The Enzyme-Linked Immunosorbent Assay (ELISA) is a widely used method for the identification and quantification of proteins, antibodies, and other biomolecules. Cellular ELISA is a specialized version of this technique that measures protein expression or post-translational modifications directly in cultured cells, eliminating the need for cell lysis in some protocols. This method is particularly useful for studying signaling pathways, cytokine secretion, and receptor activation in a high-throughput manner.
Principles of Cellular ELISA
Cellular ELISA operates similarly to traditional ELISA but is performed on intact or fixed cells in microtiter plates. The general steps include:
Cell Culture and Treatment – Cells are cultured in 96 or 384-well plates and treated with stimuli (such as drugs or cytokines).
Fixation and Permeabilization – Cells are fixed (e.g., with formaldehyde) and may be permeabilized if necessary (for intracellular targets).
Blocking – Non-specific binding sites are blocked with substances like BSA or non-fat dry milk.
Incubation with Primary Antibody – A specific antibody binds to the target protein.
Incubation with Secondary Antibody – An enzyme-conjugated secondary antibody (e.g., HRP or AP) binds to the primary antibody.
Detection – A substrate (e.g., TMB for HRP) generates a colorimetric, fluorescent, or chemiluminescent signal proportional to the amount of target.
Types of Cellular ELISA
Direct ELISA** – Utilizes an enzyme-conjugated primary antibody.
Indirect ELISA** – Employs both primary and secondary antibodies for increased sensitivity.
Sandwich ELISA** – Requires two antibodies (capture and detection) for high specificity.
Phospho-ELISA** – Detects phosphorylated proteins (e.g., p-ERK, p-AKT).
Applications of Cellular ELISA
Drug Discovery and Screening – Measures the effects of compounds on protein expression or phosphorylation.
Signal Transduction Studies – Quantifies the activation of kinases (e.g., JAK-STAT, MAPK pathways).
Cytokine and Secreted Protein Detection – Assesses inflammatory responses (e.g., TNF-α, IL-6).
Toxicological Studies – Evaluates cell health (e.g., caspase-3 for apoptosis).
Ligand-Receptor Binding Assays – Studies the activation of GPCRs or tyrosine kinases.
Advantages Over Traditional ELISA
✔ Maintains cellular context – Measures proteins in situ, preventing lysis artifacts.
✔ Compatible with high-throughput screening – Adaptable to 384-well plates for screening.
✔ Quantitative and sensitive – Detects low-abundance proteins (in the pg/mL range).
✔ Multiplexing potential – Some kits allow simultaneous detection of multiple targets.
Limitations
❌ Requires optimization – Fixation/permeabilization conditions must be standardized.
❌ Limited to adherent cells – Suspension cells may require specific coatings.
❌ Risk of signal saturation – High protein levels may exceed the linear detection range.
Step-by-Step Protocol
Materials Needed
Cultured cells (preferably adherent)
96-well microtiter plate (coated for cell culture)
Fixation buffer (4% paraformaldehyde)
Permeabilization buffer (0.1% Triton X-100)
Blocking buffer (5% BSA in PBS)
Primary and secondary antibodies
ELISA substrate (e.g., TMB for HRP)
Plate reader (absorbance/fluorescence)
Procedure
Cell Culture – Seed 10,000 to 50,000 cells per well and incubate overnight.
Cell Treatment – Apply experimental conditions (e.g., drug doses).
Cell Fixation – Add 4% paraformaldehyde (15 minutes at room temperature), then wash with PBS.
Permeabilization (if needed) – Use 0.1% Triton X-100 (10 minutes).
Blocking – Incubate with 5% BSA (1 hour at room temperature).
Primary Antibody – Add antibody (dilution 1:1000, 2 hours or overnight at 4°C).
Secondary Antibody – Incubate with HRP-conjugated secondary antibody (1 hour at room temperature).
Detection – Add substrate, stop the reaction (if needed), and read absorbance.
Data Analysis
Normalize based on cell number (e.g., using crystal violet staining).
Compare treated groups to controls using standard curves for absolute quantification if needed.
Conclusion
Cellular ELISA is a powerful tool for studying protein expression and its changes in a cellular environment. Its compatibility with high-throughput screening makes it valuable in drug development and biomedical research. By optimizing fixation conditions, antibody concentrations, and detection methods, sensitive and reproducible results can be achieved. Future advancements may include multiplex cellular ELISA platforms for simultaneous detection of multiple analytes in a single well.