KLK11 Antibody, HRP conjugated

What is KLK11 and why is it a significant research target?

KLK11 (Kallikrein 11) is a serine protease with two alternative splicing isoforms, commonly referred to as the brain type and prostate type. The significance of KLK11 as a research target stems from its clinical relevance as a biomarker in cancer detection. Elevated serum levels of hK11 have been found in approximately 70% of women with ovarian cancer and 60% of men with prostate cancer, making it a valuable biomarker for diagnosis and monitoring of these malignancies . KLK11 belongs to the kallikrein family of proteases, which are involved in various physiological processes including tissue remodeling, inflammation, and cancer progression. Research targeting KLK11 contributes to understanding cancer pathogenesis and developing diagnostic tools for early cancer detection.

What are the main applications for KLK11 Antibody, HRP conjugated?

KLK11 Antibody, HRP conjugated is specifically designed for multiple immunological applications in research settings. The primary applications include:

• Western Blotting (WB): Recommended dilution range of 1:300-5000, optimal for protein detection and quantification

• Enzyme-Linked Immunosorbent Assay (ELISA): Used at dilutions of 1:500-1000 for sensitive quantitative detection

• Immunohistochemistry with paraffin-embedded sections (IHC-P): Effective at dilutions of 1:200-400 for tissue localization studies

• Immunohistochemistry with frozen sections (IHC-F): Optimal at 1:100-500 dilution

The HRP conjugation eliminates the need for secondary antibody incubation, streamlining experimental protocols and potentially reducing background signals in sensitive applications. This antibody is particularly useful in studies examining KLK11 expression in cancer tissues and for biomarker validation experiments.

How should KLK11 Antibody, HRP conjugated be stored and handled to maintain optimal activity?

Proper storage and handling of KLK11 Antibody, HRP conjugated is essential for maintaining its immunoreactivity and enzyme activity. The recommended storage conditions are:

• Store at -20°C in the original buffer containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol

• Aliquot the antibody into multiple small volumes upon receipt to avoid repeated freeze-thaw cycles, which can significantly degrade antibody performance

• When working with the antibody, thaw aliquots completely before use and keep on ice during the experiment

• For short-term storage of opened/diluted material (up to one month), refrigeration at 2-8°C is acceptable

The addition of BSA and glycerol in the storage buffer helps stabilize the antibody, while Proclin300 serves as a preservative. When designing experiments, it's advisable to prepare working dilutions fresh on the day of use rather than storing diluted antibody solutions for extended periods, as this can lead to reduced signal intensity and increased background.

What is the expected reactivity profile of KLK11 Antibody, HRP conjugated?

The KLK11 Antibody, HRP conjugated has a defined reactivity profile that researchers should consider when designing experiments. Based on the product specifications:

• Confirmed reactivity with human KLK11 protein

• Predicted cross-reactivity with mouse, rat, and dog KLK11 based on sequence homology analysis

• No significant cross-reactivity with other proteins has been reported

The antibody specifically recognizes epitopes within the human KLK11 protein, with some products targeting the C-terminal region (AA 233-250) . When working with non-human samples, validation experiments are recommended to confirm reactivity, as predicted cross-reactivity is based on sequence analysis rather than experimental validation. Some antibody products are raised against a synthetic peptide derived from the human KLK11 sequence (range 201-282/282), which determines its epitope specificity .

How can I optimize Western blot protocols using KLK11 Antibody, HRP conjugated for detecting low abundance KLK11 in clinical samples?

Optimizing Western blot protocols for detecting low abundance KLK11 in clinical samples requires several strategic modifications:

• Sample Preparation Enhancement:

  • Employ immunoprecipitation techniques prior to Western blotting to concentrate KLK11 from serum or tissue lysates

  • Use protease inhibitor cocktails during sample preparation to prevent degradation of KLK11

  • For secreted KLK11, consider concentrating cell culture supernatants using centrifugal filter units

• Blocking and Antibody Incubation:

  • Test different blocking agents (5% non-fat milk vs. 3-5% BSA) to determine optimal signal-to-noise ratio

  • Extend primary antibody incubation time to overnight at 4°C using a dilution at the lower end of the recommended range (1:300-1:500)

  • Include 0.05% Tween-20 in wash buffers to reduce non-specific binding

• Detection Enhancement:

  • Utilize enhanced chemiluminescence (ECL) substrates specifically designed for high sensitivity detection

  • Consider extended exposure times during imaging, starting with standard exposures and increasing incrementally

  • If background is problematic, implement more rigorous washing steps and optimize antibody concentration

• Controls and Validation:

  • Always include positive control samples with known KLK11 expression

  • Use recombinant KLK11 protein as a reference standard

  • Consider running gradient gels to achieve better separation of proteins in the relevant molecular weight range

The HRP conjugation provides direct enzymatic signal generation, eliminating potential variability introduced by secondary antibodies. For extremely low abundance targets, signal amplification systems compatible with HRP can be employed after the primary antibody incubation step.

What are the critical considerations when developing an ELISA assay for KLK11 detection using HRP conjugated antibodies?

Developing an effective ELISA assay for KLK11 detection requires careful consideration of multiple parameters:

• Assay Format Selection:

  • The sandwich ELISA format is preferred for KLK11 detection, utilizing a capture antibody pre-coated on the plate and an HRP-conjugated detection antibody

  • This format provides superior specificity compared to direct or competitive ELISA formats

• Antibody Pair Optimization:

  • Ensure the capture and HRP-conjugated detection antibodies recognize distinct, non-overlapping epitopes on KLK11

  • Test different antibody concentrations to determine optimal signal-to-noise ratios

  • For the HRP-conjugated antibody, dilutions between 1:500-1:1000 typically provide optimal results

• Assay Performance Characteristics:

  • Standard curve range: Commercial KLK11 ELISA kits typically cover 0.156-10 ng/mL

  • Detection sensitivity: Target sensitivity should be around 0.055-0.094 ng/mL to detect physiologically relevant KLK11 levels

  • Follow a 4-hour protocol similar to commercial kits for optimal time efficiency

• Protocol Optimization:

  • Follow the sandwich ELISA workflow: coating → blocking → sample incubation → detection antibody → substrate → stop solution

  • Use recombinant KLK11 protein to establish a standard curve

  • Implement appropriate quality controls including blanks and samples with known KLK11 concentrations

• Data Analysis:

  • Use 4-parameter logistic regression for standard curve fitting

  • Read absorbance at 450nm with a reference wavelength of 620nm to correct for plate imperfections

For research applications requiring high sensitivity, signal amplification systems compatible with HRP (such as tyramide signal amplification) can be incorporated to lower the detection threshold beyond what is possible with standard chromogenic substrates.

How can I troubleshoot non-specific binding when using KLK11 Antibody, HRP conjugated in immunohistochemistry applications?

Non-specific binding in immunohistochemistry with KLK11 Antibody, HRP conjugated can significantly impact result interpretation. Here's a systematic approach to troubleshooting:

• Optimization of Blocking Conditions:

  • Extend blocking time to 1-2 hours at room temperature

  • Test different blocking agents (5% normal serum from the same species as the secondary antibody, 3-5% BSA, or commercial blocking reagents)

  • Consider adding 0.1-0.3% Triton X-100 to blocking solution to improve antibody penetration

• Antibody Dilution Optimization:

  • Test a dilution series within the recommended range (1:100-1:500 for IHC-P)

  • Perform a titration experiment to identify the optimal concentration that maximizes specific signal while minimizing background

• Antigen Retrieval Modifications:

  • Compare heat-induced epitope retrieval methods (citrate buffer pH 6.0 vs. EDTA buffer pH 8.0-9.0)

  • Optimize retrieval time and temperature

  • For formalin-fixed tissues, ensure adequate fixation time (not too long or too short)

• Washing Protocol Enhancement:

  • Increase the number and duration of washing steps

  • Use TBS-T (TBS with 0.1% Tween-20) instead of PBS to reduce non-specific ionic interactions

  • Ensure complete buffer exchanges between washes

• Controls Implementation:

  • Include negative controls (omitting primary antibody, using isotype control)

  • Use tissues with known KLK11 expression patterns as positive controls

  • Consider peptide competition assays to confirm specificity

• Signal Development Optimization:

  • Shorten DAB development time to reduce background

  • Consider alternative chromogens if DAB gives high background

  • Counterstain appropriately to maximize contrast

If non-specific binding persists despite these measures, consider using a non-conjugated primary antibody with a compatible secondary antibody system, which sometimes provides better control over sensitivity and specificity.

What methodological approaches should be used to validate KLK11 as a biomarker in cancer studies using HRP-conjugated antibodies?

Validating KLK11 as a cancer biomarker using HRP-conjugated antibodies requires a multi-faceted approach:

• Multi-platform Expression Analysis:

  • Utilize Western blotting (dilution 1:300-5000) to assess KLK11 protein levels across cancer cell lines and patient-derived samples

  • Perform IHC-P (dilution 1:200-400) on tissue microarrays containing normal and cancerous tissues to evaluate expression patterns

  • Develop quantitative ELISA (dilution 1:500-1000) for serum/plasma KLK11 measurement

• Clinical Sample Analysis:

  • Establish reference ranges in healthy individuals stratified by age and sex

  • Compare KLK11 levels between cancer patients and matched controls

  • Correlate KLK11 expression with clinical parameters (staging, grading, prognosis)

  • Create a standardized protocol for sample collection and processing

• Correlation with Existing Biomarkers:

  • Analyze KLK11 in conjunction with established cancer biomarkers

  • Calculate sensitivity, specificity, positive predictive value, and negative predictive value

  • Perform ROC curve analysis to determine optimal cutoff values

• Functional Validation:

  • Investigate the biological significance of KLK11 in cancer progression using cell models

  • Correlate KLK11 expression with cancer cell behaviors (proliferation, migration, invasion)

  • Confirm antibody specificity via gene knockdown/knockout approaches

• Retrospective and Prospective Clinical Studies:

  • Analyze archived samples with known outcomes to assess prognostic value

  • Design prospective studies to validate KLK11 as a predictive biomarker for specific treatments

These methodological approaches should be implemented with particular attention to pre-analytical variables (sample collection, storage conditions) and analytical validation (precision, accuracy, specificity). The existing literature suggests particular relevance for ovarian cancer (70% elevation) and prostate cancer (60% elevation), making these cancer types primary candidates for KLK11 biomarker validation studies .

What are the principles behind sandwich ELISA for KLK11 detection and how does the HRP conjugation contribute to assay performance?

The sandwich ELISA for KLK11 detection operates on specific molecular principles that leverage HRP conjugation for superior performance:

Fundamental Principle:
The sandwich ELISA utilizes two antibodies that bind to different epitopes on the KLK11 molecule. This dual-recognition approach significantly enhances specificity compared to single-antibody methods .

Step-by-Step Methodology:

• Capture Phase: A microplate is pre-coated with an antibody specific to KLK11 that immobilizes the target protein from samples

• Recognition Phase: A biotin-conjugated detection antibody specific to KLK11 is added, binding to a different epitope than the capture antibody

• Signal Generation Phase: Avidin conjugated to HRP is added, which binds with high affinity to the biotin on the detection antibody

• Visualization Phase: TMB substrate solution is added, which is catalyzed by HRP to produce a blue color that turns yellow after adding the stop solution

• Quantification Phase: Absorbance is measured at 450nm, with the optical density proportional to KLK11 concentration

Contribution of HRP Conjugation:
The HRP (Horseradish Peroxidase) conjugation provides several advantages:

• Enzymatic Amplification: Each HRP molecule can convert multiple substrate molecules, providing signal amplification

• Stable Signal Development: Controlled reaction kinetics allow for precise timing of the assay

• Compatibility with Common Substrates: HRP works efficiently with TMB, providing a colorimetric readout measurable on standard plate readers

• Reduced Complexity: Direct conjugation (either to detection antibody or via avidin-biotin system) eliminates additional incubation steps

Performance Characteristics:
Commercial KLK11 ELISA kits utilizing this principle achieve:

• Detection Range: 0.156-10 ng/mL

• Sensitivity: 0.055-0.094 ng/mL

• Reaction Duration: Approximately 4 hours

• Specificity: Minimal cross-reactivity with other proteins

The sandwich ELISA principle coupled with HRP-based detection provides an optimal balance of sensitivity, specificity, and practical workflow efficiency for KLK11 quantification in research and clinical samples.

How should I design experimental controls when using KLK11 Antibody, HRP conjugated in multiple applications?

Designing robust experimental controls for KLK11 Antibody, HRP conjugated applications is critical for result validation and troubleshooting. Here's a comprehensive control strategy for various applications:

Western Blotting Controls:

• Positive Controls:

  • Cell lines with known KLK11 expression (ovarian or prostate cancer cell lines)

  • Recombinant human KLK11 protein as a molecular weight reference

  • Tissue lysates from organs known to express KLK11 (prostate, brain)

• Negative Controls:

  • Cell lines with minimal KLK11 expression

  • KLK11 knockdown/knockout samples

  • Secondary-only control (omitting primary antibody)

• Loading Controls:

  • Pan-cellular markers (β-actin, GAPDH, α-tubulin)

  • Compartment-specific markers if examining subcellular fractions

Immunohistochemistry Controls:

• Tissue Controls:

  • Positive tissue controls: prostate or ovarian cancer tissues with known KLK11 expression

  • Negative tissue controls: tissues known not to express KLK11

  • Multi-tissue arrays to assess expression patterns across various tissue types

• Technical Controls:

  • No primary antibody control (using only detection system)

  • Isotype control (irrelevant antibody of same isotype and concentration)

  • Peptide competition/blocking control using the immunizing peptide

ELISA Controls:

• Standard Curve:

  • Recombinant KLK11 protein at known concentrations (typically 0.156-10 ng/mL)

  • Zero standard (buffer only)

• Sample Validation:

  • Spike-and-recovery experiments (adding known amounts of recombinant KLK11 to samples)

  • Dilution linearity testing (serial dilutions of positive samples)

  • Internal control samples with established KLK11 concentrations

• Assay Controls:

  • Blank wells (all reagents except sample/standard)

  • Non-specific binding control (capture antibody omitted)

Universal Control Elements:

• Batch-to-batch consistency controls (reference samples run across experiments)

• Time-course stability controls (repeated measurements of the same sample)

• Replicate samples to assess technical variability

What are the critical parameters to consider when interpreting KLK11 expression data in cancer biomarker studies?

Understanding these parameters is essential for determining whether KLK11 can serve as a reliable biomarker in specific clinical contexts and for designing studies that will generate reproducible and clinically meaningful results.

How can KLK11 antibody specificity be validated to ensure reliable research findings?

Validating KLK11 antibody specificity is a multi-step process essential for generating trustworthy research data:

• Molecular Verification Techniques:

  • Western Blot Analysis: Confirm single band of expected molecular weight (~35 kDa for mature KLK11)

  • Mass Spectrometry Validation: Immunoprecipitate KLK11 and verify identity by peptide sequencing

  • Peptide Competition Assay: Pre-incubate antibody with immunizing peptide (e.g., KLH conjugated synthetic peptide from human KLK11, range 201-282/282) to demonstrate signal elimination

  • Recombinant Protein Testing: Use purified recombinant KLK11 as positive control

• Genetic Manipulation Approaches:

  • siRNA/shRNA Knockdown: Demonstrate reduced antibody signal in cells with KLK11 knockdown

  • CRISPR/Cas9 Knockout: Show complete signal elimination in KLK11 knockout cells

  • Overexpression Models: Confirm increased signal in cells overexpressing KLK11

• Cross-Reactivity Assessment:

  • Testing Against Related Proteins: Evaluate potential cross-reactivity with other kallikrein family members

  • Multi-species Testing: Compare reactivity across species with varying sequence homology

  • Epitope Mapping: Identify the specific amino acid sequence recognized by the antibody (e.g., AA 233-250, C-Term for some antibodies)

• Application-Specific Validation:

  • Western Blotting: Verify single band at expected molecular weight at recommended dilutions (1:300-5000)

  • IHC: Compare staining patterns with published KLK11 expression data and mRNA expression patterns

  • ELISA: Perform dilution linearity and spike-recovery experiments

• Multi-antibody Verification:

  • Antibody Panel Approach: Use multiple antibodies targeting different KLK11 epitopes

  • Monoclonal/Polyclonal Comparison: Compare results between monoclonal and polyclonal antibodies

  • Orthogonal Method Correlation: Correlate antibody-based detection with mRNA expression data

This comprehensive validation process is particularly important for KLK11 research given its relevance as a potential cancer biomarker, where false positives or negatives could have significant implications for diagnostic development. The validation should be performed in relevant biological contexts, including tissue types where KLK11 plays significant roles such as prostate and ovarian tissues .

How does KLK11 expression correlate with cancer progression and what methods can detect these changes reliably?

KLK11 expression demonstrates specific patterns of correlation with cancer progression that can be reliably detected using optimized methodologies:

Expression Patterns in Cancer Progression:

• Ovarian Cancer:

  • Elevated serum levels observed in approximately 70% of women with ovarian cancer

  • Expression typically increases with advancing stage and grade

  • Potential value as an early detection biomarker when combined with other kallikreins

• Prostate Cancer:

  • Elevated serum levels detected in about 60% of men with prostate cancer

  • Expression patterns differ from PSA in some patients, suggesting complementary diagnostic value

  • May provide additional stratification information beyond Gleason score

• Other Cancers:

  • Variable expression reported in breast, colorectal, and pancreatic cancers

  • Tissue-specific expression patterns require careful validation

Reliable Detection Methods:

• Serum/Plasma Quantification:

  • Sandwich ELISA using validated antibody pairs provides most reliable quantification

  • Detection sensitivity of 0.055-0.094 ng/mL enables measurement of physiologically relevant levels

  • Sample collection standardization critical (time of day, fasting status, processing time)

• Tissue Expression Analysis:

  • IHC-P with KLK11 antibody (dilution 1:200-400) allows spatial assessment of expression

  • Digital image analysis improves quantification reliability over manual scoring

  • Multiplex immunofluorescence enables co-localization studies with other cancer markers

• Cellular Studies:

  • Western blotting (dilution 1:300-5000) for cell line and primary culture analysis

  • Flow cytometry for single-cell level quantification of KLK11 expression

  • Cell-based ELISA for high-throughput screening applications

Methodological Considerations for Reliable Detection:

• Biospecimen Handling:

  • Standardized collection protocols to minimize pre-analytical variables

  • Consistent processing timeframes to prevent protein degradation

  • Appropriate storage conditions (-80°C for long-term storage of serum/plasma)

• Control Implementation:

  • Internal reference standards across assay batches

  • Matched normal tissue/serum controls

  • Stage/grade-matched patient cohorts for comparative studies

• Data Normalization Approaches:

  • Normalization to total protein for tissue lysates

  • Use of reference ranges established in healthy populations

  • Multivariate analysis incorporating clinical parameters

These methodological approaches enable reliable assessment of KLK11 expression changes during cancer progression, providing valuable information for both basic research and clinical applications in cancer diagnosis and monitoring.

What technical challenges exist when developing KLK11-based diagnostic assays and how can they be addressed?

Developing robust KLK11-based diagnostic assays presents several technical challenges that require strategic solutions:

• Isoform Specificity Challenges:

  • Challenge: KLK11 exists in alternative splicing isoforms (brain and prostate types) , potentially confounding diagnostic interpretation

  • Solution: Design epitope-specific antibodies targeting conserved or isoform-specific regions

  • Implementation: Use antibody pairs recognizing distinct epitopes to either capture all isoforms or differentiate between them

• Sensitivity and Dynamic Range Limitations:

  • Challenge: Physiological KLK11 levels in healthy individuals can be low, requiring high sensitivity

  • Solution: Optimize sandwich ELISA configuration with appropriate antibody pairs and signal amplification

  • Implementation: Employ advanced detection systems to achieve sensitivity of 0.055-0.094 ng/mL while maintaining a dynamic range up to 10 ng/mL

• Interference from Binding Proteins:

  • Challenge: KLK11, like other kallikreins, may interact with serum inhibitors or binding proteins

  • Solution: Develop sample pre-treatment strategies to dissociate protein complexes

  • Implementation: Optimize sample buffer composition and pre-analytical processing steps

• Cross-Reactivity with Related Kallikreins:

  • Challenge: The kallikrein family includes 15 members with structural similarities

  • Solution: Rigorous antibody validation against all kallikrein family members

  • Implementation: Epitope selection focusing on unique regions with minimal homology to other kallikreins

• Pre-analytical Variables:

  • Challenge: Sample collection, processing, and storage can affect KLK11 measurements

  • Solution: Establish standardized pre-analytical protocols

  • Implementation: Develop detailed SOPs for sample handling and incorporate quality indicators

• Reference Range Establishment:

  • Challenge: Defining "normal" vs. "elevated" requires robust reference intervals

  • Solution: Analyze large cohorts of healthy individuals stratified by age, sex, and ethnicity

  • Implementation: Multi-center studies with harmonized methodologies to establish reliable cutoff values

• Assay Standardization:

  • Challenge: Ensuring comparability of results across laboratories and platforms

  • Solution: Develop international reference materials and standardization programs

  • Implementation: Incorporate traceability to reference materials in assay calibration

• Clinical Validation Complexities:

  • Challenge: Establishing clinical utility requires large, well-designed studies

  • Solution: Design studies with appropriate patient cohorts and statistical power

  • Implementation: Collaborate with clinical partners to access relevant patient populations and outcome data

• Technological Platform Selection:

  • Challenge: Choosing appropriate technology for clinical implementation

  • Solution: Evaluate multiple platforms considering sensitivity, throughput, and clinical setting

  • Implementation: Develop assays on platforms already established in clinical laboratories

By systematically addressing these technical challenges, researchers can develop KLK11-based diagnostic assays with improved performance characteristics suitable for clinical implementation, particularly for ovarian and prostate cancer applications where KLK11 has shown promising biomarker potential .

How might multiplex detection systems incorporating KLK11 Antibody, HRP conjugated enhance cancer biomarker panels?

Multiplex detection systems incorporating KLK11 Antibody, HRP conjugated offer significant potential for enhancing cancer biomarker panels through several advanced approaches:

• Integrated Kallikrein Panels:

  • Simultaneous detection of multiple kallikrein family members (KLK3/PSA, KLK2, KLK4, KLK11) provides complementary information

  • Enhanced diagnostic accuracy through multivariate kallikrein scoring algorithms

  • Improved cancer subtype characterization by kallikrein expression patterns

  • Technical implementation requires careful antibody selection to prevent cross-reactivity between closely related kallikreins

• Multiplex Immunoassay Platforms:

  • Bead-based multiplexing systems allow simultaneous quantification of KLK11 alongside other cancer biomarkers

  • Microarray formats enable high-density biomarker profiling with minimal sample volume

  • HRP-conjugated detection can be integrated through direct conjugation or secondary detection systems

  • Cross-reactivity prevention requires extensive validation of antibody specificity in the multiplex environment

• Advanced Tissue Analysis Systems:

  • Multiplex immunohistochemistry incorporating KLK11 with other tissue biomarkers

  • Spatial profiling of KLK11 expression in relation to tumor microenvironment components

  • Digital pathology integration for quantitative assessment of multiple markers

  • Tyramide signal amplification systems compatible with HRP conjugates for enhanced sensitivity

• Liquid Biopsy Integration:

  • Combining KLK11 detection with circulating tumor cell characterization

  • Integration with cell-free DNA and exosome analysis for comprehensive liquid biopsy profiles

  • Development of microfluidic platforms for integrated biomarker assessment

  • HRP-based electrochemical detection systems for point-of-care applications

• Machine Learning Enhanced Interpretation:

  • Algorithm development for interpreting complex patterns from multiplex KLK11 panels

  • Integration of clinical parameters with multiple biomarker measurements

  • Predictive modeling for treatment response based on kallikrein profiles

  • Longitudinal monitoring algorithms for detecting subtle changes in biomarker patterns

The integration of KLK11 into multiplex systems is particularly promising for ovarian and prostate cancers, where it has shown significant elevation in 70% and 60% of cases respectively . By combining KLK11 with established biomarkers, novel patterns of expression can be identified that correlate with disease progression, treatment response, and clinical outcomes.

Future research should focus on standardizing these multiplex approaches, establishing clinical validation protocols, and developing integrated data analysis pipelines that can extract meaningful clinical insights from complex biomarker patterns.