Egln3 Antibody Pair

What is EGLN3 and what are its primary biological functions?

EGLN3, also known as HPH-1, HIF-PH3, HPH-3, and PHD3, functions as a cellular oxygen sensor that catalyzes the post-translational formation of 4-hydroxyproline in hypoxia-inducible factor (HIF) alpha proteins under normoxic conditions. The protein specifically hydroxylates proline residues found in the oxygen-dependent degradation domains (both N-terminal/NODD and C-terminal/CODD) of HIF1A. Beyond its role in oxygen sensing, EGLN3 serves as an important regulator of cardiomyocyte and neuronal apoptosis. Recent research has also identified EGLN3 as a potential prognostic marker for gastric cancer . Additionally, EGLN3 plays a significant role in protein stabilization, particularly for proteins like Erk3, by protecting them from lysosomal degradation through mechanisms that depend on its hydroxylase activity .

What are the key characteristics of recombinant EGLN3 antibody pairs?

Recombinant EGLN3 antibody pairs consist of specifically designed capture and detection antibodies that target distinct epitopes of the EGLN3 protein. These antibodies are typically produced using recombinant technology, which ensures high batch-to-batch consistency, scalability, and long-term supply security . The commonly available formats include unconjugated rabbit recombinant monoclonal antibodies in PBS-only buffer (without BSA or azide) at a concentration of 1 mg/mL, making them ready for conjugation according to experimental needs . The molecular weight of the target EGLN3 protein is approximately 27 kDa, and the antibodies are designed to specifically recognize human EGLN3 with high affinity and specificity .

How do EGLN3 antibody pairs differ from single antibodies in experimental applications?

Unlike single antibodies, EGLN3 antibody pairs are specifically designed to work together in sandwich-based immunoassays where one antibody (capture) binds to the target protein while the second antibody (detection) recognizes a different epitope on the same protein. This dual-recognition approach significantly enhances specificity and sensitivity compared to single-antibody methods. For example, matched EGLN3 antibody pairs like MP00347-1 (consisting of 83321-1-PBS capture and 83321-4-PBS detection) or MP00347-2 (comprising 83321-2-PBS capture and 83321-3-PBS detection) are validated for specific applications such as cytometric bead arrays . This paired approach minimizes cross-reactivity issues and background signals, allowing for more accurate quantification of EGLN3 in complex biological samples compared to single-antibody detection methods.

What protocols should be followed for optimal EGLN3 detection in cytometric bead array assays?

For optimal detection of EGLN3 using cytometric bead array assays, researchers should implement a step-wise protocol beginning with assay optimization. The capture antibody (e.g., 83321-2-PBS, clone 240165A8) should be conjugated to beads following manufacturer protocols, while the detection antibody (e.g., 83321-3-PBS, clone 240165A7) should be appropriately labeled with a fluorescent reporter . The working detection range for EGLN3 in cytometric bead arrays is typically 2.5-40 ng/mL, but assay-specific titration is strongly recommended for determining optimal antibody concentrations in each experimental system .

A typical protocol includes:

• Conjugation of capture antibody to beads at a concentration of 1 mg/mL

• Sample incubation with conjugated beads (2-4 hours at room temperature)

• Washing steps to remove unbound protein

• Addition of detection antibody (1-2 hours incubation)

• Final washing and analysis by flow cytometry

Critical quality control steps include running standard curves with recombinant EGLN3 protein for accurate quantification and including appropriate negative controls to establish assay specificity .

What are the recommended storage conditions for maintaining EGLN3 antibody pair activity?

To maintain optimal activity of EGLN3 antibody pairs, the recommended storage conditions must be strictly followed. Both capture and detection antibodies should be stored at -80°C to preserve their binding capacity and specificity . The antibodies are supplied in PBS-only buffer (free from BSA and azide) at a concentration of 1 mg/mL, which makes them suitable for long-term storage but also ready for immediate conjugation when needed . To avoid repeated freeze-thaw cycles, which can degrade antibody performance, it is advisable to aliquot the antibodies upon receipt. For working solutions prepared from the stock, storage at 4°C is typically acceptable for up to one week, but long-term storage should always be at -80°C. Additionally, protecting the antibodies from direct light exposure is crucial, especially after conjugation with fluorescent dyes for detection purposes .

How can researchers validate the specificity of EGLN3 antibody pairs in their experimental systems?

Validating the specificity of EGLN3 antibody pairs requires a multi-faceted approach:

• Positive and negative control samples: Include samples with known EGLN3 expression levels alongside EGLN3-negative samples or EGLN3-knockdown cells (using siRNA as described in search result ).

• Western blot correlation: Confirm that the antibody pair detects a protein of the expected molecular weight (27 kDa for EGLN3) in western blot analysis .

• Cross-reactivity testing: Evaluate potential cross-reactivity with related proteins by testing the antibody pair against samples containing similar family members.

• Spike-recovery experiments: Add known quantities of recombinant EGLN3 to samples and verify recovery percentages (ideally 80-120%).

• Parallelism assessment: Serial dilutions of samples should produce results that maintain linearity and parallelism with standard curves.

• Antibody competition assays: Pre-incubation with excess unlabeled antibody should competitively reduce signal if the pair is specific.

• Knockout/knockdown validation: Compare detection in wild-type versus EGLN3 knockout or knockdown samples to confirm signal specificity .

This comprehensive validation ensures that the signal detected truly represents EGLN3 protein rather than non-specific interactions or cross-reactivity.

How can EGLN3 antibody pairs be optimized for multiplex immunoassays?

Optimizing EGLN3 antibody pairs for multiplex immunoassays requires careful consideration of multiple parameters:

• Conjugation chemistry selection: For cytometric bead arrays or multiplex imaging applications, select appropriate conjugation chemistries that maintain antibody binding capacity while providing distinct signals. The PBS-only formulation of EGLN3 antibodies (83321-2-PBS and 83321-3-PBS) makes them ideal for direct conjugation without interference from buffer components .

• Cross-reactivity mapping: When multiplexing with other targets, perform comprehensive cross-reactivity testing between all antibody pairs in the panel by running single-analyte controls alongside multiplexed samples.

• Signal optimization table:

• Bead region selection: When using cytometric bead arrays, carefully select bead regions with minimal spillover between detection channels.

• Signal amplification strategies: For low-abundance targets, consider implementing signal amplification methods like streptavidin-phycoerythrin systems or tyramide signal amplification while monitoring background levels .

The optimal working range for EGLN3 detection in cytometric bead arrays is 2.5-40 ng/mL, but researchers should verify this range in their specific multiplex system .

What are the common sources of variability in EGLN3 antibody pair assays and how can they be addressed?

Several factors can introduce variability in EGLN3 antibody pair assays:

• Antibody degradation: Store antibodies at -80°C and avoid repeated freeze-thaw cycles. The unconjugated antibodies in PBS-only buffer have specific storage requirements that must be followed to maintain activity .

• Inconsistent conjugation: Standardize conjugation protocols and validate each conjugation batch through quality control testing with reference standards.

• Sample processing variability: Implement consistent sample collection, processing, and storage protocols. Differences in sample preparation can significantly affect EGLN3 detection, particularly when working with cell lysates.

• Matrix effects: Different sample types (serum, tissue lysate, cell culture supernatant) may contain interfering substances. Address by:

  • Optimizing sample dilution factors

  • Using matrix-matched standards

  • Implementing more stringent washing protocols

  • Adding blocking agents specific to the matrix being tested

• Temperature fluctuations: Maintain consistent temperature throughout the assay procedure, as antibody-antigen binding kinetics are temperature-dependent.

• Instrument variability: For cytometric bead arrays, regular instrument calibration is essential. Keep track of instrument performance metrics and include standardized beads in each run .

• Operator technique: Standardize pipetting techniques and establish detailed protocols to minimize operator-dependent variability.

Implementing a comprehensive validation plan that includes intra-assay and inter-assay precision testing (aiming for CV <15%) will help identify and address these sources of variability.

How can researchers troubleshoot low signal or high background issues in EGLN3 detection assays?

When encountering signal problems in EGLN3 detection assays, a systematic troubleshooting approach is recommended:

Low Signal Issues:

• Antibody activity verification: Confirm antibody functionality through simpler assays like direct ELISA before attempting sandwich formats.

• Epitope accessibility: Ensure sample preparation methods don't compromise the epitopes recognized by the antibody pair. Different lysis buffers or fixation methods may affect epitope accessibility.

• Concentration adjustment: The recommended starting point is 1 mg/mL, but titration experiments may reveal optimal working concentrations for specific applications .

• Incubation optimization: Extend incubation times or adjust temperatures to enhance binding kinetics.

• Detection system verification: For fluorescence-based detection, check for photobleaching or quenching effects.

High Background Issues:

• Blocking optimization: Test different blocking reagents (BSA, casein, commercial blockers) at various concentrations to minimize non-specific binding.

• Washing stringency: Increase the number of washes or add detergents like Tween-20 at appropriate concentrations.

• Cross-reactivity investigation: Test antibodies individually to identify the source of cross-reactivity.

• Buffer optimization: Adjust salt concentration or pH to reduce non-specific interactions while maintaining specific binding.

• Sample dilution: Further dilute samples to reduce matrix effects or potential hook effects with high-concentration samples.

When using cytometric bead arrays, additional consideration should be given to bead aggregation issues and flow cytometer settings, as these can significantly impact assay performance and background levels .

How does EGLN3 hydroxylase activity impact protein stability and what implications does this have for experimental design?

EGLN3 hydroxylase activity plays a critical role in protein stability, particularly for proteins like Erk3. Research has demonstrated that EGLN3 protects Erk3 from lysosomal degradation through a mechanism dependent on its hydroxylase activity . Specifically, EGLN3 interferes with the interactions between Erk3 and proteins involved in lysosomal degradation pathways, including HSC70 and LAMP2A . When EGLN3 hydroxylase activity is inhibited, either through mutation (as in the R205K mutant) or pharmacological inhibition (using DMOG), its ability to prevent the Erk3-LAMP2A interaction is compromised, leading to increased Erk3 degradation .

This understanding has several implications for experimental design:

• Inhibitor controls: Researchers studying EGLN3-dependent processes should include controls with hydroxylase inhibitors like DMOG to distinguish between hydroxylase-dependent and hydroxylase-independent functions of EGLN3 .

• Protein stabilization considerations: When investigating proteins that potentially interact with EGLN3 (like Erk3), researchers should account for EGLN3's impact on protein stability when designing pulse-chase experiments or quantifying protein levels.

• Interaction domain mapping: The region between amino acids 74-239 of EGLN3 is crucial for its interaction with Erk3, while Erk3 interacts with EGLN3 primarily through its region spanning amino acids 340-480 . This information should guide the design of truncation mutants or peptide inhibitors for studying these interactions.

• Subcellular localization studies: Since protein degradation pathways are compartmentalized within cells, consideration of where EGLN3 interactions occur is critical for accurate interpretation of results.

What are the key considerations when studying EGLN3 interactions with binding partners using antibody-based detection methods?

When investigating EGLN3 interactions with binding partners such as Erk3, p53, HIF1A, HSC70, or LAMP2A using antibody-based detection methods, researchers should consider several critical factors:

• Epitope masking: Protein-protein interactions may mask antibody epitopes, potentially leading to false-negative results. To address this:

  • Use multiple antibody pairs recognizing different epitopes

  • Consider denaturing versus native conditions based on research goals

  • Implement complementary non-antibody-based techniques (e.g., mass spectrometry)

• Co-immunoprecipitation optimization: When studying interactions between EGLN3 and partners like Erk3, optimize lysis conditions to preserve interactions while minimizing non-specific binding .

• Controls for hydroxylase-dependent interactions: Include both wild-type EGLN3 and catalytically inactive mutants (like R205K) or use hydroxylase inhibitors (DMOG) to distinguish between hydroxylase-dependent and independent interactions .

• Subcellular fractionation considerations: EGLN3 interactions may occur in specific cellular compartments. For instance, EGLN3-p53 interactions were observed primarily in nuclei . Therefore:

  • Include appropriate subcellular fractionation steps

  • Verify the purity of fractions with compartment-specific markers

  • Consider in situ proximity ligation assays for spatial resolution of interactions

• Quantitative analysis: Implement quantitative methods like FRET, BRET, or AlphaScreen technologies alongside traditional co-IP approaches for a more comprehensive understanding of interaction dynamics.

• Domain-specific antibodies: When studying specific interaction domains (e.g., aa 74-239 of EGLN3 for Erk3 binding), consider using domain-specific antibodies that don't interfere with the interaction regions .

How can EGLN3 antibody pairs be utilized to study differential expression in pathological conditions?

EGLN3 antibody pairs offer powerful tools for studying differential expression in various pathological conditions, including cancer, cardiovascular diseases, and hypoxia-related disorders. Implementation strategies include:

• Quantitative profiling in tissue samples:

  • Use matched antibody pairs in cytometric bead arrays to precisely quantify EGLN3 levels across disease stages

  • Develop tissue microarray applications with optimized multiplex detection to correlate EGLN3 expression with other biomarkers

  • Combine with clinical outcome data to evaluate EGLN3's potential as a prognostic marker, particularly in gastric cancer

• Cellular response monitoring:

  • Track EGLN3 expression changes during hypoxia response using time-course experiments

  • Correlate EGLN3 levels with cellular phenotypes in cardiomyocyte and neuronal apoptosis models

  • Implement high-content screening approaches with automated image analysis for large-scale studies

• Mechanism investigation methodology:

  • Use siRNA knockdown approaches to modulate EGLN3 expression and monitor effects on target proteins like p53 or Erk3

  • Compare hydroxylase-dependent and independent functions using wild-type EGLN3 versus R205K mutant or DMOG treatment

  • Integrate with pathway analysis tools to position EGLN3 within regulatory networks

• Technological integration table:

• Validation in model systems:

  • Cross-validate findings from human samples in appropriate animal models

  • Utilize genetic knockout/knockin approaches to confirm antibody specificity and biological findings

  • Implement rescue experiments to confirm the specificity of observed phenotypes

By implementing these approaches, researchers can comprehensively characterize EGLN3's role in pathological conditions and potentially identify new therapeutic targets or diagnostic markers.

What innovative applications are emerging for EGLN3 antibody pairs beyond traditional immunoassays?

Several cutting-edge applications for EGLN3 antibody pairs are expanding their utility beyond conventional immunoassays:

• Proximity-based protein interaction mapping:

  • Implementing proximity ligation assays (PLA) to visualize and quantify EGLN3 interactions with proteins like Erk3, p53, HSC70, and LAMP2A with subcellular resolution

  • Developing BioID or APEX2-based proximity labeling approaches with EGLN3 antibodies for validation

  • Exploring split-enzyme complementation assays to monitor dynamic EGLN3 interactions in living cells

• Advanced imaging applications:

  • Integrating EGLN3 antibody pairs into multiplexed ion beam imaging (MIBI) or multiplexed immunofluorescence panels for spatial biology studies

  • Developing super-resolution microscopy applications to visualize EGLN3 in relation to subcellular structures

  • Implementing live-cell imaging with nanobody derivatives of existing EGLN3 antibodies

• Therapeutic monitoring applications:

  • Developing companion diagnostic assays using EGLN3 antibody pairs to monitor response to hypoxia-modulating therapies

  • Creating point-of-care testing platforms for rapid EGLN3 quantification in clinical settings

  • Establishing liquid biopsy applications for detecting EGLN3 in circulation as a potential biomarker

• Single-cell analysis integration:

  • Adapting EGLN3 antibody pairs for mass cytometry (CyTOF) panels to correlate EGLN3 with dozens of other proteins at single-cell resolution

  • Developing compatible protocols for spatial transcriptomics combined with EGLN3 protein detection

  • Creating microfluidic-based single-cell protein analysis platforms incorporating EGLN3 detection

The conjugation-ready PBS-only formulation of these antibody pairs (83321-1-PBS, 83321-2-PBS, 83321-3-PBS, 83321-4-PBS) makes them particularly suitable for these advanced applications where custom conjugation is required .

How might the study of EGLN3's role in protein stability inform novel therapeutic approaches?

The discovery that EGLN3 regulates protein stability, particularly for proteins like Erk3 through hydroxylase-dependent mechanisms, opens several potential therapeutic avenues:

• Targeted protein stabilization strategies:

  • Development of small molecules that mimic EGLN3's ability to interfere with protein-LAMP2A interactions, potentially stabilizing beneficial proteins in disease contexts

  • Design of peptide inhibitors based on the EGLN3-Erk3 interaction domains (aa 74-239 of EGLN3) that could selectively modulate protein stability

  • Creation of protein-protein interaction modulators that specifically alter EGLN3's interaction with degradation machinery components like HSC70 or LAMP2A

• Hydroxylase activity modulation:

  • Refinement of hydroxylase inhibitors beyond current compounds like DMOG to achieve greater specificity for EGLN3 over related family members

  • Development of activity-based probes for monitoring EGLN3 hydroxylase activity in living systems

  • Exploration of allosteric modulators that could fine-tune rather than completely block EGLN3 activity

• Pathology-specific applications:

  • For cancers where EGLN3 serves as a prognostic marker, development of therapeutic strategies targeting its protein-stabilizing functions

  • In cardiovascular conditions, exploration of EGLN3 modulators to influence cardiomyocyte apoptosis pathways

  • For neurological disorders, development of brain-penetrant EGLN3 modulators to affect neuronal apoptosis mechanisms

• Combination therapy approaches:

  • Integration of EGLN3-targeting strategies with existing therapies that affect the HIF pathway

  • Development of dual-targeting approaches addressing both EGLN3's hydroxylase-dependent and independent functions

  • Design of sequential therapy regimens that first modulate EGLN3 activity to prime cells for subsequent treatments

Research using EGLN3 antibody pairs will be essential for validating these approaches through target engagement studies, pharmacodynamic biomarker development, and mechanism-of-action investigations in preclinical models.

What methodological advances are needed to better characterize EGLN3's hydroxylase-dependent versus hydroxylase-independent functions?

To advance our understanding of EGLN3's dual functionality—hydroxylase-dependent and hydroxylase-independent—several methodological improvements are needed:

• Advanced protein modification detection:

  • Development of site-specific antibodies that recognize hydroxylated versus non-hydroxylated target proteins

  • Implementation of mass spectrometry methods optimized for detecting hydroxylation on specific proline residues in EGLN3 targets

  • Creation of biosensors that can monitor hydroxylation events in real-time within living cells

• Genetic model refinement:

  • Generation of knock-in models expressing catalytically inactive EGLN3 (similar to R205K) to distinguish between structural and enzymatic roles in vivo

  • Development of inducible systems for temporal control of EGLN3 variant expression

  • Creation of domain-specific deletion models based on the mapped interaction regions (e.g., aa 74-239 for Erk3 interaction)

• Interaction dynamics characterization:

  • Implementation of quantitative interaction proteomics comparing wild-type EGLN3 versus R205K interactomes under various conditions

  • Development of methods to monitor the dynamics of protein complex formation/dissolution involving EGLN3, HSC70, and LAMP2A

  • Creation of assays that can directly measure protein stability and degradation rates in the presence of various EGLN3 forms

• Pathway-specific analysis:

  • Better tools for distinguishing between lysosomal, proteasomal, and other degradation pathways affected by EGLN3

  • Development of multiplexed assays to simultaneously monitor multiple EGLN3 targets and their stability

  • Creation of computational models that can predict which proteins might be regulated by EGLN3's hydroxylase-dependent versus independent mechanisms

• Structural biology approaches:

  • Determination of high-resolution structures of EGLN3 in complex with various binding partners

  • Implementation of hydrogen-deuterium exchange mass spectrometry to map conformational changes associated with different EGLN3 functions

  • Development of cryo-EM approaches to visualize larger EGLN3-containing complexes

These methodological advances would significantly enhance our ability to dissect the complex functions of EGLN3 and potentially lead to more targeted therapeutic approaches in the future.

What are the established best practices for implementing EGLN3 antibody pairs in multi-site collaborative research?

For consistent and reliable results across multiple research sites, implementing standardized best practices for EGLN3 antibody pair usage is essential:

• Reagent standardization:

  • Utilize centralized procurement of antibody pairs to ensure all sites work with identical lots

  • Implement mandatory lot testing before distribution to verify performance metrics

  • Create and distribute reference standards for calibration across sites

  • Store all antibodies at -80°C as recommended to maintain consistent activity

• Protocol harmonization:

  • Develop detailed standard operating procedures (SOPs) with specific guidance for each application

  • Include mandatory titration steps to optimize antibody concentrations for specific applications

  • Create video demonstrations of critical technique-dependent steps

  • Implement electronic laboratory notebooks with standardized templates for data recording

• Quality control framework:

  • Establish minimum acceptance criteria for standard curves (r² > 0.98)

  • Define acceptable ranges for positive and negative controls

  • Implement inter-laboratory proficiency testing with standardized samples

  • Develop scoring systems for assay performance across sites

• Data standardization:

  • Create uniform data collection templates

  • Establish centralized data repositories with standardized formats

  • Implement automated quality checks for data submission

  • Develop consistent analysis pipelines to minimize site-specific biases

• Training and compliance:

  • Conduct centralized training workshops for key personnel

  • Implement certification requirements before independent assay performance

  • Schedule regular refresher training and updates

  • Develop troubleshooting decision trees for common issues

By implementing these best practices, multi-site collaborative research involving EGLN3 antibody pairs can achieve the high level of consistency required for meaningful cross-site comparisons and data pooling.

What integrated analytical approaches combine EGLN3 protein detection with other omics technologies?

Integrating EGLN3 protein detection with complementary omics technologies creates powerful multi-dimensional research approaches:

• Proteogenomic integration:

  • Combine EGLN3 antibody-based quantification with RNA-seq to correlate protein levels with transcript expression

  • Integrate with genomic data to identify genetic variants affecting EGLN3 expression or function

  • Couple with ribosome profiling to assess translational regulation of EGLN3 and its targets

• Post-translational modification mapping:

  • Pair EGLN3 protein quantification with phosphoproteomics to assess signaling networks affected by EGLN3 activity

  • Integrate with hydroxylation-specific proteomics to identify novel EGLN3 substrates beyond HIF1A

  • Combine with ubiquitylome analysis to comprehensively map degradation pathways influenced by EGLN3

• Metabolomic connections:

  • Correlate EGLN3 levels with metabolic profiles, particularly those related to oxygen sensing

  • Investigate connections between EGLN3 activity and TCA cycle intermediates that may influence hydroxylase function

  • Develop integrated models of how metabolic state affects EGLN3-dependent protein stability

• Spatial multi-omics integration:

  • Combine EGLN3 antibody-based imaging with spatial transcriptomics

  • Integrate with imaging mass cytometry for high-dimensional spatial protein profiling

  • Develop computational pipelines that can integrate multiple spatial data types with EGLN3 as a central node

• Temporal dynamics analysis:

  • Implement time-course experiments combining EGLN3 protein quantification with transcriptomics

  • Develop mathematical models integrating temporal protein stability data for EGLN3 targets like Erk3

  • Create visualization tools for multi-omic temporal data centered on EGLN3 function

These integrated approaches leverage the specificity of EGLN3 antibody pairs while providing broader biological context through complementary technologies, enabling more comprehensive understanding of EGLN3's role in complex biological systems.

What are the critical considerations for selecting appropriate controls in EGLN3-focused research?

Selecting appropriate controls is fundamental to generating reliable and interpretable data in EGLN3-focused research:

• Antibody specificity controls:

  • Include EGLN3 knockdown or knockout samples verified by alternative detection methods

  • Implement isotype controls matched to the antibody pairs being used

  • Include pre-absorption controls where primary antibodies are pre-incubated with recombinant EGLN3

  • Use competing antibodies targeting the same epitopes to verify binding specificity

• Functional activity controls:

  • Include catalytically inactive EGLN3 variants (e.g., R205K) when studying hydroxylase-dependent functions

  • Use pharmacological inhibitors like DMOG as complementary approaches to genetic methods

  • Implement dose-response studies with inhibitors to distinguish partial from complete inhibition effects

  • Include oxygen-level controls when studying hypoxia-dependent phenotypes

• Interaction verification controls:

  • Use domain mapping to generate negative control proteins lacking key interaction regions (e.g., EGLN3 without aa 74-239 for Erk3 interaction studies)

  • Implement both forward and reverse co-immunoprecipitation approaches

  • Include competition assays with purified proteins or peptides representing interaction domains

  • Use structural mutations that disrupt specific protein-protein interfaces

• System-level controls:

  • Consider cell type-specific differences in EGLN3 function and include appropriate cellular models

  • Address potential redundancy with other EGLN family members (EGLN1/PHD2, EGLN2/PHD1)

  • Account for microenvironmental factors like oxygen tension, pH, and metabolite availability

  • Implement time-course controls to distinguish acute from chronic effects

• Assay-specific technical controls:

  • For cytometric bead arrays, include bead-only controls and secondary-only controls

  • Implement spike-recovery experiments to verify detection in complex matrices

  • Include dilution linearity tests to confirm assay performance across concentration ranges

  • Use reference standard curves in each experimental run for accurate quantification