LTC4S Antibody, Biotin conjugated

What is LTC4S and what is its significance in inflammatory pathways?

LTC4S (Leukotriene C4 synthase) is a membrane-bound enzyme that catalyzes the conjugation of the unstable epoxide leukotriene A4 (LTA4) with glutathione to form leukotriene C4 (LTC4). This reaction represents a critical step in the biosynthesis of cysteinyl leukotrienes (cysLTs), which include LTC4, LTD4, and LTE4 . These inflammatory mediators play significant roles in various pathological conditions, most notably asthma . The enzyme functions within the 5-lipoxygenase pathway of arachidonic acid metabolism and has been shown to have higher activity compared to LTA4 hydrolase (LTA4H) in certain tissues such as abdominal aortic aneurysm (AAA) walls .

How do biotin-conjugated antibodies enhance LTC4S detection in research applications?

Biotin-conjugated antibodies targeting LTC4S facilitate sensitive and specific detection through the strong and stable interaction between biotin and streptavidin. In detection systems like ELISA, the biotinylated antibody serves as a secondary detection reagent that binds to LTC4S captured by a primary antibody . The biotin conjugation enables amplification of the detection signal when combined with HRP-streptavidin conjugates (SABC), significantly enhancing sensitivity without increasing background noise. This system allows for precise quantification of LTC4S expression levels across various experimental and clinical samples with detection limits in the picogram range .

What sample types are appropriate for LTC4S antibody applications?

LTC4S antibodies can be effectively used with a variety of biological samples, including:

• Tissue homogenates - particularly useful for comparing LTC4S expression in normal versus pathological tissues, as demonstrated in AAA wall studies

• Subcellular fractions - membrane fractions are especially relevant since LTC4S is membrane-bound, allowing for direct assessment of enzyme activity

• Cell lysates - from primary cells or cell lines expressing LTC4S

• Serum and plasma - for detection of circulating LTC4S in certain pathological conditions

• Bronchoalveolar lavage fluid - particularly relevant for asthma and respiratory inflammation research

Sample preparation protocols should be optimized to preserve the structural integrity of LTC4S, especially when dealing with membrane fractions where ultracentrifugation techniques may be necessary to isolate enzyme-rich preparations .

What is the optimal protocol for using biotin-conjugated LTC4S antibodies in sandwich ELISA?

The optimal protocol for sandwich ELISA using biotin-conjugated LTC4S antibodies involves several critical steps:

• Microplate preparation: Use plates pre-coated with anti-LTC4S capture antibody

• Sample addition: Add properly diluted samples and standards to appropriate wells

• Primary incubation: Typically at 37°C for 60-90 minutes

• Washing: Remove unbound components using appropriate buffer (PBS-Tween)

• Secondary antibody application: Add biotin-conjugated detection antibody specific to LTC4S

• Secondary incubation: Usually 60 minutes at 37°C

• Washing: Remove unbound secondary antibody

• Signal development: Add HRP-Streptavidin Conjugate (SABC) followed by TMB substrate solution

• Signal detection: Measure absorbance at 450nm after adding stop solution

• Analysis: Calculate LTC4S concentration using standard curve

The entire assay typically takes approximately 4 hours to complete . Temperature control and precise timing are critical for reproducible results. For competitive ELISA formats detecting LTC4 rather than the enzyme itself, the protocol differs slightly but still utilizes biotin-labeled components with a total assay time of approximately 2 hours .

How can researchers validate the specificity of LTC4S antibodies in experimental systems?

Validating antibody specificity for LTC4S requires a multi-faceted approach:

• Positive and negative controls:

  • Use recombinant LTC4S protein as a positive control

  • Compare with samples from LTC4S knockout models or cells as negative controls

• Cross-reactivity testing:

  • Test against related proteins, particularly other glutathione S-transferases

  • Verify no signal generation in systems known to lack LTC4S expression

• Functional validation:

  • Correlate antibody detection with enzyme activity measurements

  • Conduct activity assays with LTA4 substrate and glutathione to verify the detected protein is functionally active

• Western blot analysis:

  • Confirm a single band at the expected molecular weight (~18 kDa for human LTC4S)

  • Perform peptide competition assays to demonstrate binding specificity

• Immunoprecipitation followed by mass spectrometry:

  • Use the antibody to pull down the target protein

  • Confirm identity via peptide sequencing

What are the critical parameters for optimizing LTC4S detection sensitivity?

To achieve optimal sensitivity when detecting LTC4S using biotin-conjugated antibodies, researchers should consider these critical parameters:

• Antibody concentration: Titrate the biotin-conjugated antibody to determine optimal working concentration that maximizes signal-to-noise ratio

• Sample preparation: For membrane proteins like LTC4S, proper extraction methods are crucial:

  • Use appropriate detergents for solubilization

  • Consider ultracentrifugation (100,000 × g for 60 minutes at 4°C) for subcellular fractionation to isolate membrane-bound LTC4S

• Blocking conditions: Optimize blocking buffer composition to minimize background while preserving specific binding

• Incubation parameters:

  • Temperature (typically 37°C)

  • Time (60-90 minutes for most steps)

  • Gentle agitation to ensure even distribution of reagents

• Washing stringency: Balance between removing nonspecific binding while preserving specific interactions

• Signal amplification: Utilize optimal streptavidin-HRP concentration and appropriate substrate development time

• Standard curve preparation: Use a wide range of standards (e.g., 15.625-1000pg/ml) to ensure accurate quantification across various sample concentrations

How can biotin-conjugated LTC4S antibodies be used to investigate the relationship between enzyme structure and function?

Biotin-conjugated LTC4S antibodies provide valuable tools for exploring structure-function relationships through various approaches:

• Site-specific antibodies: Develop biotin-conjugated antibodies targeting different epitopes to probe the accessibility of functional domains in various conformational states of LTC4S

• Immunoprecipitation studies: Use antibodies to isolate native LTC4S complexes from biological samples to:

  • Identify binding partners through co-immunoprecipitation

  • Study post-translational modifications affecting enzyme activity

  • Investigate oligomeric states of the enzyme in situ

• Immuno-electron microscopy: Use biotin-conjugated antibodies with gold-labeled streptavidin to visualize LTC4S localization at the ultrastructural level, particularly its association with nuclear membranes and endoplasmic reticulum

• Comparative analysis of mutant forms: Apply antibodies to detect and quantify expression of wild-type versus mutated LTC4S (such as the Trp-116 mutants), correlating structural changes with altered enzyme activity

• Conformational epitope mapping: Use panels of antibodies to detect structural changes upon substrate binding or in different physiological conditions

These approaches complement crystallographic studies like those revealing LTC4S complexed with product analogs such as S-hexyl-, 4-phenyl-butyl-, and 2-hydroxy-4-phenyl-butyl-glutathione .

What insights can be gained from comparative analysis of LTC4S versus LTA4H expression using antibody-based techniques?

Comparative analysis of LTC4S and LTA4H expression using antibody-based techniques can reveal critical insights into leukotriene pathway regulation:

• Pathway balance: Quantitative immunoassays can determine the relative expression of LTC4S versus LTA4H, which compete for the same substrate (LTA4), directing metabolism toward either cysteinyl leukotrienes (LTC4S) or LTB4 (LTA4H)

• Cell-specific expression patterns: Immunohistochemistry using specific antibodies can map the distribution of both enzymes across different cell types in tissues, revealing which cells predominantly produce cysLTs versus LTB4

• Disease-specific alterations: Changes in the LTC4S/LTA4H ratio in pathological conditions can be quantified, as demonstrated in AAA tissue where significantly higher LTC4S activity compared to LTA4H was observed

• Regulatory mechanisms: Co-immunoprecipitation using antibodies against either enzyme can identify shared or distinct regulatory proteins that control their relative activities

• Temporal dynamics: Time-course studies using antibody detection can reveal how the balance between these competing enzymes shifts during inflammatory responses or disease progression

This comparative approach can significantly enhance our understanding of inflammatory diseases where the balance between different leukotriene classes plays a pathogenic role.

How do researchers measure LTC4S activity in correlation with protein expression detected by antibodies?

Correlating LTC4S enzyme activity with protein expression levels detected by antibodies requires complementary experimental approaches:

• Enzyme activity assays:

  • Direct measurement of LTC4 formation from LTA4 and glutathione using HPLC coupled with enzyme immunoassay (EIA)

  • Determination of kinetic parameters (kcat and Km) for both substrates (GSH and LTA4)

  • Quantification based on the ratio of peak areas compared with internal standards like prostaglandin B2

• Protein quantification:

  • ELISA using biotin-conjugated LTC4S antibodies to determine absolute protein levels

  • Western blot analysis with densitometry for relative expression quantification

  • Flow cytometry for cell-specific expression analysis

• Correlation methods:

  • Calculate specific activity (enzyme activity per unit protein)

  • Regression analysis comparing activity versus expression across samples

  • Analysis of activity/expression ratios under different experimental conditions

• Confounding factors:

  • Account for post-translational modifications affecting enzyme activity

  • Consider subcellular localization, as only properly localized enzyme will be active

  • Factor in the presence of endogenous inhibitors or activators

For example, in AAA tissue studies, both LTC4S activity (measured by LTC4 production) and expression levels were elevated, with activity measurements showing approximately 2-fold higher formation of LTC4 (2.14 pmol/μg protein) compared to LTB4 (1.10 pmol/μg protein) .

What are emerging applications of LTC4S antibodies in studying inflammatory disease mechanisms?

LTC4S antibodies are increasingly being applied to investigate novel aspects of inflammatory disease mechanisms:

• Single-cell analysis of inflammatory heterogeneity:

  • Mass cytometry (CyTOF) incorporating LTC4S antibodies to identify specific inflammatory cell subsets

  • Single-cell RNA-seq combined with protein detection using antibodies to correlate transcriptional programs with LTC4S expression

• Spatial transcriptomics and proteomics:

  • In situ hybridization combined with LTC4S immunodetection to map expression patterns in tissue microenvironments

  • Multiplexed immunofluorescence to analyze co-expression with other inflammatory mediators

• Therapeutic antibody development:

  • LTC4S neutralizing antibodies as potential therapeutics for asthma and other inflammatory conditions

  • Testing the efficacy of therapeutic antibodies using biotin-conjugated LTC4S antibodies as pharmacodynamic markers

• Cardiovascular inflammation research:

  • Studying the role of LTC4S in vascular inflammation, particularly in conditions like abdominal aortic aneurysm where predominant formation of cysteinyl-leukotrienes has been observed

  • Correlating LTC4S expression with markers of cardiovascular disease progression

• Precision medicine approaches:

  • Using LTC4S antibodies to develop companion diagnostics for leukotriene-modifying drugs

  • Stratifying patients based on LTC4S expression levels to predict therapeutic responses

These emerging applications highlight the expanding role of LTC4S antibodies beyond traditional research applications into translational and clinical domains.