LPO Antibody

What is LPO and Why are LPO Antibodies Essential for Research?

Lactoperoxidase (LPO) is a heme peroxidase enzyme secreted from mammary, salivary, and other mucosal glands including the lungs, bronchii, and nose. It functions as a natural first line of defense against antibacterial and antiviral agents. In humans, lactoperoxidase is encoded by the LPO gene, which is part of a gene cluster on chromosome 17 .

LPO antibodies are critical research tools that enable:

• Detection and quantification of LPO expression across different tissue types

• Investigation of antimicrobial defense mechanisms

• Analysis of peroxidase enzyme activity in various physiological and pathological conditions

• Examination of mucosal immunity in gastrointestinal, respiratory, and mammary tissues

The molecular weight of LPO observed in Western blot applications is approximately 80 kDa, slightly larger than the calculated molecular weight of 75679 Da, likely due to post-translational modifications and retention of leader sequences .

Common Molecular Properties of LPO

What Applications are Most Suitable for LPO Antibody Use in Laboratory Research?

LPO antibodies have demonstrated efficacy in multiple laboratory applications, with specific protocols optimized for each technique. Based on validated experimental data, the following applications have proven most reliable:

Western Blot (WB)

LPO antibodies can effectively detect the protein in reducing conditions. Optimal protocols include:

• Sample preparation: 30 μg of protein under reducing conditions

• Gel conditions: 5-20% SDS-PAGE gel at 70V (stacking)/90V (resolving)

• Transfer: Nitrocellulose membrane at 150 mA for 50-90 minutes

• Blocking: 5% non-fat milk/TBS for 1.5 hour at room temperature

• Primary antibody: 0.5 μg/mL overnight at 4°C

• Detection: Enhanced Chemiluminescent detection (ECL) systems

Immunofluorescence (IF) and Immunocytochemistry (ICC)

For cellular localization studies, the following parameters yield optimal results:

• Antigen retrieval: Enzymatic retrieval for 15 minutes

• Blocking: 10% goat serum

• Primary antibody concentration: 5 μg/mL incubated overnight at 4°C

• Secondary antibody: Fluorophore-conjugated anti-rabbit IgG at 1:100 dilution

• Counterstaining: DAPI for nuclear visualization

Immunohistochemistry (IHC)

For tissue section analysis:

• Fixation: 10% phosphate-buffered formalin overnight

• Detection system: Biotin-streptavidin-HRP complex

• Visualization: 3,3′-diaminobenzidine

• Counterstaining: Mayer's hematoxylin for tissue architecture

These applications collectively enable comprehensive analysis of LPO expression and localization across various experimental systems.

How Can Researchers Verify the Specificity of an LPO Antibody?

Verifying antibody specificity is crucial for generating reliable research data. For LPO antibodies, several validation approaches have been established:

Positive and Negative Controls

• Positive control: Tissues known to express LPO (salivary glands, mammary tissue, colon epithelium)

• Negative control: Tissues with minimal LPO expression (such as liver or lung tissue in wild-type mice)

• Cell line controls: Transfected versus untransfected cell lines (e.g., CMT-93 cells)

Cross-Reactivity Testing

A critical consideration is potential cross-reactivity with other peroxidases, particularly thyroid peroxidase (TPO). Studies have demonstrated that:

• Human LPO-specific antibodies (e.g., 10376-1-AP) do not recognize human TPO

• TPO-specific monoclonal antibodies recognizing linear epitopes (mAb 47, mAb A4, and ab76935) do not bind to bovine LPO

Preabsorption Experiments

To validate specificity, antibodies can be preabsorbed with purified LPO protein before application in experimental procedures. A significant reduction in signal following preabsorption confirms specificity for the target antigen .

Multiple Antibody Validation

Using multiple antibodies targeting different epitopes of LPO can provide additional confidence in specificity. Consistent results across different antibodies strongly support specific detection of the target protein.

What are Best Practices for Storage and Handling of LPO Antibodies?

Proper storage and handling of LPO antibodies are essential for maintaining functionality and extending shelf life. Based on manufacturer recommendations and research protocols, the following practices should be followed:

Storage Conditions

• Lyophilized antibodies: Store at -20°C for up to one year from receipt date

• Reconstituted antibodies: Store at 4°C for up to one month or aliquot and store at -20°C for up to six months

• Avoid repeated freeze-thaw cycles which significantly degrade antibody performance

Reconstitution Protocol

• Add 0.2 ml of distilled water to lyophilized antibody to yield a concentration of 500 μg/ml

• Allow complete dissolution before use

• Note that each vial typically contains buffer components (e.g., 4 mg Trehalose, 0.9 mg NaCl, 0.2 mg Na2HPO4)

Working Dilution Optimization

Optimal working dilutions vary by application:

• Western blot: 0.5 μg/mL (approximately 1:1000 dilution)

• Immunofluorescence: 5 μg/mL (approximately 1:100 dilution)

• Immunohistochemistry: Dilutions should be optimized for each tissue type

Quality Control Considerations

• Always include positive and negative controls in each experiment

• Monitor for batch-to-batch variations by maintaining reference samples

• Document lot numbers and maintain consistency within experimental series when possible

How Does LPO Expression Vary Across Different Tissues and Under Different Physiological Conditions?

LPO expression demonstrates significant tissue-specific and condition-dependent variations. Comprehensive studies using validated antibodies have revealed important patterns:

Tissue-Specific Expression

Analysis of gastrointestinal tissues shows distinct expression patterns:

• Colon: Highest expression levels in both normal and inflammatory conditions

• Rectum: Moderate to high expression

• Ileum: Lower but detectable expression

• Stomach: Minimal detection

• Liver/Lung: Typically negative for LPO expression

Strain-Dependent Variations

Significant strain-dependent differences have been observed in mouse models:

• 129 strain mice: 4.7-8.2 fold higher LPO protein levels in colon compared to B6 strain

• This variation exists independent of disease status

Quantitative Expression Data

The following table summarizes relative LPO mRNA expression levels across different tissues and mouse strains:

Note: DKO refers to glutathione peroxidase double knockout mice (GPx1/2-deficient)

These expression patterns suggest tissue-specific regulatory mechanisms and potential roles for LPO in intestinal homeostasis.

What Methodological Considerations Apply When Using LPO Antibodies in Inflammation or Oxidative Stress Studies?

When investigating LPO in inflammation or oxidative stress contexts, several methodological considerations become particularly important:

Control for Inflammatory Status

• Always characterize inflammatory status of tissues using established markers

• Consider that inflammation per se does not necessarily accelerate LPO DNA hypermethylation in mouse colon

• Include both inflamed and non-inflamed tissues from the same model system

Epigenetic Regulation Analysis

LPO gene expression is subject to epigenetic regulation:

• The LPO intragenic CpG island can undergo aberrant hypermethylation

• LPO gene expression can be suppressed by Bmi1, a component of Polycomb repressive complex 1 (PRC1)

• Consider assessing methylation status alongside protein expression

Knockout Model Considerations

When using knockout models:

• GPx1/2-double knockout (DKO) mice develop spontaneous ileocolitis

• Disease severity is strain-dependent (B6 strain: mild; 129 strain: severe)

• The glutathione peroxidase status significantly affects LPO expression patterns

Quantification Methods

For reliable quantification:

• Western blot: Normalize LPO signal to appropriate housekeeping proteins (e.g., β-actin)

• RT-PCR: Use multiple reference genes for normalization

• Consider both protein and mRNA levels, as post-transcriptional regulation may occur

How Can Researchers Distinguish Between LPO and Other Peroxidases in Immunohistochemical Studies?

Distinguishing between different peroxidases, particularly LPO and TPO, presents a significant challenge in immunohistochemical studies due to structural similarities. Several approaches can help ensure specificity:

Antibody Selection and Validation

• Use antibodies that have been experimentally validated for specificity

• Human LPO-specific antibody (10376-1-AP) has been demonstrated not to cross-react with human TPO

• TPO-specific monoclonal antibodies recognizing linear epitopes (mAb 47, mAb A4, and ab76935) do not bind to bovine LPO

Epitope Analysis

• Consider the specific epitope recognized by the antibody

• For polyclonal antibodies, validate using multiple monoclonal antibodies to different epitopes

• Antibodies generated against synthetic peptides from middle regions of human LPO show good specificity

Preabsorption Controls

• Perform preabsorption controls with purified LPO protein

• Test cross-absorption with other peroxidases to confirm specificity

• Include both positive and negative controls in every experimental setup

Tissue-Specific Expression Patterns

Different peroxidases exhibit distinct tissue expression patterns:

• LPO: Predominantly in mammary, salivary, and mucosal glands

• TPO: Predominantly in thyroid tissue

• These tissue-specific patterns can serve as internal controls

Subcellular Localization

• LPO is often secreted or associated with apical membranes in epithelial cells

• TPO typically shows apical membrane staining in thyroid follicular cells

• Careful analysis of subcellular localization can provide additional differentiation

What Advanced Techniques are Available for Studying LPO Localization and Function?

Advanced techniques can provide deeper insights into LPO localization, interactions, and functions beyond standard immunodetection methods:

High-Resolution Imaging Techniques

• Super-resolution microscopy: Enables visualization of LPO at subcellular compartments

• Correlative light and electron microscopy (CLEM): Combines immunofluorescence with ultrastructural analysis

• Live-cell imaging: Can track LPO trafficking in real-time using fluorescently tagged antibodies

Molecular Interaction Studies

• Proximity ligation assay (PLA): Detects protein-protein interactions in situ

• Co-immunoprecipitation: Identifies LPO binding partners

• FRET-based approaches: Measures molecular proximity between LPO and potential interactors

Functional Activity Assays

• In situ activity assays: Measure peroxidase activity in tissue sections

• Combined immunodetection and activity staining: Correlates protein presence with enzymatic function

• Enzyme kinetics: Quantifies LPO activity under different experimental conditions

Multi-omics Integration

• Combined antibody-based detection with:

  • Transcriptomics: Correlates protein expression with mRNA levels

  • Proteomics: Places LPO in broader protein networks

  • Epigenomics: Links DNA methylation status with LPO expression levels

How Can Biophysics-Informed Modeling Improve LPO Antibody Design for Enhanced Specificity?

Recent advances in computational modeling have opened new avenues for antibody design and optimization, particularly for achieving enhanced specificity:

Computational Approaches for Antibody Design

• Biophysics-informed models can disentangle multiple binding modes associated with specific ligands

• By identifying distinct binding modes for each potential ligand, researchers can predict and generate antibody variants with enhanced specificity

• These approaches extend beyond the limitations of traditional experimental selection methods

Experimental Validation of Computational Designs

The effectiveness of computational design has been demonstrated through:

• Phage display experiments involving selection against diverse combinations of closely related ligands

• Validation of computationally designed antibody variants not present in initial libraries

• Generation of antibodies with customized specificity profiles

Optimization Strategies for LPO-Specific Antibodies

To generate LPO-specific antibodies that avoid cross-reactivity with other peroxidases:

• Complementarity determining regions (CDRs), particularly CDR3, can be systematically varied

• Energy functions associated with each binding mode can be optimized

• For specific binding: Minimize energy functions associated with desired ligand while maximizing those for undesired ligands

• For cross-specific binding: Jointly minimize energy functions associated with multiple desired ligands

Applications of Enhanced Specificity Antibodies

Highly specific LPO antibodies enable:

• More precise localization studies in tissues with multiple peroxidases

• Accurate quantification of LPO in complex biological samples

• Reliable functional studies with reduced interference from related enzymes

• Development of diagnostic applications requiring high specificity