mpo1 Antibody

What is myeloperoxidase (MPO) and why are MPO antibodies important research tools?

Myeloperoxidase is a 140kDa cationic protein primarily found in azurophilic granules of neutrophils and monocytes, accounting for up to 5% of total neutrophil protein. It generates antimicrobial chlorinated oxygen species from hydrogen peroxide produced during the neutrophil respiratory burst, playing a crucial role in the immune system's response to infections .

MPO antibodies are important research tools for several reasons:

• They enable detection and localization of MPO in various sample types

• They facilitate the study of neutrophil function and neutrophil extracellular traps (NETs)

• They allow investigation of ANCA-associated vasculitis pathogenesis

• They help distinguish between different types of acute leukemias

In research contexts, MPO antibodies like CPTC-MPO-1 are characterized mouse monoclonal antibodies that recognize specific MPO epitopes, allowing for consistent and reproducible experimental results when properly validated .

How do I select the appropriate anti-MPO antibody for my specific research application?

Selection of an appropriate anti-MPO antibody depends on several methodological considerations:

Table 1: Selection Criteria for Anti-MPO Antibodies in Research Applications

For optimal results, examine the antibody's characterization data. For example, CPTC-MPO-1 shows positive results in immunohistochemistry but negative results in immunofluorescence applications according to Human Protein Atlas evaluations , making it suitable for IHC but not IF studies.

The "five pillars" of antibody validation should guide your selection: (1) genetic strategies (knockout/knockdown controls), (2) orthogonal strategies (correlating with antibody-independent methods), (3) multiple antibody strategies (comparing independent antibodies), (4) recombinant expression strategies, and (5) immunocapture mass spectrometry .

What sample types can be effectively analyzed with anti-MPO antibodies?

Anti-MPO antibodies have been successfully used with various sample types, each requiring specific methodological considerations:

Tissue samples:

• Formalin-fixed paraffin-embedded (FFPE) tissue sections, particularly from spleen, bone marrow, and inflammatory sites

• Fresh-frozen tissue sections for applications where epitopes may be sensitive to fixation

• Tissue microarrays for high-throughput analysis

Cell samples:

• Neutrophil preparations (primary or cultured)

• Leukemia cell lines (e.g., HL-60, MOLT-4)

• Transfected cell lines expressing MPO

Biological fluids:

• Serum samples for detection of anti-MPO autoantibodies

• Neutrophil lysates for Western blot applications

• Purified MPO protein for standard curves and controls

Example protocol for immunohistochemistry: "Tissue Micro-Array (TMA) core of colon cancer showing cytoplasmic staining using Antibody CPTC-MPO-2. Titer: 1:1000" .

For optimal results with cell samples: "Myeloperoxidase/MPO was detected in immersion fixed MOLT-4 human acute lymphoblastic leukemia cell line using 8 µg/mL Mouse Anti-Human Myeloperoxidase/MPO Monoclonal Antibody for 3 hours at room temperature" .

What are recommended protocols for Western blot applications using anti-MPO antibodies?

When conducting Western blot analysis with anti-MPO antibodies, researchers should follow these methodological guidelines:

Sample preparation:

• Lyse cells in appropriate buffer (e.g., RIPA with protease inhibitors)

• For neutrophils, use caution to prevent degranulation during isolation

• Load 20-30μg of total protein per lane

Recommended protocol based on validated experiments:

• Separate proteins on 10-12% SDS-PAGE gels under reducing conditions

• Transfer to PVDF membrane (nitrocellulose alternatives may reduce signal)

• Block with 5% non-fat milk or BSA in TBST (1 hour, room temperature)

• Incubate with primary anti-MPO antibody at optimized dilution (typically 0.5-1.0 μg/mL) overnight at 4°C

• Wash 3x with TBST

• Incubate with appropriate HRP-conjugated secondary antibody

• Wash and develop using standard chemiluminescence methods

Example of expected results: "Western blot shows lysates of HL-60 human acute promyelocytic leukemia cell line, human neutrophil cells, and mouse spleen tissue. PVDF membrane was probed with 0.5 µg/mL of Goat Anti-Human/Mouse Myeloperoxidase/MPO Antigen Affinity-purified Polyclonal Antibody followed by HRP-conjugated Anti-Goat IgG Secondary Antibody. A specific band was detected for Myeloperoxidase/MPO at approximately 60 kDa" .

What controls should be included when using anti-MPO antibodies in research?

Robust experimental design with appropriate controls is critical for generating reliable data with anti-MPO antibodies:

Positive controls:

• HL-60 cells (human promyelocytic leukemia cell line with high MPO expression)

• Isolated human or mouse neutrophils

• Mouse spleen tissue sections

• Recombinant MPO protein

Negative controls:

• MPO-knockout cell lines (for genetic validation)

• Isotype control antibodies (matched to the primary antibody class)

• Cell lines known to be negative for MPO (e.g., some lymphoid lines)

• Secondary antibody-only controls to assess background

Additional validation controls:

• Peptide competition assays (pre-incubation with immunizing peptide should abolish specific signal)

• RNA expression correlation (compare protein detection with mRNA levels via RNAScope or similar techniques)

• Multiple antibody validation (compare results with different anti-MPO antibodies targeting different epitopes)

For immunohistochemistry: "Formalin-fixed paraffin-embedded tissue sections of mouse spleen were probed for MPO mRNA (ACD RNAScope Probe). Adjacent tissue section was processed for immunohistochemistry using anti-mouse MPO polyclonal antibody. Specific staining was localized to cell surface" .

What are the specific epitopes recognized by anti-MPO antibodies and how does this impact experimental design?

Understanding the epitope specificity of anti-MPO antibodies is crucial for experimental design and interpretation of results:

Key epitopes identified in MPO:

• Amino acids 213-222 (WTPGVKRNGF) - recognized by 33-58% of antibodies

• Amino acids 511-522 (RLDNRYQPMEPN) - recognized by 58.3% of antibodies

• Amino acids 393-402, 437-446, 479-488, and 717-726 - all reactive to the heavy chain structure

• Amino acids 91-100 (GSASPMELLS) - within the pro-peptide structure

Impact on experimental design:

• Epitope accessibility varies by technique - certain epitopes may be masked during fixation or denaturation

• Conformational epitopes may be lost in Western blot but preserved in immunoprecipitation

• Multiple antibodies targeting different epitopes should be used for confirmation of results

The distribution of epitope recognition shows high variability among patients with ANCA-associated vasculitis: "Males displayed a more diverse repertoire of antibody specificities than females, on average targeting 3.7 specificities compared with 1.2 in females" .

For researchers developing or selecting antibodies, structural considerations are important: "To visualize the location of the seven significant and common epitopes, to determine surface availability of these epitopes and to assess the proximity of these epitopes to functional regions of the protein we referred to the crystal structure model of MPO" .

How can researchers validate the specificity of anti-MPO antibodies to ensure experimental rigor?

Rigorous validation of anti-MPO antibodies is essential for experimental reproducibility and reliability. The following methodological approaches are recommended:

Comprehensive validation framework (based on the "five pillars" approach):

• Genetic validation:

  • Use MPO-knockout cell lines as negative controls

  • Employ siRNA/shRNA knockdown of MPO expression

  • Compare with overexpression systems

• Orthogonal validation:

  • Correlate antibody-based detection with mass spectrometry data

  • Compare with mRNA expression (qPCR, RNA-seq, or RNAScope)

  • Validate against enzymatic activity assays for MPO

• Independent antibody validation:

  • Compare results using antibodies targeting different MPO epitopes

  • Use antibodies from different host species or different clones

  • Compare monoclonal vs. polyclonal antibody performance

Example validation approach: "YCharOS also performs a number of other assays, emphasizing immunohistochemistry and Western Blots in rodent brains but also including KO mice, and samples from human brains when possible... They have generated antibodies directed towards more than 800 target proteins, using their system of immunohistochemistry, Western Blots, and immunofluorescence characterization" .

The importance of validation is underscored by concerning findings: "Shockingly, it also revealed that an average of ~12 publications per protein target included data from an antibody that failed to recognize the relevant target protein!" .

How do different fixation and sample preparation methods affect MPO antibody binding and signal integrity?

Different fixation and sample preparation methods can significantly impact antibody binding to MPO, affecting experimental outcomes:

Effects of fixation on MPO epitope accessibility:

"The p (peripheral)-ANCA staining pattern is an artefact produced when the MPO released from the granules by the ethanol used in fixation of the cells is attracted to the nucleus" .

Methodological recommendations:

• For IHC on FFPE tissues, antigen retrieval is critical (citrate buffer pH 6.0 or EDTA buffer pH 8.0)

• For Western blotting, reducing conditions are generally recommended

• For flow cytometry, permeabilization is necessary for intracellular MPO detection

• For immunofluorescence of NETs, fixation timing is critical to preserve extracellular structures

Example of fixation impact: "Using this substrate, anti-PR3 antibodies produce a granular cytoplasmic staining pattern, which is referred to as cANCA. In comparison, due to an artefact that is a result of the fixation process, anti-MPO antibodies display a perinuclear pattern (pANCA)" .

What methodological approaches can overcome cross-reactivity issues with anti-MPO antibodies?

Cross-reactivity is a significant concern with MPO antibodies, particularly because MPO shares structural similarities with other peroxidases. Researchers can employ several methodological strategies to address this issue:

Strategies to mitigate cross-reactivity:

• Peptide competition assays:

  • Pre-incubate antibody with purified MPO or immunizing peptide

  • Include graduated concentrations of competitor to demonstrate specificity

  • Compare with non-relevant peptide controls

• Expanded control panel:

  • Test antibody against related proteins (e.g., eosinophil peroxidase)

  • Include samples from MPO-knockout models

  • Test across multiple tissue/cell types to identify non-specific binding patterns

• Optimized assay conditions:

  • Titrate antibody concentration to minimize non-specific binding

  • Adjust blocking reagents (5% BSA often superior to milk for peroxidase detection)

  • Modify washing stringency to reduce background

Evidence of specificity testing: "In direct ELISAs, less than 1% cross-reactivity with recombinant mouse EPPO is observed" and "No cross-reactivity with recombinant human Eosinophil Peroxidase is observed" .

For advanced applications, computational analysis can improve antibody specificity: "Using data from phage display experiments, we show that the model successfully disentangles these modes, even when they are associated with chemically very similar ligands" .

How can anti-MPO antibodies be effectively used to study neutrophil extracellular traps (NETs)?

Neutrophil extracellular traps (NETs) represent an important area of immunological research where anti-MPO antibodies serve as crucial tools. Here are methodological considerations for using these antibodies in NET studies:

Optimized protocol for NET visualization using anti-MPO antibodies:

• Sample preparation:

  • Isolate neutrophils using density gradient centrifugation

  • Stimulate NETs with PMA (100nM), calcium ionophore, or pathogens

  • Fix carefully (4% PFA, 10 minutes) to preserve extracellular structures

• Immunostaining approach:

  • Block with 3-5% BSA in PBS (1 hour, room temperature)

  • Co-stain with anti-MPO antibody (5-10 μg/mL) and DNA markers (DAPI)

  • Include additional NET markers (e.g., citrullinated histone H3)

  • Use confocal microscopy for optimal visualization

• Quantification methods:

  • Count NET-forming neutrophils (cells showing MPO-DNA colocalization)

  • Express as percentage of total neutrophil population

  • Measure NET area using image analysis software

Example results from validated experiments: "Immunofluorescence staining of myeloperoxidase (MPO, green), citrullinated histone H4 (citH4, red), extracellular DNA (DAPI, blue) and their merged images are shown... Based on nuclear morphology and co-staining with MPO and citH4, NET-forming PMNs were identified and their numbers quantitated compared to the total cell population (expressed as percentage of total)" .

This approach allows quantitative assessment of NET formation in various experimental conditions, including pathogen exposure and pharmacological interventions.

How do anti-MPO monoclonal antibodies differ from autoantibodies detected in ANCA-associated vasculitis?

It's critical for researchers to understand the distinction between research tools (anti-MPO monoclonal antibodies) and the autoantibodies detected in clinical samples:

Comparative characteristics:

"One study generated multiple human–mouse MPO chimera to examine regions of antibody specificity, while another found that MPO-ANCA recognize epitopes on..." .

Clinical detection systems:

• Indirect immunofluorescence on ethanol-fixed neutrophils (perinuclear pattern)

• Antigen-specific solid-phase immunoassays (ELISA, fluorescence immunoassay)

• Multiplex bead-based assays

"The presence of PR3-ANCA and MPO-ANCA can be detected using antigen-specific immunoassays or indirect immunofluorescence (IIF). IIF is typically performed using ethanol-fixed neutrophils" .

What are the most advanced methodologies for using anti-MPO antibodies in multiplexed immunoassays?

Multiplexed immunoassays represent an advanced application of anti-MPO antibodies that allows simultaneous detection of multiple analytes. Here are methodological approaches:

Cutting-edge multiplexed approaches:

• Microsphere-based multiplex assays:

  • "Myeloperoxidase (MPO) antigen is covalently coupled to polystyrene microspheres that are impregnated with fluorescent dyes to create a unique fluorescent signature"

  • "The microspheres are washed to remove unbound conjugate, and bound conjugate is detected by laser photometry"

  • "A primary laser reveals the fluorescent signature of each microsphere to distinguish it from microspheres that are labeled with other antigens"

• Multi-epitope profiling:

  • Use multiple anti-MPO antibodies targeting different epitopes

  • Combine with antibodies against other neutrophil markers (elastase, proteinase 3)

  • Apply machine learning algorithms to patterns for improved specificity

• Mass cytometry (CyTOF) applications:

  • Label anti-MPO antibodies with rare earth metals

  • Combine with >40 other cellular markers simultaneously

  • Provides single-cell resolution of MPO expression in heterogeneous populations

These advanced techniques allow researchers to place MPO expression and activity in broader cellular and pathophysiological contexts.

How can researchers troubleshoot inconsistent results when using anti-MPO antibodies?

When faced with inconsistent results, researchers should systematically troubleshoot using the following methodological framework:

Structured troubleshooting approach:

• Antibody validation issues:

  • Verify antibody specificity using knockout/knockdown controls

  • Test multiple antibody lots if inconsistency correlates with lot changes

  • Consider epitope masking in your specific application

• Sample preparation variables:

  • Ensure consistent fixation times and conditions

  • Verify protein denaturation conditions for Western blot

  • Check for proteolytic degradation during sample processing

• Technical optimization:

  • Titrate antibody concentration systematically

  • Adjust blocking reagents (BSA vs. milk)

  • Modify incubation times and temperatures

  • Consider alternative detection systems

• Experimental design improvements:

  • Include additional positive and negative controls

  • Implement orthogonal detection methods

  • Ensure biological replicates span potential variables

The importance of validation is underscored by recent findings: "It has been estimated that ~50% of commercial antibodies fail to meet even basic standards for characterization, and this problem is thought to result in financial losses of $0.4–1.8 billion per year in the United States alone" .

What are the considerations for developing highly specific anti-MPO recombinant antibodies?

The development of recombinant anti-MPO antibodies represents an advanced research direction with significant advantages over traditional monoclonal antibodies:

Methodological considerations for recombinant antibody development:

• Selection strategy:

  • Phage display libraries offer high-throughput screening capabilities

  • "Our approach involves the identification of different binding modes, each associated with a particular ligand against which the antibodies are either selected or not"

  • "Using data from phage display experiments, we show that the model successfully disentangles these modes, even when they are associated with chemically very similar ligands"

• Epitope targeting:

  • Focus on accessible, conserved regions for consistent detection

  • Target unique epitopes to minimize cross-reactivity with related peroxidases

  • Consider engineering antibodies against multiple epitopes: "The decapeptides that make up the defined epitope sequences had an average pI of 6.45, while the average pI for the remaining decapeptides equalled 7.11"

• Validation requirements:

  • "These data demonstrate the means for both identifying useful reagents and removing bad ones"

  • "This study showed the value of recombinant antibodies, demonstrating that on average they outperformed both monoclonal and polyclonal antibodies in all the assays used"

• Format considerations:

  • scFv fragments for improved tissue penetration

  • Full IgG format for applications requiring Fc-mediated functions

  • Fusion proteins for specialized applications