Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets
February 10th, 2020
Blood-based biomarkers for neurodegenerative diseases are essential for implementing large-scale clinical studies. A reliable and validated blood test should require a small sample volume as well as be a less invasive sampling method, affordable, and reproducible. This paper demonstrates that high-throughput capillary electrophoresis immunoassay satisfies criteria for potential biomarker development.
This protocol can be used to optimize both protein and primary antibody concentrations in a single assay plate, and to perform two assays using a single sample preparation. The main advantage of this technique is that it allows 2 1/2 days of lab work including data analysis, to be completed in 3 1/2 hours. This technique provides a high throughput assay format for developing a blood-based biomarker for ALS and is an ideal method for conducting large clinical trials.
This method can provide insight into target protein profiles during disease prognosis and can facilitate the monitoring of the effects of drugs of interesting clinical trials. After collecting 100 microliters of human platelet lysates from ALS patients and healthy subjects, fill out in-house generated templates for the capillary layout and sample preparation. The sample mixture preparation table is dynamic and will automatically calculate how much volume should be removed from the source.
Place all the assay reagents on ice. Except the standard pack, which should remain at room temperature. To prepare the fluorescent master mix, add 40 microliters of deionized water to a clear tube of DTT, and add 20 microliters of 10X sample buffer.
And 20 microliters of the prepared 400 millimolar DTT solution to the pink tube from the assay kit. To prepare the biotinylated ladder, add 16 microliters of deionized water, two microliters of 10X sample buffer and two microliters of the prepared 400 millimolar DTT solution to the white tube provided in the kit. After gentle mixing, transfer the ladder solution into a 200 microliter PCR tube before denaturing.
Add 1.5 microliters of 10X sample buffer and 148.5 microliters of deionized water to a 600 microliter microcentrifuge tube and vortex to mix, before placing the tube on ice. Add the antibodies of interest directly the diluent as indicated in the table and flush the pipette tip multiple times in a homogenous antibody solution. To prepare the capillary sample mix, open all of the PCR tubes and add 1.6 microliter aliquots of fluorescent 5X sample buffer to each tube as indicated in the table using a reverse pipetting technique.
Immediately closing each tube as the buffer is added. When all of the buffer has been added, open all of the tubes and add a 0.1X sample buffer in volumes as indicated in the table, immediately closing each tube as the buffer is added. Next, open all of the tubes and add the protein samples as indicated in the table and immediately close each tube cap.
When all of the samples have been added, briefly centrifuge all of the tubes in a benchtop centrifuge. Vortex to mix and centrifuge the samples again. At the end of the second centrifugation, place all of the tubes into a thermocycler with a heated lid.
And denature the samples under the indicated conditions. At the end of the denaturation, centrifuge, mix and centrifuge the samples again and place all the tubes in a tube rack on ice. Using the figure as a guide, add 10 microliters of streptavidin horseradish peroxidase to well D1, 10 microliters of the appropriate secondary antibody to wells D2 through D5, 10 microliters of antibody diluent to each well in row B and to well C1, 10 microliters of the appropriate primary antibody to wells C2 through C25, five microliters of biotinylated ladder from PCR tube number one to well A1, three microliters of sample to rows A2 through A25 and 15 microliters of freshly-prepared luminol peroxide to each well of row E.Pipetting the sample mix and the reagent into the tiny well in the correct orders is critical to the success of experiment, so it should be done very carefully.
Then add 500 microliters of wash buffer to each designated wash buffer well. And spin down the plate contents by centrifugation. To perform the capillary analysis on the assay plate, connect the capillary analyzer to the online system and click Instrument and Connect in the analyzer software.
Select the instrument serial number within the popup menu, and click Connect. Select the Assay tab, and select New Assay, enter the assay parameters and save the file name and location. Then confirm that the blinking blue indicator in the analyzer is a solid blue and remove the capillary cartridge from its packaging.
Remove the protective seal from the assay plate and visually observe the pre-filled wells for air bubbles. Place the assay plate in the plate holder and holding the capillary cartridge at an angle, insert the cartridge into the capillary slot and close the analyzer door. Then click Start in the analyzer software.
The use of a whole platelet lysate mixture in the assay may reduce the signal intensity of the target proteins and contribute to a high background signal. Therefore, in this assay, clear supernatant was used after rupturing the platelets. A linear dynamic range for the platelet cytosol protein concentration was established at 0.2 to 0.8 milligrams per milliliter, and an essay template was adopted so that both the protein concentration and the primary antibody titration were able to be performed in a single assay.
The high dynamic range detection profile delivers a significantly wider dynamic range due to the greater sensitivity of the capillary assay, resulting in a better detection and quantification over a larger sample concentration range. Individual exposure times can also be defined. Using the optimized assay conditions, this analysis of platelet lysate cytosolic fractions obtained from ALS patients using two sets of antibodies allow the identification of disease-specific TDP-43 and its phosphorylated derivative within the samples.
The total TDP-43 could then be quantified using the calibration curve. And the intra and inter-run assay variabilities were tested in pooled human ALS platelet cytosolic fractions. This method allows the use of primary antibodies, facilitating the identification of up to five different target proteins within the same sample in a single assay run.
Not only does this technique substantially reduce the protein analysis time, it requires microliter sample volumes and generates results with a high degree of reproducibility. Some of the chemicals used in this assay and all human samples are considered biohazardous, so appropriate personal protective equipment should always be used when performing these experiments.
Overview
This study focuses on the development of blood-based biomarkers for neurodegenerative diseases, specifically using a high-throughput capillary electrophoresis immunoassay. The goal is to create a less invasive, affordable, and reproducible method for clinical studies, with implications for monitoring disease progression and treatment effects in conditions like ALS.
Key Study Components
Area of Science
- Neurodegenerative diseases
- Biomarker development
- Clinical trial methodologies
Background
- Blood-based biomarkers can significantly enhance neurodegenerative disease research.
- Current testing methods often require large sample sizes and are invasive.
- High-throughput methods can improve efficiency in clinical studies.
- This protocol aims to streamline the process of biomarker development.
Purpose of Study
- To establish a reliable protocol for assessing biomarkers in blood samples.
- To minimize the time and resources needed for lab analysis.
- To facilitate the identification of disease-specific proteins.
Methods Used
- The study utilized a high-throughput capillary electrophoresis immunoassay.
- Human platelet lysates from ALS patients and healthy subjects served as the primary biological model.
- Key steps included optimizing protein and antibody concentrations and using a single sample preparation for multiple assays.
- The entire lab work process, including data analysis, was reduced to 3.5 hours.
Main Results
- The method successfully identified TDP-43 and its phosphorylated derivative from patient samples.
- A linear dynamic range was established for protein concentration, enhancing detection sensitivity.
- This protocol allows simultaneous assessment of multiple target proteins, improving efficiency in biomarker quantification.
Conclusions
- This study demonstrates an effective method for developing blood-based biomarkers in neurodegenerative diseases.
- The high-throughput approach significantly reduces analysis time and enhances the feasibility of large clinical trials.
- This could lead to better tracking of disease progression and treatment effects in studies involving ALS and other neurodegenerative conditions.
Frequently Asked Questions
Executive Industry Relevance
Capillary electrophoresis immunoassay (CEI) enables high-throughput, reproducible quantification of disease-relevant proteins in human platelets, supporting blood-based biomarker discovery for neurodegenerative diseases like ALS. By reducing assay time from 2.5 days to 3.5 hours and requiring microliter sample volumes, CEI enhances feasibility for large clinical trials and longitudinal monitoring. This method facilitates target protein profiling and drug effect assessment, improving predictive confidence in early discovery and translational workflows.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by quantifying pan TDP-43 and phosphorylated TDP-43 in platelet lysates.
- Operational Value: Supports biological de-risking through reproducible, multiplexed protein detection in a single sample preparation.
- Predictive Value: Facilitates portfolio triage by providing quantitative readouts for target engagement and disease-modulating effects.
Screening & Assay Development
- Scientific Value: Delivers high sensitivity and linear dynamic range (0.2–0.8 mg/mL) for accurate biomarker quantification in clinical samples.
- Operational Value: Enables automation-ready workflows with capacity to analyze up to 24 samples per run, improving throughput and reproducibility.
- Strategic Value: Reduces reagent consumption and hands-on time, supporting scalable assay development for biomarker screening campaigns.
Translational & Preclinical Research
- Scientific Value: Provides disease-relevant biological system (human platelet lysate) for translational continuity from discovery to preclinical validation.
- Operational Value: Enables longitudinal monitoring of biomarker changes in response to therapeutic interventions.
- Predictive Value: Supports risk-adjusted advancement decisions by delivering quantitative, reproducible protein expression data.
Pipeline & Workflow Integration
CEI integrates into the discovery continuum by enabling hypothesis testing in early biology, supporting assay readiness in screening, and providing quantitative analytics for lead optimization and preclinical evaluation.
- Discovery Biology: Supports target validation through specific detection of TDP-43 isoforms in human platelets.
- Screening: Delivers standardized, reproducible outputs with high dynamic range for reliable compound or biomarker evaluation.
- Analytics: Generates quantifiable protein expression data via calibration curves, enabling inter- and intra-run variability assessment.
- Translational Research: Connects to preclinical workflows through use of human-derived samples relevant to neurodegenerative disease pathology.
- Enterprise Reuse: Establishes a reusable platform for multiplexed protein analysis across multiple targets and sample sets.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target biomarker identification and reduces mechanistic ambiguity in neurodegenerative pathways.
- Operational Value: Enhances standardization, reproducibility, and sample efficiency through automated, high-throughput format.
- Strategic Value: Improves go/no-go decision-making by enabling rapid, cost-effective assessment of target modulation in clinical samples.
- Portfolio Impact: Supports risk-adjusted prioritization through longitudinal biomarker tracking in ALS and related indications.
Implementation Considerations
- Requires expertise in capillary electrophoresis, immunoassay design, and fluorescent detection systems.
- Dependent on access to capillary analyzer, thermocycler, and compatible reagent kits (e.g., streptavidin-HRP, biotinylated ladder).
- Necessitates cross-team standardization for sample preparation, antibody titration, and data analysis protocols.
- Adaptation considerations include sample type (e.g., plasma, CSF) and potential background interference from unrefined lysates.
- Practical limitations include high background with crude samples and inability to use gradient gels, as noted in source material.
