PRTN3 Antibody, HRP conjugated

What is PRTN3 and why is it a significant target for antibody-based detection in research?

PRTN3 (Proteinase 3) is a serine protease with a canonical length of 256 amino acid residues and a molecular weight of approximately 27.8 kDa in humans. It belongs to the Peptidase S1 protein family and is primarily expressed in polymorphonuclear leukocytes. PRTN3 plays important biological roles by degrading extracellular matrix components including elastin, fibronectin, laminin, vitronectin, and collagen types I, III, and IV .

PRTN3 has become a significant research target for several reasons:

• It serves as a crucial autoantigen in ANCA-Associated Vasculitis (AAV), where anti-neutrophil cytoplasmic autoantibodies target PRTN3

• PRTN3 protein expression is significantly elevated in certain cancers, particularly lung adenocarcinoma (LUAD)

• It functions as a biological marker for promyelocytes in hematological research

• Alternative transcripts from the PRTN3 gene locus may produce different protein isoforms with distinct biological functions

For these reasons, HRP-conjugated PRTN3 antibodies are valuable tools for investigating PRTN3's role in normal physiology and various pathological conditions.

What experimental applications are most suitable for HRP-conjugated PRTN3 antibodies?

HRP-conjugated PRTN3 antibodies are versatile tools across multiple experimental applications:

• Western Blotting: The HRP conjugation enables sensitive chemiluminescent detection of PRTN3 protein in various sample types. Western blotting has confirmed elevated PRTN3 expression in LUAD tissue samples and can detect both canonical and alternative forms of PRTN3 .

• ELISA: HRP-conjugated PRTN3 antibodies are instrumental in developing sensitive enzyme-linked immunosorbent assays to quantify PRTN3 levels in biological fluids or to detect anti-PRTN3 autoantibodies in patient samples. This approach has been successfully employed to measure elevated anti-PRTN3 IgG and IgM autoantibodies in LUAD patients .

• Immunohistochemistry (IHC): These antibodies enable visualization of PRTN3 expression in tissue sections, allowing researchers to study its distribution and abundance in different cell types. IHC has demonstrated significantly higher PRTN3 protein expression in LUAD tissues compared to para-carcinoma and normal control tissues .

• Immunofluorescence (IF): HRP-conjugated primary antibodies can be used for immunofluorescence staining after substrate development. This application has confirmed immunoreactivity of LUAD plasma to PRTN3 in LUAD cells .

When selecting an application, researchers should consider the cellular localization of PRTN3 (cell membrane, cytoplasm, and secreted forms) and optimize protocols accordingly .

What controls should be included when designing experiments with HRP-conjugated PRTN3 antibodies?

Robust experimental design with appropriate controls is essential when working with HRP-conjugated PRTN3 antibodies:

Positive Controls:

• Commercial recombinant PRTN3 protein at known concentrations

• Cell lines with validated PRTN3 expression (e.g., polymorphonuclear leukocytes)

• For autoantibody detection, include monoclonal anti-PRTN3 antibody as demonstrated in validation studies

Negative Controls:

• Isotype control antibodies (matched to the PRTN3 antibody class and species)

• Samples from tissues known to have minimal PRTN3 expression

• For blocking experiments, pre-incubation with recombinant PRTN3 protein

Technical Controls:

• HRP substrate-only controls to assess background signal

• Secondary antibody-only controls (when using unconjugated primary antibodies)

• Loading controls for western blotting (housekeeping proteins)

Research has shown that PRTN3 expression varies significantly between tissue types, with higher expression in LUAD compared to para-carcinoma tissues and normal controls . This differential expression pattern can serve as an internal validation for antibody specificity in tissue-based experiments.

What are the optimal sample preparation techniques for PRTN3 detection using HRP-conjugated antibodies?

Sample preparation is critical for successful PRTN3 detection and varies by application:

For Western Blotting:

• Use lysis buffers containing protease inhibitors to prevent PRTN3 degradation

• Consider subcellular fractionation to separate membrane, cytoplasmic, and secreted forms of PRTN3

• For detection of alternative transcripts or protein isoforms, optimize protein separation conditions to resolve the canonical 27.8 kDa PRTN3 from variant forms

For ELISA:

• For plasma/serum samples, dilution ratios between 1:100 and 1:500 have been validated for anti-PRTN3 autoantibody detection

• Fresh or properly stored samples are essential as freeze-thaw cycles may affect protein stability

For Immunohistochemistry:

• Formalin-fixed, paraffin-embedded (FFPE) tissues have been successfully used for PRTN3 detection in LUAD tissue arrays

• Antigen retrieval methods should be optimized, as PRTN3 epitopes may be sensitive to fixation-induced masking

For Immunofluorescence:

• A549 cells have been successfully used for detecting PRTN3 immunoreactivity

• Fixation with 4% paraformaldehyde followed by permeabilization is typically effective

Researchers should note that post-translational modifications, particularly glycosylation, have been reported for PRTN3 . These modifications may affect antibody binding and should be considered during sample preparation.

How should researchers interpret variations in PRTN3 detection across different samples?

When interpreting variations in PRTN3 detection, researchers should consider several biological and technical factors:

Biological Factors:

• PRTN3 expression correlates with pathological grade in LUAD, with stronger expression in G2 and G3 LUAD tissues compared to G1

• Alternative transcripts from the PRTN3 gene locus may produce different protein isoforms with distinct mobility patterns in gel electrophoresis

• Developmental regulation may affect PRTN3 expression patterns; an alternative promoter within intron 1 of the PRTN3 gene is active in bone marrow, leukemia cell lines, and after GM-CSF treatment

Technical Considerations:

• Quantify relative expression using appropriate normalization controls

• For Western blotting, PRTN3 may present as multiple bands due to alternative transcripts, post-translational modifications, or proteolytic processing

• When analyzing autoantibody responses, consider both IgG and IgM antibodies against PRTN3, as both have diagnostic value in conditions like LUAD

Interpretation Table for PRTN3 Expression Patterns:

How can researchers differentiate between alternative PRTN3 transcripts and protein isoforms?

The PRTN3 gene locus produces multiple transcripts through alternative promoter usage and mRNA processing, presenting a complex challenge for researchers:

Methodological Approach:

• Transcript-Specific PCR: Design primers targeting unique regions of alternative transcripts, such as:

  • Primers spanning the alternative 3' UTR containing the 68-bp repeat with a 12-bp spacer identified in some patients with AAV

  • Primers detecting transcripts originating from the alternative transcription start site within intron 1 (PRTN3-002)

• Western Blotting Strategy:

  • Use gradient gels (10-15%) to resolve closely related protein isoforms

  • The canonical PR3 and alternative myeloblastin (MBN) protein have different N-terminal sequences while sharing high homology elsewhere

  • A 24 kDa protein (p24 PR3/MBN) has been detected using patient sera and may represent an antigenic isoform

• Epitope Mapping:

  • Employ multiple antibodies targeting different PRTN3 epitopes

  • N-terminal directed antibodies can distinguish between PR3 and MBN forms

Research Applications:

• Northern blotting has demonstrated that some AAV patients express irregular PRTN3 transcripts 100-400 nucleotides larger than the canonical transcript

• Patients positive for the alternative PRTN3-002 transcript showed significantly greater levels of total PR3 mRNA than negative patients

• Different protein isoforms may have distinct biological functions, with PR3 having antimicrobial properties and MBN promoting myeloid proliferation

This differentiation is particularly important in autoimmune conditions where alternative autoantigen forms may drive distinct immune responses.

What methodological approaches enable multiplex detection of PRTN3 alongside other neutrophil markers?

Advanced research often requires simultaneous detection of multiple biomarkers to understand complex biological processes. For PRTN3, several multiplex approaches have been validated:

Multiplex Immunoassays:

• Design antibody panels with minimal cross-reactivity:

  • PRTN3 can be multiplexed with other neutrophil granule proteins

  • Select antibodies with minimal spectral overlap when using fluorescent detection systems

  • For HRP-conjugated antibodies, sequential detection with different substrates can be employed

• Validation protocol for multiplex detection:

  • Single-marker controls to establish baseline signals

  • Titration experiments to determine optimal antibody concentrations

  • Blocking steps to minimize non-specific binding

Multiparametric Flow Cytometry:

• HRP-conjugated PRTN3 antibodies can be used in fixed/permeabilized cell preparations

• Combined with surface markers to identify specific neutrophil subpopulations

Multiplex PCR for Transcript Variants:

• Design primer sets to simultaneously detect canonical and alternative PRTN3 transcripts

• Nested PCR approaches have confirmed the presence of larger 3′UTR variants in patients

For validating multiplex approaches, researchers should include samples with known expression patterns of PRTN3, such as polymorphonuclear leukocytes (positive control) and non-myeloid cells (negative control).

How can HRP-conjugated PRTN3 antibodies contribute to biomarker development for early cancer detection?

Recent research has revealed promising applications for PRTN3 antibodies in cancer biomarker development, particularly for lung adenocarcinoma:

Key Methodological Approaches:

• Plasma Autoantibody Detection:

  • ELISA assays using recombinant PRTN3 protein can detect elevated anti-PRTN3 IgG and IgM autoantibodies in LUAD patients

  • The AUC for anti-PRTN3 IgG autoantibodies in diagnosing early LUAD was 0.782 from normal controls and 0.761 from benign pulmonary nodules

  • Western blotting can confirm plasma immune responses to PRTN3 in LUAD patients

• Tissue Expression Analysis:

  • Immunohistochemistry has demonstrated significantly higher PRTN3 protein expression in LUAD tissues compared to controls

  • Expression levels correlate with pathological grade, with stronger expression in G2 and G3 LUAD

• Combination Biomarker Panels:

  • When combined with CEA (carcinoembryonic antigen), anti-PRTN3 autoantibodies significantly improved diagnostic accuracy for early LUAD compared to CEA alone

  • This combinatorial approach addresses the limited sensitivity of individual biomarkers

Research Applications Table:

These methodologies provide a framework for developing PRTN3-based cancer detection assays with potential for clinical translation.

What are the challenges in detecting PRTN3 in complex biological specimens, and how can they be overcome?

Detecting PRTN3 in complex biological specimens presents several technical challenges that researchers must address:

Challenge 1: Presence of Multiple Isoforms

• The PRTN3 gene locus produces alternative transcripts through different promoter usage and 3′UTR processing

• Solution: Use antibodies targeting conserved epitopes present in all known isoforms, or employ multiple antibodies targeting different regions

Challenge 2: Post-Translational Modifications

• PRTN3 undergoes glycosylation which may mask epitopes or alter antibody binding

• Solution: Validate antibody performance with both native and recombinant PRTN3 proteins; consider deglycosylation treatments when appropriate

Challenge 3: Interfering Factors in Plasma/Serum

• Naturally occurring anti-PRTN3 antibodies exist in normal individuals

• High background in immunoassays can complicate interpretation

• Solution: Optimize blocking conditions and include appropriate dilution series; use non-primary antibody controls

Challenge 4: Tissue-Specific Expression Variability

• PRTN3 expression varies significantly across tissues and pathological states

• Solution: Include tissue-matched controls and quantitative standards; normalize expression to appropriate reference markers

Challenge 5: Cross-Reactivity with Related Proteases

• PRTN3 shares homology with other serine proteases

• Solution: Verify antibody specificity through competitive binding assays with related proteins; validate critical findings with orthogonal detection methods

Advanced Protocol Optimization:

• For weakly expressed variants, consider signal amplification systems compatible with HRP

• For tissue analysis, implement antigen retrieval optimization

• For autoantibody detection, include absorption steps with irrelevant antigens to reduce non-specific binding

What are common sources of background and non-specific binding when using HRP-conjugated PRTN3 antibodies?

When working with HRP-conjugated PRTN3 antibodies, several factors can contribute to background and non-specific binding:

Common Background Sources and Solutions:

• Endogenous Peroxidase Activity

  • Problem: Tissues, particularly those rich in granulocytes (which express PRTN3), contain endogenous peroxidases

  • Solution: Implement peroxidase quenching steps (e.g., 3% H₂O₂ treatment for 10-15 minutes) before primary antibody incubation

• Cross-Reactivity with Related Proteases

  • Problem: PRTN3 belongs to the Peptidase S1 family with structural similarities to other serine proteases

  • Solution: Validate antibody specificity through competitive binding assays; use tissue from PRTN3-deficient systems as negative controls

• Non-Specific Protein Interactions

  • Problem: HRP-conjugated antibodies may bind non-specifically to highly charged or sticky components

  • Solution: Optimize blocking solutions (consider protein-free blockers for some applications); include detergents like Tween-20 in wash buffers

• Natural Anti-PRTN3 Antibodies

  • Problem: Healthy individuals may have naturally occurring antibodies against PRTN3, complicating autoantibody assays

  • Solution: Establish proper baseline levels using sufficient normal controls; consider absorption steps to remove non-specific antibodies

• Alternative Transcript Products

  • Problem: PRTN3 gene produces multiple transcript variants that may confound interpretation

  • Solution: Use antibodies validated against all known isoforms or employ multiple antibodies targeting different epitopes

Background Reduction Strategy:

• Titrate antibody concentration to find optimal signal-to-noise ratio

• Increase washing duration and frequency

• Consider signal amplification systems for specific detection of low-abundance targets

• For tissue samples, implement appropriate antigen retrieval methods

What approaches can resolve discrepancies between different PRTN3 detection methods?

When faced with discrepancies between different PRTN3 detection methods, researchers should implement a systematic troubleshooting approach:

Step 1: Characterize the Discrepancy

• Document specific differences in results between methods

• Determine if discrepancies are quantitative (intensity differences) or qualitative (presence/absence)

• Assess reproducibility of each method independently

Step 2: Method-Specific Considerations

Step 3: Biological Explanations

• Alternative transcripts: The PRTN3 gene produces multiple mRNA variants through alternative promoter usage and 3′UTR processing

• Protein isoforms: PR3 and myeloblastin (MBN) are structurally distinct proteins encoded by the PRTN3 locus

• Post-translational modifications: PRTN3 undergoes glycosylation which may affect detection

• Disease-specific alterations: Patients with AAV show dysregulation of PRTN3 expression and processing

Case Study Resolution Approach:
When Northern blot analysis detected abnormal-sized PRTN3 transcripts in AAV patients that weren't initially characterized by standard PCR, researchers implemented nested PCR with multiple primer sets to identify and sequence the variant transcripts, revealing a 68-bp repeat with a unique 12-bp spacer in the 3′UTR .

How does sample storage and handling affect the performance of HRP-conjugated PRTN3 antibodies?

Proper sample storage and handling are critical for maintaining PRTN3 integrity and optimizing antibody performance:

Protein Samples (Cell/Tissue Lysates):

• Store at -80°C with protease inhibitors to prevent degradation

• Avoid repeated freeze-thaw cycles that can degrade PRTN3 and affect epitope integrity

• For western blotting, prepare fresh samples when possible, as PRTN3 may undergo proteolytic processing during storage

Plasma/Serum Samples:

• For anti-PRTN3 autoantibody detection, plasma samples should be processed promptly and stored at -80°C

• Standardize collection tubes and processing times across experimental groups

• Consider aliquoting samples to avoid repeated freeze-thaw cycles

Fixed Tissue Samples:

• Optimal fixation conditions should be established for PRTN3 detection (overfixation can mask epitopes)

• FFPE tissues have been successfully used for PRTN3 detection in LUAD tissue arrays

• For archived samples, implement appropriate antigen retrieval methods

HRP-Conjugated Antibodies:

• Follow manufacturer's storage recommendations (typically 4°C for short-term, -20°C with glycerol for long-term)

• Avoid repeated freeze-thaw cycles that can reduce HRP activity

• Check for precipitation before use and centrifuge if necessary

• Consider adding stabilizing proteins (BSA) for diluted antibody solutions

Impact of Sample Age on Detection Sensitivity:

How can HRP-conjugated PRTN3 antibodies contribute to understanding the role of PRTN3 in cancer progression?

Recent research has revealed an unexpected relationship between PRTN3 and cancer, particularly lung adenocarcinoma, opening new avenues for investigation:

Methodological Approaches for Cancer Research:

• Tissue Microarray Analysis

  • HRP-conjugated PRTN3 antibodies enable high-throughput screening of PRTN3 expression across tumor stages

  • IHC studies have shown significantly higher PRTN3 protein expression in LUAD tissues compared to controls, with expression correlating with pathological grade

  • This approach can identify potential associations between PRTN3 expression and clinical outcomes

• Dual Staining Protocols

  • Combine PRTN3 staining with cancer stem cell markers or proliferation markers

  • Investigate co-localization with immune cell infiltrates to understand tumor microenvironment interactions

  • Develop multiplexed IHC protocols to characterize the cellular context of PRTN3 expression

• Functional Studies with Validated Antibodies

  • Use HRP-conjugated PRTN3 antibodies in neutralization experiments to assess functional roles

  • Develop blocking strategies to investigate PRTN3's contribution to tumor progression

  • Monitor changes in PRTN3 expression following therapeutic interventions

Research Questions Addressable with PRTN3 Antibodies:

• Does PRTN3 expression correlate with tumor invasiveness through its extracellular matrix-degrading properties?

• Can PRTN3 serve as a prognostic marker in LUAD and other cancers?

• Is the relationship between anti-PRTN3 autoantibodies and LUAD causative or consequential?

• Does targeting PRTN3 have therapeutic potential in PRTN3-expressing tumors?

Research has shown that the AUC of anti-PRTN3 IgG autoantibodies for diagnosing early LUAD from normal controls was 0.782, suggesting significant potential for early cancer detection applications .

What methodological advances are improving the sensitivity and specificity of PRTN3 detection?

Technological innovations continue to enhance the capabilities of PRTN3 detection systems:

Advanced Detection Technologies:

• Signal Amplification Systems

  • Tyramide signal amplification (TSA) with HRP-conjugated antibodies can significantly increase detection sensitivity

  • Polymer-based detection systems reduce background while enhancing signal strength

  • These approaches are particularly valuable for detecting low-abundance PRTN3 variants

• Digital Pathology and Image Analysis

  • Automated quantification of PRTN3 staining improves objectivity and reproducibility

  • Machine learning algorithms can identify subtle patterns in PRTN3 expression

  • These methods have enhanced the ability to correlate PRTN3 expression with clinical parameters

• Single-Cell Technologies

  • Flow cytometry with HRP-conjugated PRTN3 antibodies enables quantitative assessment at the single-cell level

  • Mass cytometry (CyTOF) allows multiplexed protein detection including PRTN3

  • Single-cell RNA-seq can be integrated with protein data to correlate PRTN3 transcript variants with protein expression

• Proximity Ligation Assays

  • These techniques enable detection of protein-protein interactions involving PRTN3

  • Can help identify binding partners relevant to PRTN3's role in disease processes

Sensitivity Comparison Table:

These methodological advances are particularly valuable for studying the alternative transcripts from the PRTN3 gene locus, which may be expressed at lower levels or in specific cellular contexts .

How can researchers investigate the relationship between PRTN3 dysregulation and autoimmune conditions?

The dysregulation of PRTN3 plays a significant role in autoimmune conditions, particularly ANCA-Associated Vasculitis. HRP-conjugated PRTN3 antibodies enable several methodological approaches to investigate these relationships:

Research Methodologies:

• Transcript Variant Analysis

  • Northern blotting and PCR-based approaches have identified irregular PRTN3 transcripts in AAV patients

  • These include transcripts with alternative 3′UTRs containing a 68-bp repeat with a 12-bp spacer

  • Alternative transcription start site within intron 1 of the PRTN3 gene has been identified in active disease

• Protein Isoform Characterization

  • Western blotting with HRP-conjugated antibodies can detect both canonical PR3 and alternative forms like myeloblastin (MBN)

  • A p24 PR3/MBN protein has been detected using patients' sera, suggesting it may be antigenic

  • Experimental protocol should include antibodies targeting different epitopes to distinguish between isoforms

• Autoantibody Profiling

  • ELISA assays can measure anti-PRTN3 IgG and IgM autoantibodies in patient samples

  • Western blotting confirms the presence of plasma immune responses to PRTN3

  • Competitive binding assays can characterize epitope specificity of autoantibodies

• Functional Studies

  • Investigate whether alternative PRTN3 isoforms have different functional properties

  • PR3 has been reported to have antimicrobial functions while MBN promotes myeloid proliferation

  • These functional differences may contribute to disease pathogenesis

Experimental Model Systems:

• Cell lines treated with GM-CSF express the alternative PRTN3-002 transcript, providing an in vitro model

• Leukemia cell lines express alternative PRTN3 transcripts and can serve as research models

• Normal human bone marrow samples can be used to study developmental regulation of PRTN3 expression