KLK11 has been linked to ovarian, prostate, and colorectal cancers. HRP-conjugated antibodies are critical for quantifying KLK11 protein levels in these contexts:
Oxaliplatin Resistance: KLK11 overexpression in colorectal cancer (CRC) cells correlates with resistance to oxaliplatin (L-OHP). Knockdown of KLK11 via RNAi restores chemosensitivity by suppressing the PI3K/AKT pathway and enhancing apoptosis .
Biomarker Potential: Elevated KLK11 serum levels are observed in ~70% of ovarian cancer and ~60% of prostate cancer cases, suggesting utility as a diagnostic marker .
Cross-Reactivity: Polyclonal antibodies may bind to homologous kallikreins (e.g., KLK1-KLK15). Pre-adsorption with non-specific peptides may improve specificity .
Isoform Specificity: Antibodies targeting regions like KLK11 201–282 may preferentially detect isoform 1 (brain-type), while C-terminal antibodies may bind both isoforms .
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.
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.
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.
The KLK11 Antibody, HRP conjugated has a defined reactivity profile that researchers should consider when designing experiments. Based on the product specifications:
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 .
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.
Developing an effective ELISA assay for KLK11 detection requires careful consideration of multiple parameters:
Assay Format Selection:
Antibody Pair Optimization:
Assay Performance Characteristics:
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.
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:
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.
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 .
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:
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.
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:
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:
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:
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
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.
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:
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 .
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:
Prostate Cancer:
Other Cancers:
Variable expression reported in breast, colorectal, and pancreatic cancers
Tissue-specific expression patterns require careful validation
Reliable Detection Methods:
Serum/Plasma Quantification:
Tissue Expression Analysis:
Cellular Studies:
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.
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 .
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.