Cell-Based ELISA Protocol

November 1, 2025

Introduction

The Cell-Based ELISA (CB-ELISA) is a powerful immunoassay designed to measure protein expression levels directly in cultured cells without the need for lysate preparation. This approach maintains the native cellular architecture, enabling quantitative assessment of surface and intracellular targets under physiological or stimulated conditions.

CB-ELISA is especially useful for studying cell signaling, receptor activation, drug response, and protein phosphorylation in adherent cell models. The assay can be colorimetric, fluorescent, or luminescent, depending on the detection chemistry used.

Materials & Reagents

96-well tissue culture-treated plate (flat-bottom)
Adherent cells (e.g., HeLa, HEK293, CHO, or primary cells)
Appropriate culture medium (e.g., DMEM or RPMI-1640 with 10% FBS)
Fixative: 4% paraformaldehyde (PFA) in PBS
Permeabilization buffer (0.1% Triton X-100 in PBS, if needed)
Blocking buffer: 1% BSA or 5% normal serum in PBS
Primary antibody specific to the target protein
HRP-conjugated or fluorescently labeled secondary antibody
Substrate solution (e.g., TMB, fluorescence, or chemiluminescent substrate)
ELISA plate reader (absorbance, fluorescence, or luminescence mode)

Step-by-Step Protocol

Cell Seeding: Seed 1–2 × 10⁴ adherent cells per well in a 96-well tissue culture plate. Incubate overnight at 37°C with 5% CO₂ to allow adherence and recovery.
Treatment: Expose cells to desired treatments (e.g., drug compounds, cytokines, or growth factors) for appropriate durations.
Fixation: Gently remove the medium and add 4% paraformaldehyde for 15 minutes at room temperature to preserve cellular structure. Wash 3× with PBS to remove residual fixative.
Permeabilization (Optional): For intracellular targets, treat cells with 0.1% Triton X-100 in PBS for 5 minutes at room temperature. Wash 3× with PBS.
Blocking: Add 100 µL of blocking buffer per well and incubate for 1 hour at room temperature to prevent non-specific antibody binding.
Primary Antibody Incubation: Add 100 µL of diluted primary antibody in blocking buffer and incubate for 1–2 hours at room temperature or overnight at 4°C. Wash 3× with PBS.
Secondary Antibody Incubation: Add HRP- or fluorescence-labeled secondary antibody diluted in blocking buffer and incubate for 1 hour. Wash 3× with PBS to remove unbound antibody.
Detection: Add substrate solution (e.g., TMB for colorimetric assays) and incubate until color develops. Stop reaction with 1N HCl (for TMB), and read absorbance at 450 nm using a microplate reader. For fluorescence or chemiluminescence, read according to the instrument settings.
Data Analysis: Normalize data to cell number or total protein, if required. Calculate relative protein expression levels compared to untreated or control samples.

Tips / Notes

Use tissue culture-treated plates to ensure optimal cell adhesion.
Do not allow cells to dry at any step, as it can cause high background or cell detachment.
Include untreated control wells to normalize signal variation.
For multiplex detection, use fluorescently labeled secondary antibodies with non-overlapping emission spectra.
Validate fixation and permeabilization conditions for each cell type to preserve antigen integrity.

Troubleshooting

Weak Signal: Increase antibody concentration, extend incubation time, or verify target protein expression levels.
High Background: Improve blocking efficiency, increase wash steps, or reduce secondary antibody concentration.
Uneven Signal: Ensure consistent seeding density and uniform plate incubation conditions.
Cell Loss: Handle plates gently during washing and avoid strong aspiration.

Internal Links

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Keywords

cell-based ELISA, adherent cells, in-cell ELISA, protein quantification, receptor phosphorylation, antibody incubation, cellular signaling, fixed-cell assay

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