PTGS2 Antibody, Biotin conjugated

What is PTGS2 and why is it significant in research applications?

PTGS2 (Prostaglandin-Endoperoxide Synthase 2), also known as cyclooxygenase-2 (COX2), is a key enzyme in prostaglandin biosynthesis. This enzyme plays a critical role in inflammation and pain signaling pathways. Research significance stems from its overexpression in numerous pathological conditions including cancer, arthritis, and cardiovascular diseases .

The protein has a calculated molecular weight of 69kDa but is typically observed at 72kDa in its glycosylated form (gPTGS2) . Cellular localization includes caveola, cytoplasm, endoplasmic reticulum, endoplasmic reticulum lumen, and neuron projections , making it an important target for studying cellular responses to inflammatory stimuli in various tissues.

In colorectal cancer research, distinct PTGS2 expression patterns between tumor cells and surrounding stroma suggest independent regulatory mechanisms within the same tumor microenvironment , highlighting its complex role in cancer progression.

What are the primary considerations when selecting a PTGS2 antibody for specific experimental applications?

When selecting a PTGS2 antibody, researchers should consider:

• Epitope specificity: Different antibodies target distinct regions of PTGS2. For example, some target AA 187-425, while others recognize regions such as AA 501-604 . Epitope selection can impact detection sensitivity in different applications.

• Species reactivity: Confirm compatibility with your experimental model. Available antibodies show varying reactivity profiles (human-only vs. human/mouse/rat cross-reactivity) .

• Clonality: Polyclonal antibodies may offer broader epitope recognition, while monoclonal antibodies typically provide higher specificity for individual epitopes .

• Validated applications: Verify that the antibody has been validated for your specific application:

  • Western blotting (WB)

  • Immunohistochemistry (IHC)

  • Immunoprecipitation (IP)

  • Immunocytochemistry (ICC)

  • ELISA

• Positive control recommendations: Many manufacturers specify positive controls (e.g., LPS-stimulated cell lines such as HeLa or RAW 264.7) .

• Conjugation status: Consider whether direct detection (biotin-conjugated) or indirect detection (unconjugated primary with labeled secondary) better suits your experimental design .

What advantages does biotin conjugation offer for PTGS2 antibody applications?

Biotin conjugation provides several methodological advantages for PTGS2 detection:

• Signal amplification: The high-affinity biotin-streptavidin interaction (Kd ≈ 10^-15 M) enables significant signal enhancement compared to conventional secondary antibody systems.

• Multiplexed detection options: Biotin-conjugated PTGS2 antibodies can be combined with differently labeled primary antibodies for simultaneous detection of multiple targets, critical for co-localization studies examining PTGS2 expression in specific cellular populations .

• Streptavidin flexibility: Researchers can select from various streptavidin conjugates (HRP, fluorophores, quantum dots) without changing the primary antibody preparation.

• Reduced background: Elimination of species cross-reactivity issues that can occur with secondary antibody systems.

• Workflow efficiency: Direct detection reduces experimental time and potential variability introduced by additional incubation and washing steps.

How do I optimize Western blotting protocols for detecting glycosylated PTGS2?

Optimizing Western blotting for glycosylated PTGS2 (gPTGS2) requires careful method refinement:

Focus specifically on the 72 kDa gPTGS2 band for quantification when studying the glycosylated form . This approach has demonstrated high sensitivity, enabling detection in 96/100 colorectal cancer samples versus only 11/100 matched normal mucosa samples .

What positive and negative controls should be included when using PTGS2 antibodies?

Appropriate controls are essential for validating PTGS2 antibody performance:

Positive Controls:

• LPS-stimulated cell lines: HeLa+LPS and RAW 264.7+LPS show reliable PTGS2 induction .

• Tissue samples: Rat brain provides consistent PTGS2 expression .

• Recombinant PTGS2 protein: Can serve as a standard for quantification .

• Known PTGS2-expressing tumors: Colorectal cancer samples with documented PTGS2 overexpression .

Negative Controls:

• Antibody omission: Primary antibody replaced with buffer or non-immune IgG.

• Blocking peptide competition: Pre-incubation of antibody with immunizing peptide should abolish specific staining.

• Normal matched tissue: For cancer studies, adjacent normal tissue often shows minimal PTGS2 expression .

• Destaining controls: When performing multiplexed IHC, verification of complete antibody removal between staining rounds using secondary antibody only .

Procedural Controls:

• Loading controls: β-actin for Western blotting ensures equal protein loading .

• Multiple antibody validation: Testing antibodies targeting different PTGS2 epitopes to confirm specificity.

• Dilution series: Establishing optimal antibody concentration for maximum signal-to-noise ratio.

How can I differentiate between tumor and stromal PTGS2 expression in complex tissue samples?

Differentiating between tumor and stromal PTGS2 expression requires complementary methodological approaches:

• Immunohistochemical scoring:

  • Independently score tumor-associated and stroma-associated PTGS2 expression

  • Quantify percent of PTGS2-positive cells in hot spots using image analysis software (e.g., Image Scope 12.3)

  • Research indicates potential independence between expression patterns (correlation coefficient of 0.334 between tumor and stromal PTGS2)

• Cell type characterization through serial sections:

  • Perform IHC on adjacent sections for:

    • PTGS2

    • CD68 (general macrophage marker)

    • CD163 (M2 macrophage marker)

  • Quantify correlation between PTGS2 and macrophage markers (CD68/PTGS2: r=0.422, CD163/PTGS2: r=0.316)

• Double-fluorescent staining:

  • Co-stain single tissue sections for PTGS2 and mesenchymal markers (e.g., vimentin)

  • Capture images using fluorescence microscopy or scanning systems

• Multiplexed IHC approach:

  • Sequential staining, imaging, destaining, and reprobing of identical tissue sections

  • Example multiplexes: CD68–iNOS–PTGS2 and ARG1–MRC1–CD163–PTGS2

  • Destaining protocol: ETOH washings (5 min 50% ETOH, 10 min 100% ETOH, 5 min 50% ETOH) followed by antibody removal with guanidine-based stripping solution

These approaches reveal distinct regulatory mechanisms of PTGS2 induction between tumor cells and different stromal populations within the same microenvironment.

What methodologies enable precise PTGS2 quantification in translational research studies?

Translational research requires robust quantification methods for PTGS2:

• Western blot quantification:

  • Use standard curves with recombinant PTGS2 protein

  • Quantify the 72kDa gPTGS2 band specifically

  • Express results in absolute terms (e.g., pg of protein in 30 μg of tissue lysate)

  • Ensure reproducibility through replicate analyses (correlation coefficient r=0.907)

• Immunohistochemical quantification:

  • Digital scanning of slides using specialized scanners (e.g., Leica AT2)

  • Automated hotspot identification and quantification

  • Expression as percentage of PTGS2-positive cells

• Multiplexed analysis for cellular context:

  • Image analysis using color thresholds in ImageJ

  • Extraction of 8-bit black&white masks for each marker

  • Colocalization quantification using JACoP plug-in

  • Correlation analysis between PTGS2 and cellular markers

• Quality control measures:

  • Include both positive and negative controls in each experiment

  • Perform technical replicates to ensure methodology reliability

  • Validate antibody performance across multiple applications

These approaches have demonstrated high detection rates in clinical samples (96% in colorectal cancer tissues) with significant differentiation between tumor and normal tissues (median values: 156.86 pg vs. 0.00 pg) .

How can biotin-conjugated PTGS2 antibodies be utilized in DNA-protein interaction studies?

While the search results don't specifically address biotin-conjugated PTGS2 antibodies in DNA-protein interaction studies, methodological principles can be applied from related techniques:

• Chromatin immunoprecipitation (ChIP) adaptations:

  • Use biotin-conjugated PTGS2 antibodies to immunoprecipitate PTGS2-associated chromatin

  • Capture complexes with streptavidin-coated magnetic beads

  • Analyze DNA using PCR, sequencing, or arrays to identify PTGS2-associated genomic regions

• DNA pull-down assay optimization:

  • Generate biotinylated DNA sequences of interest (e.g., PTGS2 promoter regions)

  • PCR amplification using biotinylated primers as demonstrated for PTGS2 promoter studies

  • Verify biotin labeling efficiency using HRP-conjugated streptavidin

  • Incubate biotinylated DNA with nuclear extracts and capture with streptavidin beads

  • Analyze pull-down proteins by Western blotting using PTGS2 antibodies

• Promoter interaction studies:

  • Create truncated promoter constructs to map binding regions

  • Examples from PTGS2 research include promoter fragments P0 (0–2000 bp), P1 (0–981 bp), P2 (499–1434 bp), and P3 (1052–2000 bp)

  • Perform qualitative proteome analysis of precipitated proteins using HPLC–MS/MS

  • Identify specific proteins binding to different promoter regions through Venn diagram analysis

These approaches can help elucidate transcriptional regulation mechanisms affecting PTGS2 expression in different cellular contexts.

What considerations are important when using PTGS2 antibodies in multiplexed imaging studies?

Multiplexed imaging with PTGS2 antibodies requires careful methodological planning:

• Sequential staining protocol development:

  • Select compatible chromogens (e.g., AEC) or fluorophores

  • Develop robust destaining and stripping protocols

  • Example multiplex combinations: CD68–iNOS–PTGS2 and ARG1–MRC1–CD163–PTGS2

• Complete antibody removal verification:

  • Perform controls using only secondary antibody between staining rounds

  • No signal should be detected after proper stripping

• Nuclear counterstaining strategy:

  • Apply hematoxylin counterstaining only in the first run to avoid cumulative staining

• Image acquisition standardization:

  • Use slide scanning systems for consistent image capture

  • Maintain identical exposure settings across samples

  • Capture images after each staining round before proceeding to destaining

• Quantitative image analysis:

  • Use color thresholding to identify positive staining

  • Extract data as 8-bit black&white masks

  • Quantify colocalization using specialized plugins (e.g., JACoP)

  • Analyze numerous fields for statistical validity (e.g., 7 samples–36 fields–108 images)

• Cross-platform validation:

  • Confirm multiplexed findings with conventional single-staining approaches

  • Correlate imaging data with biochemical measurements

This methodology enables comprehensive characterization of PTGS2 expression within specific cellular subpopulations in complex tissues.

How can I assess potential PTGS2 antibody cross-reactivity with related cyclooxygenase family members?

Thorough validation of PTGS2 antibody specificity against related family members requires systematic analysis:

• Sequence alignment analysis:

  • Compare epitope regions between PTGS1 (COX-1) and PTGS2 (COX-2)

  • Identify potential cross-reactive sequences

  • Select antibodies targeting unique PTGS2 regions (e.g., antibodies recognizing AA 187-425 vs. AA 501-604)

• Recombinant protein testing:

  • Test antibody reactivity against purified PTGS1 and PTGS2 proteins

  • Perform competitive binding assays with immunizing peptides

  • Quantify relative affinity for each protein

• Knockout/knockdown validation:

  • Use PTGS2 knockout cell lines or siRNA knockdown

  • Verify disappearance of the target band/signal

  • Confirm persistent detection of PTGS1 if present

• Western blot discrimination:

  • PTGS1 and PTGS2 have different molecular weights (70kDa vs. 72kDa)

  • Run both proteins on the same gel for direct comparison

  • Look for the presence of single vs. multiple bands

• Cell/tissue panel screening:

  • Test antibodies on samples with known differential expression

  • For example, unstimulated vs. LPS-stimulated cells (PTGS2 is inducible while PTGS1 is constitutive)

  • Quantify signal ratios between different conditions

• Immunohistochemical pattern analysis:

  • Compare staining patterns with published literature

  • PTGS1 and PTGS2 typically show different tissue distribution patterns

  • Evaluate staining in tissues known to express predominantly one isoform

What are the optimal conditions for using biotin-conjugated PTGS2 antibodies in flow cytometry?

For flow cytometry applications with biotin-conjugated PTGS2 antibodies:

• Sample preparation optimization:

  • Use gentle fixation (1-2% paraformaldehyde) to preserve epitope accessibility

  • Permeabilization is essential as PTGS2 is predominantly intracellular

  • Test different permeabilization reagents (saponin vs. Triton X-100) for optimal results

• Signal amplification strategy:

  • Select appropriate streptavidin conjugates (PE, APC, or BV421)

  • Titrate streptavidin concentration to maximize signal-to-noise ratio

  • Consider multilayer approaches for further amplification if needed

• Control implementation:

  • Include FMO (fluorescence minus one) controls

  • Use isotype-matched biotin-conjugated antibodies as negative controls

  • Include positive controls (LPS-stimulated cells) with known PTGS2 expression

• Endogenous biotin blocking:

  • Pre-block with unconjugated streptavidin followed by biotin

  • Alternatively, use avidin/biotin blocking kits

• Multiparameter panel design:

  • Position PTGS2 detection in channels with sufficient sensitivity

  • Consider compensation requirements with other fluorochromes

  • Include markers to identify specific cell populations (e.g., CD68 for macrophages)

This approach enables quantitative analysis of PTGS2 expression in heterogeneous cell populations and correlation with other cellular parameters.

How do I troubleshoot inconsistent results when using PTGS2 antibodies in different experimental systems?

Troubleshooting inconsistent PTGS2 antibody performance requires systematic evaluation:

Systematic validation across applications should show consistent results. Research has demonstrated high reproducibility in PTGS2 detection with correlation coefficients of r=0.907 between replicate analyses .

What is the optimal strategy for using PTGS2 antibodies to distinguish between M1 and M2 macrophage populations?

Distinguishing M1/M2 macrophages in relation to PTGS2 expression requires sophisticated methodological approaches:

• Multiplex staining strategy:

  • First multiplex: CD68 (pan-macrophage)–iNOS (M1)–PTGS2

  • Second multiplex: ARG1 (M2)–MRC1 (M2)–CD163 (M2)–PTGS2

• Sequential staining protocol:

  • Use AEC as chromogenic substrate for consistent results

  • Apply stringent destaining protocol between markers:

    • ETOH washings (5 min 50% ETOH, 10 min 100% ETOH, 5 min 50% ETOH)

    • Antibody removal with guanidine-based stripping solution (6 M Gn-HCl, 0.2% NP-40, 10 mM DTT, 20 mM Tris-HCl, pH7.5; 37°C, 20 min)

• Comprehensive image analysis:

  • Scan slides after each staining round

  • Extract positive staining using ImageJ color thresholds

  • Generate 8-bit black&white masks for each marker

  • Quantify marker colocalization using JACoP plugin

• Statistical analysis:

  • Calculate correlation coefficients between PTGS2 and macrophage markers

  • For example: CD68/PTGS2 r=0.422, p=0.0000586; CD163/PTGS2 r=0.316, p=0.00324

  • Compare M1 vs. M2 correlations with PTGS2 expression

This approach has successfully revealed distinct associations between PTGS2 expression and different macrophage phenotypes in colorectal cancer, providing insights into inflammatory processes in the tumor microenvironment .

What methods can verify PTGS2 antibody binding to native versus denatured protein conformations?

Verification of antibody binding to different protein conformations requires multiple complementary techniques:

• Native vs. denaturing Western blotting:

  • Compare antibody performance under denaturing (SDS-PAGE) vs. native (non-denaturing) conditions

  • Assess differences in binding efficiency and band patterns

  • Some antibodies (like those targeting AA 187-425) may have conformation-dependent recognition

• Immunoprecipitation testing:

  • Ability to immunoprecipitate native PTGS2 indicates recognition of folded protein

  • Verify by subsequent Western blotting with a different PTGS2 antibody

• Cross-application validation:

  • Compare antibody performance across:

    • Western blotting (denatured protein)

    • IHC (partially preserved structure depending on fixation)

    • Immunocytochemistry (better-preserved structure)

    • Flow cytometry (largely native conformation)

• Epitope mapping:

  • Linear epitopes (e.g., AA 187-425, AA 501-604) may be recognized in both native and denatured states

  • Conformational epitopes will show significant performance differences between applications

• Application-specific recommendations:

  • Follow manufacturer guidelines for optimal dilutions in different applications

  • For example, CAB3560 antibody recommends 1:500-1:2000 for WB but 1:50-1:200 for IHC-P

This comprehensive validation ensures selection of the appropriate antibody based on the experimental technique and required conformation sensitivity.

How can PTGS2 antibodies contribute to biomarker development in cancer research?

PTGS2 antibodies provide valuable tools for cancer biomarker development:

• Differential expression analysis:

  • High detection rate in cancerous tissues (96/100 CRC samples) versus low detection in normal tissues (11/100)

  • Significant quantitative differences (median values: 156.86 pg vs. 0.00 pg)

  • Wide dynamic range of expression (0.00-1515.64 pg) enabling patient stratification

• Cellular context characterization:

  • Independent scoring of tumor-associated versus stroma-associated PTGS2

  • Correlation with macrophage markers (CD68, CD163) and mesenchymal markers (vimentin)

  • Identification of specific cellular sources within the microenvironment

• Prognostic and predictive applications:

  • Standardized quantification methods enable correlation with clinical outcomes

  • Potential for identifying patient subgroups likely to benefit from COX-2 inhibitors

  • Combined analysis with other markers to develop multiparameter signatures

• Therapeutic target validation:

  • Monitoring PTGS2 expression changes in response to experimental therapies

  • Correlation between protein levels and enzymatic activity to guide drug development

  • Assessment of inhibitor specificity against PTGS1 vs. PTGS2

• Technical advantages:

  • Reproducible quantification (r=0.907 between replicate analyses)

  • Compatible with routine clinical specimens (FFPE tissues)

  • Amenable to both manual and automated analysis platforms

These approaches have demonstrated significant promise in colorectal cancer research and can be extended to other cancer types where inflammation plays a significant role.

What approaches can assess PTGS2 post-translational modifications using specific antibodies?

Investigating PTGS2 post-translational modifications requires specialized methodology:

• Glycosylation analysis:

  • Focus on the 72kDa glycosylated PTGS2 (gPTGS2) band in Western blotting

  • Compare with the 69kDa calculated (non-glycosylated) size

  • Use glycosidase treatments to confirm glycosylation status

  • Quantify glycosylation ratio between tumor and normal tissues

• Phosphorylation studies:

  • Use phospho-specific PTGS2 antibodies if available

  • Alternatively, perform immunoprecipitation with total PTGS2 antibodies followed by phospho-protein detection

  • Treat samples with phosphatases to confirm specificity

• Ubiquitination detection:

  • Immunoprecipitate PTGS2 using validated antibodies

  • Probe with anti-ubiquitin antibodies in Western blotting

  • Use proteasome inhibitors to enhance detection of ubiquitinated forms

• SUMOylation analysis:

  • Similar approach to ubiquitination using anti-SUMO antibodies

  • Compare molecular weight shifts to identify modified forms

• Combined approaches for comprehensive PTM analysis:

  • Sequential immunoprecipitation with different modification-specific antibodies

  • Mass spectrometry analysis of immunoprecipitated PTGS2

  • Correlation of modified forms with functional activity or protein stability

These approaches can reveal how post-translational modifications regulate PTGS2 function in different physiological and pathological contexts.

How can researchers optimize PTGS2 antibody-based proximity ligation assays to study protein interactions?

Proximity Ligation Assay (PLA) optimization for PTGS2 interaction studies:

• Antibody selection criteria:

  • Primary antibodies must be from different host species

  • For PTGS2, rabbit antibodies can be paired with mouse/rat antibodies against potential interaction partners

  • Validate each primary antibody individually before combination

• Protocol optimization:

  • Fixation: Test different fixatives (4% PFA vs. methanol) to preserve protein interactions

  • Permeabilization: Optimize detergent type and concentration to maintain interaction while enabling antibody access

  • Blocking: Thorough blocking of non-specific binding sites is critical for specificity

• Controls implementation:

  • Positive control: Known PTGS2 interaction partners

  • Negative control: Omission of one primary antibody

  • Biological control: Cell lines with PTGS2 knockdown

  • Technical control: Perform conventional co-immunoprecipitation for validation

• Quantification approaches:

  • Count discrete PLA puncta per cell

  • Normalize to cell number or area

  • Compare signal distribution between subcellular compartments

• Multiplex possibilities:

  • Combine PLA with immunofluorescence for additional markers

  • Correlate interaction frequency with cell type or activation state

  • Particularly valuable for tissue sections to provide cellular context

This technique could provide valuable insights into PTGS2 interactions with transcription factors like RUNX1 or with components of inflammatory signaling pathways in different cellular contexts.

What are the considerations for using PTGS2 antibodies in three-dimensional tissue imaging?

Optimizing PTGS2 antibody use in 3D tissue imaging requires specialized approaches:

• Tissue preparation considerations:

  • Optimize fixation to balance antigen preservation with tissue transparency

  • Consider tissue clearing techniques compatible with immunolabeling

  • Control thickness for adequate antibody penetration (typically 50-100μm sections)

• Antibody penetration strategies:

  • Extended incubation times (24-72 hours)

  • Use of detergents to enhance penetration

  • Consider Fab fragments for better tissue penetration

  • Perform titration studies to determine optimal concentration

• 3D-specific labeling approaches:

  • For biotin-conjugated antibodies, test smaller streptavidin conjugates for better penetration

  • Consider signal amplification systems specific for thick sections

  • Use directly conjugated primary antibodies to reduce background

  • Include nuclear counterstains for spatial reference

• Imaging parameters:

  • Select microscopy techniques appropriate for thick sections (confocal, light-sheet, or two-photon)

  • Optimize z-step size for adequate sampling without photobleaching

  • Adjust laser power to balance signal detection and tissue penetration

• Data analysis considerations:

  • 3D reconstruction and visualization software

  • Quantitative analysis of spatial distribution patterns

  • Co-localization analysis in three dimensions

This approach would enhance understanding of PTGS2 distribution in complex tissues like tumors, where the spatial relationship between tumor cells and stromal elements is critical .

How can PTGS2 antibodies be applied to study enzyme activity versus protein expression levels?

Distinguishing PTGS2 activity from expression requires complementary approaches:

• Antibody selection strategy:

  • Use antibodies recognizing total PTGS2 protein regardless of activity state

  • Where available, consider conformation-specific antibodies that may differentiate active versus inactive forms

  • Combine with activity-based probes if accessible

• Correlation methodology:

  • Measure PTGS2 protein expression by Western blotting or IHC

  • In parallel, assess enzymatic activity using biochemical assays

  • Calculate correlation coefficients between expression and activity

  • Identify discrepancies suggesting post-translational regulation

• Inhibitor studies:

  • Treat samples with selective PTGS2 inhibitors

  • Confirm sustained protein expression despite activity inhibition

  • Useful for distinguishing protein presence from functional activity

• Cellular localization analysis:

  • Assess PTGS2 distribution between active compartments (ER, nuclear envelope) and inactive pools

  • Use subcellular fractionation followed by Western blotting

  • Alternatively, employ high-resolution microscopy with organelle markers

• Physiological response measurement:

  • Quantify downstream products (prostaglandins) as functional readouts

  • Correlate with PTGS2 protein levels detected by antibodies

  • Identify conditions where expression and activity are dissociated

This integrated approach provides deeper insights into PTGS2 regulation beyond simple protein expression, critical for understanding drug resistance mechanisms and developing more effective therapeutic strategies.

What emerging technologies are enhancing PTGS2 antibody applications in research?

Recent technological advances are expanding PTGS2 antibody applications:

• Mass cytometry (CyTOF):

  • Metal-conjugated antibodies enable simultaneous detection of 40+ markers

  • Valuable for comprehensive immune cell phenotyping in relation to PTGS2 expression

  • Overcomes spectral overlap limitations of conventional flow cytometry

• CODEX multiplexed imaging:

  • Sequential staining cycles with DNA-barcoded antibodies

  • Enables visualization of dozens of markers on a single tissue section

  • Expansion of the multiplexed approach demonstrated with PTGS2 and macrophage markers

• Spatial transcriptomics integration:

  • Combining antibody-based protein detection with spatial RNA mapping

  • Correlates PTGS2 protein expression with transcriptional profiles

  • Provides insights into post-transcriptional regulation mechanisms

• Nanobody and recombinant antibody technology:

  • Smaller binding molecules with enhanced tissue penetration

  • Site-specific biotin conjugation for consistent performance

  • Recombinant production ensures batch-to-batch consistency

• Automated image analysis with AI:

  • Deep learning algorithms for objective quantification of PTGS2 staining

  • Pattern recognition for cellular context identification

  • Standardized scoring systems for clinical translation

These emerging technologies will enhance our understanding of PTGS2 biology in complex tissues and disease states, potentially leading to more precise diagnostic and therapeutic approaches.

How do different antibody formats impact PTGS2 detection sensitivity and specificity?

Antibody format significantly influences PTGS2 detection performance:

The literature demonstrates successful application of both monoclonal antibodies (D5H5 mAb , CAB3560 ) and polyclonal antibodies for PTGS2 detection, with selection depending on the specific research requirements. For quantitative applications requiring high reproducibility, monoclonal antibodies demonstrated excellent performance with correlation coefficients of r=0.907 between replicate analyses .