Bioanalysis for Foundations and Patient Groups

Struggling with a Bioanalysis Vendor for your Complement Assays? Dash’s Automation Delivers Consistent Results and High Throughput.

The complement system represents one of the most technically demanding areas of immunological research, where assay variability can derail therapeutic programs and manual workflows create bottlenecks that slow drug development. For companies developing complement-targeted therapeutics or investigating complement-mediated pathologies, three biomarkers have emerged as critical: C3a (alternative and classical pathway activation), sC5b-9 (terminal complement complex formation), and Factor Bb (alternative pathway-specific activation).

Yet measuring these analytes reliably remains a persistent challenge. At Dash Bio, we've solved this problem through fully automated bioanalysis that delivers 10x faster turnaround with demonstrably lower variability and higher data quality than traditional manual approaches.

C3a: Capturing Early Pathway Activation

C3a is a 77-amino acid anaphylatoxin released during C3 convertase activity, making it a sensitive marker of complement activation through both classical and alternative pathways. The analytical challenge lies in its instability and the presence of endogenous carboxypeptidases that rapidly convert C3a to C3a-desArg, confounding quantification. Accurate measurement requires:

• Immediate sample stabilization with specific protease inhibitors
• Careful timing between sample collection and analysis
• Assays capable of distinguishing C3a from its degradation products
• Dynamic range sufficient to capture both basal levels (typically 100-300 ng/mL) and acute activation (potentially >10,000 ng/mL)

Manual protocols struggle with the time-dependent variability introduced by inconsistent processing speeds and serial dilution preparation.

sC5b-9: Quantifying Terminal Pathway Activity

The soluble terminal complement complex (sC5b-9, also called SC5b-9 or TCC) represents the final stage of complement activation—the formation of the membrane attack complex in its soluble, vitronectin-bound form. This biomarker is particularly valuable for monitoring complement inhibitor efficacy, especially for C5-targeting therapeutics.

The technical challenges include:

• Complex formation kinetics that require precise timing
• Conformational epitopes that demand carefully validated antibodies
• Matrix effects from serum proteins that interfere with detection
• Need for high sensitivity to detect both physiological levels (~100-300 ng/mL) and pathological activation

Factor Bb: Alternative Pathway Specificity

Factor Bb, the catalytic fragment generated when Factor B is cleaved by Factor D in the alternative pathway C3 convertase, provides pathway-specific information that C3a cannot. This is critical when distinguishing alternative pathway-driven diseases from classical pathway activation.

Analytical hurdles include:

• Short half-life of the C3bBb complex (t½ ~90 seconds at 37°C)
• Quantification of a cleavage product rather than an intact protein
• Need for assays that don't cross-react with uncleaved Factor B
• Temperature sensitivity requiring consistent sample handling

The manual execution of Factor Bb assays is particularly problematic because timing variability directly translates to measurement error.

The Dash Bio Advantage: Fully Automated Bioanalysis

Our automated platform fundamentally changes the equation for complement biomarker quantification. Rather than accepting the inherent variability of manual pipetting, timing, and serial dilutions, we've engineered reproducibility into every step.

From Kit to Custom: A Case Study in Assay Evolution

We recently partnered with a therapeutic developer who was struggling with inconsistent complement activation data using standard ELISA kits. Their manual workflow produced coefficient of variation (CV) values often exceeding 20%, making it impossible to detect subtle but meaningful differences between treatment groups. More concerning, they couldn't reliably reproduce results between runs—a critical failure for regulated bioanalysis.

The Challenge: The partner needed to measure complement activation in response to their therapeutic candidate across hundreds of samples, with serial dilutions to establish dose-response relationships and time-course experiments requiring precise stimulation and quench timing. Manual execution meant:

• Variable pipetting between operators
• Inconsistent incubation times when processing multiple plates
• Serial dilution errors that compounded across dilution series
• Sample-to-sample timing differences during stimulation protocols

Our Solution: We started with the commercial kit then bridged it to our own assay using full automation on our 384-well high-throughput system. The transformation involved:

1. Luminescence-Based Detection for Superior Dynamic Range

We converted the colorimetric readout to a luminescence-based system, dramatically expanding the dynamic range. This eliminated the need for multiple dilution runs to capture both low and high complement activation samples—a single dilution series now covers the entire biological range. The improved signal-to-noise ratio of chemiluminescence also reduced CVs to consistently <10%, and often <5% for mid-range values.

2. Automated Serial Dilution Execution

Our liquid handling system executes precise serial dilutions with sub-microliter accuracy. Every dilution is identical across all samples, eliminating operator-dependent variability. The automation maintains consistent mixing at each dilution step, critical for complement proteins that can aggregate or settle.

3. Programmed Stimulation and Quench Timing

For stimulation experiments, timing is everything. Our system executes programmed stimulation protocols where each well receives stimulus at precisely the defined interval, followed by automated quench addition at the exact timepoint. This complex choreography—impossible to execute consistently by hand across 384 wells—becomes reproducible routine on our platform.

The automation handles:

• Simultaneous stimulation of appropriate well groups
• Temperature-controlled incubation during activation periods
• Precise quench timing that stops complement activation identically across all samples
• Integration of appropriate controls at defined positions

4. Complete Process Integration

From sample receipt to data delivery, our platform integrates:

• Automated sample thawing with temperature verification
• Barcode tracking for chain of custody
• Quality control sample integration at defined frequencies
• Real-time monitoring of assay performance metrics
• Automated data reduction with predefined acceptance criteria

The Quantitative Impact: 10x Faster, Demonstrably Better

The results speak to the power of automation in complex immunoassays:

Turnaround Time: What previously required 5-7 days for a full study now completes in 12-18 hours. This isn't just about working faster—it's about maintaining sample integrity and reducing freeze-thaw cycles that degrade complement proteins.

Variability Reduction: Inter-assay CVs dropped from 18-25% (manual) to 5-8% (automated). Intra-assay CVs improved from 12-15% to 3-6%. This improvement directly translates to increased statistical power and the ability to detect smaller treatment effects.

Data Quality Metrics:

• Parallelism of dilution curves: >95% of samples show acceptable parallelism (previously ~70%)
• Standard curve performance: R² consistently >0.99 (previously 0.95-0.98)
• Quality control recovery: 90-110% with CV <8% across all runs
• Outlier rate: <2% of wells flagged (previously 8-12%)

Throughput: The 384-well format combined with automation enables processing of 1,500+ samples per week for each biomarker, with full QC documentation—a 10-fold increase over manual capacity.

Technical Specifications That Matter

Our complement biomarker platform delivers:

C3a Quantification:

• Lower limit of quantification (LLOQ): 5 ng/mL
• Dynamic range: 5-5,000 ng/mL in a single dilution series
• Sample volume: 25 μL (compared to 100 μL for manual)
• Intra-assay CV: <6%
• Inter-assay CV: <8%

sC5b-9 Quantification:

• LLOQ: 10 ng/mL
• Dynamic range: 10-10,000 ng/mL
• Specificity: No cross-reactivity with C5a, C3a, or individual complex components
• Automated incubation timing ensures consistent complex detection
• Intra-assay CV: <5%
• Inter-assay CV: <7%

Factor Bb Quantification:

• LLOQ: 0.5 μg/mL
• Dynamic range: 0.5-500 μg/mL
• Temperature-controlled processing eliminates heat-induced degradation
• Automated timing prevents ex vivo activation artifacts
• Intra-assay CV: <6%
• Inter-assay CV: <9%

Beyond Assay Execution: Integrated Expertise

Automation handles the mechanical complexity, but interpretation requires immunological expertise. Our team provides:

Method Development and Validation: We don't just run assays—we develop and validate them to meet your program's needs, whether exploratory research or GLP bioanalysis for regulatory submissions.

Matrix Optimization: Complement assays perform differently in serum, plasma (EDTA vs. heparin), and experimental matrices. We optimize and validate for your specific sample type.

Interference Testing: We proactively test for interference from therapeutic antibodies, complement inhibitors, or other biologics in your samples.

Data Interpretation Support: Understanding complement activation patterns requires immunological context. We help distinguish biologically meaningful changes from analytical variation.

Real-World Applications

Our automated complement platform supports:

Therapeutic Development Programs:

• PK/PD studies correlating drug exposure with complement inhibition
• Dose-ranging studies where precise C3a, sC5b-9, and Factor Bb measurements inform dose selection
• Time-course studies tracking complement activation with automated sampling precision

Mechanistic Research:

• Dissecting pathway-specific contributions using C3a + Factor Bb profiles
• Terminal pathway assessment via sC5b-9 in disease models
• Screening complement modulators in high-throughput formats

Biomarker Strategy Development:

• Establishing complement activation signatures in disease states
• Validating complement biomarkers for patient stratification
• Monitoring complement-mediated adverse events in clinical programs
  

The Case for Automation

Manual complement assays may work for small pilot studies, but they fail when precision, throughput, and consistency matter. If you're developing complement-targeted therapeutics, investigating complement-mediated pathology, or simply tired of questioning whether your complement data is real or artifact, automation isn't a luxury—it's essential.

At Dash Bio, our fully automated platform transforms complement biomarker quantification from a bottleneck into a competitive advantage. We deliver data you can trust, at the speed your program demands, with the quality that regulatory agencies expect.

Contact Dash Bio to discuss how our automated complement bioanalysis platform can accelerate your research and development programs. We're happy to share detailed validation data and discuss custom assay development for your specific application.