sensitivity |
| Computational correction | Algorithms to correct for photobleaching | Extends useful imaging duration |
These advancements have made FITC-conjugated antibodies more reliable and sensitive, expanding their utility in challenging applications such as single-molecule detection and in vivo imaging with nanocarrier systems .
