LTA4H Antibody, FITC conjugated

What is LTA4H and what cellular functions does it regulate?

LTA4H (Leukotriene A-4 hydrolase) is a zinc-dependent enzyme with dual functionality, exhibiting both epoxide hydrolase and aminopeptidase activities. It plays a crucial role in the metabolism of leukotrienes, which are bioactive lipids derived from arachidonic acid essential for mediating inflammatory responses and host defense mechanisms. Located primarily in the cytosol, LTA4H's epoxide hydrolase function catalyzes the conversion of leukotriene A4 to leukotriene B4, a potent lipid mediator that acts as a chemoattractant, guiding immune cells to sites of inflammation and infection. Additionally, its aminopeptidase activity contributes to regulatory functions in immune responses by processing peptide substrates. The human LTA4H gene encodes a protein consisting of 610 amino acids, and its proper functioning is vital for maintaining immune homeostasis and responding effectively to pathogenic challenges .

What are the primary research applications for FITC-conjugated LTA4H antibodies?

The primary research applications for FITC-conjugated LTA4H antibodies include ELISA (Enzyme-Linked Immunosorbent Assay), immunofluorescence (IF), flow cytometry (FCM), and immunohistochemistry (IHC-P) . FITC conjugation allows for direct visualization of LTA4H in cells and tissues without the need for secondary antibodies. In flow cytometry applications, these antibodies can be used to identify cells expressing LTA4H after permeabilization, as demonstrated in studies where researchers separated cells expressing LTA4H into leukocytes (CD45 high) and non-leukocytes (CD45 low) . Additionally, FITC-conjugated LTA4H antibodies can be utilized in multi-parameter flow cytometry experiments to simultaneously detect other markers, enhancing the depth of analysis in immunological research.

How should researchers properly store and handle FITC-conjugated LTA4H antibodies?

To maintain optimal functionality of FITC-conjugated LTA4H antibodies, researchers should adhere to specific storage and handling protocols. Upon receipt, the antibody should be stored at either -20°C or -80°C for long-term preservation . It is crucial to avoid repeated freeze-thaw cycles as this can lead to protein denaturation and loss of antibody functionality. The antibody is typically provided in a protective buffer containing preservative (0.03% Proclin 300) and stabilizers (50% Glycerol in 0.01M PBS, pH 7.4), which helps maintain its structure and activity .

For handling during experiments, researchers should minimize exposure to light due to the photosensitive nature of the FITC fluorophore. Aliquoting the antibody into smaller volumes prior to freezing is recommended to prevent repeated freeze-thaw cycles. When working with the antibody, maintain cold temperatures (on ice) and return to storage promptly after use to preserve the integrity of both the antibody and the fluorescent conjugate.

What validation methods confirm the specificity of LTA4H antibodies?

Validation of LTA4H antibodies involves multiple complementary techniques to ensure specificity and reliability in research applications. Protein G purification, as employed for commercially available antibodies, yields >95% pure antibody preparations, removing potential contaminants that could affect specificity . ELISA validation, a commonly tested application, confirms antigen recognition and binding efficiency .

In more comprehensive validation approaches, researchers may employ Western blotting to verify that the antibody recognizes LTA4H protein at the expected molecular weight. Immunoprecipitation (IP) can establish that the antibody successfully pulls down LTA4H protein from cell lysates . Flow cytometry validation demonstrates the antibody's ability to detect cellular LTA4H after proper permeabilization procedures, as seen in studies distinguishing between leukocytes (CD45 high) and non-leukocytes (CD45 low) .

Cross-reactivity testing against species other than the immunogen (human) should be performed if researchers intend to use the antibody in non-human models. The commercially available antibodies are known to recognize human, mouse, and rat LTA4H protein , making them versatile tools for comparative studies.

How can researchers develop a custom ELISA for quantifying LTA4H in biological samples?

Developing a custom ELISA for quantifying LTA4H in biological samples requires careful optimization of multiple parameters. Based on published methodologies, researchers should follow this approach:

• Antibody Selection: Select two different LTA4H antibodies with distinct epitope recognition (e.g., Novus EPR5713 as coating antibody and R&D antibody as biotinylated detection antibody) to create a sandwich ELISA format .

• Standard Curve Preparation: Purchase recombinant LTA4H (such as Creative BioMart recombinant murine LTA4H with His-tag) for generating a standard curve. Prepare serial dilutions of known quantities to establish a calibration curve .

• Sample Processing: When analyzing complex biological samples such as BALF (bronchoalveolar lavage fluid) or lung homogenate soup, ensure proper sample dilution and processing to minimize matrix effects.

• Assay Protocol:

  • Coat ELISA plates with the capture antibody (e.g., Novus EPR5713) at optimized concentration

  • Block non-specific binding sites

  • Add 50 μL of samples in duplicates alongside standards

  • Incubate with biotinylated detection antibody

  • Apply streptavidin-HRP conjugate

  • Develop with appropriate substrate

  • Measure optical density at 450 nm using a microplate reader

• Data Analysis: Analyze samples against the standard curve, using appropriate software (such as Concert-Triad Serves software version 2.0.0.12) for quantification, ensuring standard curve linearity and appropriate detection limits .

This methodology allows for specific and sensitive quantification of LTA4H in various biological samples, enabling researchers to correlate enzyme levels with physiological or pathological states.

What are the implications of LTA4H as a biomarker in allergen immunotherapy (AIT), and how can researchers assess its predictive value?

LTA4H has emerged as a promising biomarker for predicting the efficacy of allergen immunotherapy (AIT), with significant implications for personalized medicine approaches in allergy treatment. Research has shown that serum LTA4H levels are upregulated specifically in AIT responders but not in non-responders, suggesting its potential as an early prediction marker for treatment outcomes .

To assess LTA4H's predictive value in AIT, researchers should follow these methodological approaches:

• Patient Stratification: Clearly define criteria for classifying patients as AIT responders versus non-responders based on standardized clinical outcomes.

• Sample Collection: Obtain serum samples before initiating AIT and at defined intervals (e.g., after 1 year of treatment) to track changes in LTA4H levels.

• Biomarker Evaluation:

  • Utilize LC-MS/MS-based proteomics for initial discovery phase

  • Validate findings using ELISA on larger patient cohorts

  • Compare LTA4H predictive value against established markers (e.g., allergen-specific IgE and IgG4)

• ROC Analysis: Generate receiver operating characteristic (ROC) curves to determine the predictive accuracy of LTA4H. Research has demonstrated an AUC value of 0.844 (95% confidence interval: 0.727 to 0.962) for LTA4H in distinguishing responders from non-responders, surpassing the predictive value of traditional markers like allergen-specific IgE and IgG4 .

• Multivariate Analysis: Combine LTA4H measurements with other clinical and molecular parameters to develop comprehensive predictive models for AIT outcomes.

This approach enables researchers to evaluate LTA4H as a biomarker for early prediction of AIT efficacy, potentially allowing clinicians to identify patients most likely to benefit from therapy and adjust treatment strategies accordingly.

How can researchers effectively utilize flow cytometry to analyze LTA4H expression in heterogeneous cell populations?

Flow cytometric analysis of LTA4H expression in heterogeneous cell populations requires precise protocols to ensure accurate identification and quantification. Based on published methodologies, researchers should implement the following approach:

• Cell Isolation and Processing: Prepare single-cell suspensions from tissues of interest, ensuring gentle processing to maintain cellular integrity and antigen expression.

• Surface Marker Staining: First stain cells with antibodies against lineage-specific surface markers (e.g., CD45 for leukocytes) to distinguish different cell populations. In studies of inflammatory conditions, include markers such as Ly6G and CD11b to identify neutrophil populations (Ly6G high, CD11b high) .

• Cell Permeabilization: Since LTA4H is primarily a cytosolic enzyme, permeabilize cells using an appropriate flow cytometry permeabilization buffer (such as R&D buffer) after surface marker staining to allow antibody access to intracellular LTA4H .

• LTA4H Staining: Incubate permeabilized cells with FITC-conjugated LTA4H antibody at optimized concentration and time. Include appropriate isotype controls to determine background fluorescence.

• Multi-parameter Analysis: Perform multi-color detection using a flow cytometer capable of detecting the fluorophores used. Gate cells as follows:

  • Separate leukocytes from non-leukocytes based on CD45 expression (CD45 high vs. CD45 low)

  • Further categorize leukocyte subpopulations based on additional markers

  • Analyze LTA4H expression within each defined population

• Data Analysis: Use specialized software (such as FlowJo version 8.8.6) to quantify LTA4H expression across different cell types, allowing for comparative analysis between experimental groups .

This methodological approach enables researchers to characterize cell type-specific expression patterns of LTA4H in complex biological samples, providing insights into its differential expression and potential functional roles in various cell populations during physiological and pathological processes.

What are the dual enzymatic activities of LTA4H and how can researchers differentiate and measure each function in experimental settings?

LTA4H possesses two distinct enzymatic activities—epoxide hydrolase and aminopeptidase—that play crucial roles in inflammation regulation. Differentiating and measuring these activities requires specialized methodological approaches:

• Epoxide Hydrolase Activity Measurement:

  • This activity converts leukotriene A4 (LTA4) to leukotriene B4 (LTB4)

  • Quantification can be performed by incubating purified LTA4H or biological samples with synthetic LTA4 substrate

  • The resulting LTB4 production can be measured using:

    • HPLC coupled with UV detection

    • Enzyme immunoassay (EIA) specific for LTB4

    • LC-MS/MS for precise quantification

  • Inhibitors like bestatin can be used to confirm specificity of the reaction

• Aminopeptidase Activity Measurement:

  • This activity involves the cleavage of N-terminal amino acids from peptide substrates

  • Researchers can use synthetic chromogenic or fluorogenic substrates (such as alanine-p-nitroanilide)

  • Measure the release of chromogenic/fluorogenic groups spectrophotometrically or fluorometrically

  • Specific peptide substrates like Pro-Gly-Pro can be used to assess aminopeptidase activity on physiologically relevant substrates

• Distinguishing Between Activities:

  • Selective inhibitors: Bestatin preferentially inhibits the aminopeptidase activity while minimally affecting the epoxide hydrolase function

  • Site-directed mutagenesis: Specific mutations can selectively impair one activity while preserving the other

  • pH dependency: The two activities have different pH optima, allowing selective measurement under different pH conditions

• Functional Assessment in Disease Models:

  • In cigarette smoke-induced emphysema models, the aminopeptidase activity alleviates neutrophilic inflammation by clearing Proline-Glycine-Proline (PGP)

  • In inflammation studies, researchers can distinguish the activities by examining P4N-induced LTB4 production (epoxide hydrolase activity) and its downstream effects on cytokine expression, which can be suppressed by bestatin

Understanding and separately measuring these dual functions is critical for research into inflammation-related diseases and for developing targeted therapeutic strategies that might modulate one activity while preserving the other.

How does LTA4H activation influence immune cell signaling cascades, and what methods can researchers use to track these pathways?

LTA4H activation triggers complex immune signaling cascades with profound effects on inflammatory responses. Research has revealed that LTA4H activation, particularly its epoxide hydrolase function, leads to LTB4 production, which subsequently initiates multiple downstream signaling events. Researchers can track and analyze these pathways using the following methodological approaches:

• Primary Signaling Events Tracking:

  • Measure LTB4 production using EIA or LC-MS/MS following LTA4H activation

  • Analyze LTB4 receptor (BLT1/BLT2) expression and activation using flow cytometry and calcium mobilization assays

  • Monitor G-protein coupled receptor signaling through GTPγS binding assays

  • Assess receptor internalization using fluorescently-labeled ligands or antibodies

• Downstream Cytokine/Chemokine Production:

  • Quantify proinflammatory mediators (TNF-α, IL-1β, IL-6, IL-8) using ELISA or multiplex cytokine arrays

  • Validate the direct relationship between LTA4H activity and cytokine production using specific inhibitors like bestatin

  • Employ qRT-PCR to measure cytokine mRNA expression levels following LTA4H activation

• Signaling Pathway Analysis:

  • Investigate activation of NF-κB pathway using electrophoretic mobility shift assays (EMSA) or reporter assays

  • Analyze MAPK pathway activation through phospho-specific western blotting (p38, ERK, JNK)

  • Study JAK-STAT signaling using phospho-flow cytometry or western blotting

• Specialized Pathway Tracking:

  • For B-cell activation pathways, monitor the LTA4H → LTB4 → activin A → BAFF signaling cascade:

    • Measure activin A using ELISA

    • Track BAFF production using ELISA or flow cytometry

    • Confirm the pathway using specific inhibitors at each step

  • Analyze the ALK4/Smad3 pathway activation through:

    • Phospho-specific detection of Smad3

    • Smad3 nuclear translocation assays

    • Target gene expression analysis

• In Vivo Pathway Validation:

  • Use genetic approaches (knockout/knockin mice) or pharmacological approaches (specific activators like P4N or inhibitors like bestatin) to manipulate LTA4H activity

  • Trace the impact on downstream mediators in disease models

  • Employ cell-specific depletion (e.g., liposomal clodronate for macrophages) to identify the cellular sources and targets of the pathway components

These methodological approaches allow researchers to comprehensively analyze how LTA4H activation influences immune signaling networks, providing insights into its role in inflammation regulation and potential therapeutic interventions targeting this pathway.

What controls should be implemented when using FITC-conjugated LTA4H antibodies in flow cytometry and immunofluorescence?

Implementing appropriate controls is critical for generating reliable and interpretable data when using FITC-conjugated LTA4H antibodies. Researchers should incorporate the following control strategies:

• Antibody Specificity Controls:

  • Isotype control: Use FITC-conjugated IgG of the same isotype (IgG1 kappa for monoclonal or IgG for polyclonal) from the same host species (rabbit) at the same concentration to establish background fluorescence

  • Blocking control: Pre-incubate the antibody with recombinant LTA4H protein (such as the immunogen used: recombinant Human Leukotriene A-4 hydrolase protein 107-311AA) to confirm binding specificity

  • Knockdown/knockout samples: When possible, use samples from LTA4H-deficient systems to verify antibody specificity

• Fluorescence Controls:

  • Unstained cells: Establish autofluorescence baseline

  • Single-color controls: When performing multi-parameter analysis, include single-stained samples for compensation setup

  • Fluorescence-minus-one (FMO) controls: Include samples with all fluorophores except FITC to determine precise gating boundaries

• Permeabilization Controls:

  • Surface vs. intracellular staining: Since LTA4H is primarily cytosolic, compare permeabilized to non-permeabilized samples to confirm proper access to intracellular compartments

  • Permeabilization efficiency control: Include a known intracellular marker to verify successful permeabilization

• Cell Population Controls:

  • For flow cytometry, use CD45 staining to distinguish leukocytes (CD45 high) from non-leukocytes (CD45 low) as demonstrated in published protocols

  • Include cells known to express high levels of LTA4H (e.g., neutrophils or monocytes) as positive controls

  • Include cell types with minimal LTA4H expression as negative controls

• Technical Controls:

  • Instrument calibration: Use calibration beads to ensure consistent fluorescence detection

  • Viability dye: Include a viability marker to exclude dead cells, which can bind antibodies non-specifically

  • Concentration optimization: Perform titration experiments to determine optimal antibody concentration for maximum signal-to-noise ratio

Implementing these comprehensive controls enables researchers to generate robust and reproducible data when using FITC-conjugated LTA4H antibodies in flow cytometry and immunofluorescence applications.

How can researchers optimize FITC-conjugated LTA4H antibody performance in challenging experimental conditions?

Optimizing FITC-conjugated LTA4H antibody performance in challenging experimental conditions requires addressing several technical considerations:

• Low Signal Intensity Solutions:

  • Increase antibody concentration after performing careful titration experiments

  • Extend incubation time (e.g., overnight at 4°C instead of 1-2 hours)

  • Enhance permeabilization efficiency for intracellular staining using optimized buffers

  • Use signal amplification methods such as biotin-streptavidin systems or tyramide signal amplification

  • For fluorescence microscopy, use anti-FITC antibodies conjugated to brighter fluorophores

• High Background Reduction Strategies:

  • Implement more stringent blocking with 5-10% serum from the same species as the secondary antibody

  • Add 0.1-0.3% Triton X-100 to wash buffers to reduce non-specific binding

  • Pre-absorb antibodies with tissue homogenates from the species being studied

  • Use additional blocking agents such as bovine serum albumin (BSA) or casein

  • Increase washing steps in number and duration

• Photobleaching Prevention:

  • Minimize exposure to light during all handling steps

  • Use anti-fade mounting media containing agents like p-phenylenediamine or proprietary anti-fade formulations

  • For flow cytometry, analyze FITC-stained samples first or use lower laser power

  • Consider using the antibody in conjunction with photostabilizing buffer systems

• Sample-specific Optimizations:

  • For fixed tissues: Optimize fixation time (over-fixation can mask epitopes)

  • For FFPE samples: Implement enhanced antigen retrieval methods (heat-induced or enzymatic)

  • For samples with high autofluorescence (e.g., lung tissue):

    • Use Sudan Black B (0.1-0.3%) to quench autofluorescence

    • Employ spectral unmixing on confocal microscopes

    • Consider alternative detection systems like alkaline phosphatase for problematic samples

• Buffer Compatibility:

  • The antibody is provided in buffer containing 50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300

  • Ensure experimental buffers are compatible with this formulation

  • When using the antibody in buffers of different composition, dialyze or dilute sufficiently to minimize buffer effects

By systematically implementing these optimization strategies, researchers can enhance the performance of FITC-conjugated LTA4H antibodies even in challenging experimental conditions, ensuring reliable and reproducible results.

How can LTA4H antibodies contribute to inflammation-related disease research, particularly in neutrophilic inflammation models?

LTA4H antibodies serve as critical tools for investigating the dual role of LTA4H in inflammation-related diseases, particularly in neutrophilic inflammation models. Research has demonstrated that LTA4H's aminopeptidase activity alleviates neutrophilic inflammation in cigarette smoke (CS)-induced emphysema models by clearing Proline-Glycine-Proline (PGP), highlighting the complex role of this enzyme in inflammatory regulation .

Methodological approaches for utilizing LTA4H antibodies in inflammation research include:

• Neutrophilic Inflammation Model Analysis:

  • Use FITC-conjugated LTA4H antibodies in flow cytometry to quantify LTA4H expression in neutrophil populations (identified as Ly6G high, CD11b high cells) under inflammatory conditions

  • Compare LTA4H expression between neutrophils from control versus cigarette smoke-exposed lung tissues

  • Correlate LTA4H levels with neutrophil activation markers and pro-inflammatory mediator production

• Dual Enzymatic Activity Differentiation:

  • Develop immunohistochemical or flow cytometric methods using LTA4H antibodies in conjunction with activity-based probes to distinguish between the pro-inflammatory epoxide hydrolase activity and the anti-inflammatory aminopeptidase activity

  • Apply selective inhibitors of each function to parse their distinct contributions to disease processes

• ELISA-based Biomarker Development:

  • Implement custom-developed LTA4H ELISA assays using paired antibodies (e.g., Novus EPR5713 for coating and R&D antibodies for detection) to quantify LTA4H in bronchoalveolar lavage fluid (BALF) and lung homogenates

  • Correlate LTA4H levels with disease severity indicators and neutrophil counts

• Mechanistic Studies:

  • Employ LTA4H antibodies in immunoprecipitation experiments to isolate LTA4H-interacting proteins that might modulate its function in inflammatory conditions

  • Use the identified interaction partners to elucidate regulatory mechanisms controlling the balance between pro- and anti-inflammatory LTA4H functions

Through these approaches, researchers can leverage LTA4H antibodies to gain deeper insights into the complex role of this enzyme in inflammatory diseases, potentially leading to novel therapeutic strategies targeting specific LTA4H functions or expression patterns in conditions characterized by neutrophilic inflammation.

What role might LTA4H play in cancer immunology, and how can researchers investigate this using appropriate antibody-based techniques?

LTA4H has emerged as a significant player in cancer immunology, with potential implications for tumor growth regulation and the development of immunotherapeutic approaches. Research indicates that LTA4H activation can influence antitumor immune responses through complex signaling cascades. Researchers can investigate these roles using the following antibody-based techniques:

• Tumor Microenvironment Characterization:

  • Employ multicolor flow cytometry with FITC-conjugated LTA4H antibodies to quantify expression across different immune cell populations within the tumor microenvironment

  • Use immunohistochemistry or multiplex immunofluorescence with LTA4H antibodies to map spatial distribution of LTA4H-expressing cells in relation to tumor cells and other immune infiltrates

  • Correlate LTA4H expression patterns with tumor progression and response to therapies

• Mechanistic Investigation of LTA4H in Antitumor Immunity:

  • Studies have shown that small-molecule drugs like P4N can inhibit tumor growth by activating LTA4H, leading to LTB4 production and subsequent induction of antitumor autoantibodies

  • Use flow cytometry with LTA4H antibodies to track changes in enzyme expression following treatment with LTA4H modulators

  • Implement ELISA to measure LTB4 production as a functional readout of LTA4H activation

  • Correlate LTA4H activation with downstream signaling events, including:

    • Production of proinflammatory cytokines (TNF-α, IL-8)

    • Activation of the activin A/BAFF pathway

    • B-cell activation and antibody production

• Macrophage-Centered Analysis:

  • Research has identified macrophages as direct targets for LTA4H modulators in antitumor responses

  • Use antibody-based cell sorting followed by functional assays to isolate and characterize LTA4H-expressing macrophages from tumor tissues

  • Perform macrophage depletion experiments (using methods like liposomal clodronate) combined with LTA4H functional assays to validate the role of macrophage-expressed LTA4H in tumor control

• Signaling Pathway Dissection:

  • Implement phospho-flow cytometry using LTA4H antibodies in combination with phospho-specific antibodies to track activation of downstream signaling molecules

  • Develop proximity ligation assays using LTA4H antibodies to identify and quantify protein-protein interactions within signaling complexes

  • Use chromatin immunoprecipitation (ChIP) assays to identify transcriptional targets regulated by LTA4H-initiated signaling cascades

• Therapeutic Response Correlation:

  • Quantify LTA4H expression in patient samples using antibody-based techniques and correlate with response to immunotherapies

  • Develop predictive models incorporating LTA4H status to identify patients who might benefit from combination therapies targeting the LTA4H pathway

These methodological approaches enable researchers to comprehensively investigate the multifaceted roles of LTA4H in cancer immunology, potentially leading to novel therapeutic strategies that harness this pathway for enhanced antitumor immunity.