ELISA (Enzyme-Linked Immunosorbent Assay) is the gold standard for quantitative protein detection in biological samples. This guide walks you through the complete ELISA workflow, from plate preparation to data analysis, with practical tips for each step. For a comparison of ELISA formats (direct, indirect, sandwich, competitive), see our ELISA Formats Guide.
Workflow Overview (Sandwich ELISA)
1. Coat capture antibody
2. Block
3. Add samples & standards
4. Add detection antibody
5. Add enzyme conjugate (if biotinylated detection)
6. Add substrate & read
7. Data analysis
1. Plate Coating
Coat a 96-well ELISA plate with capture antibody diluted in coating buffer (typically 100 mM carbonate/bicarbonate buffer, pH 9.6, or PBS, pH 7.4). The optimal coating concentration depends on the antibody, but 1–10 μg/mL is a common starting range.
| Parameter |
Recommended |
Notes |
| Coating volume |
100 μL/well |
Covers the bottom of a standard flat-bottom 96-well plate |
| Incubation |
Overnight at 4°C |
Or 2 h at 37°C for faster protocol; overnight gives more uniform coating |
| Plate type |
High-binding polystyrene |
Nunc MaxiSorp or equivalent; do not use tissue culture-treated plates |
After coating, wash 3× with wash buffer (PBS + 0.05% Tween-20, or PBS-T).
2. Blocking
Blocking fills unoccupied protein-binding sites on the plate, preventing non-specific binding of detection reagents.
| Blocking Reagent |
Concentration |
Best For |
| BSA in PBS |
1–3% |
Most sandwich ELISAs; biotin-streptavidin detection |
| Non-fat dry milk in PBS |
5% |
Standard blocking; avoid with biotin detection (milk contains biotin) |
| Commercial blocking buffer |
As directed |
Optimized for low background; convenient |
Protocol: Add 200–300 μL blocking buffer per well. Incubate 1–2 h at room temperature (RT) or overnight at 4°C. Wash 3× with PBS-T.
3. Sample & Standard Preparation
Prepare a standard curve using serial dilutions of purified recombinant protein at known concentrations. The standard range depends on the expected analyte concentration in your samples.
Standard curve best practice: Use at least 7 standard concentrations in 2-fold serial dilutions, plus a blank (zero standard). Prepare fresh standards for each experiment. Dilute standards in the same matrix as your samples (e.g., 1% BSA/PBS for serum samples diluted in BSA/PBS).
Sample preparation tips:
Serum/plasma: centrifuge to remove debris; dilute as needed in sample diluent
Cell culture supernatant: centrifuge to remove cells; use neat or diluted
Cell/tissue lysate: use appropriate lysis buffer; centrifuge at 12,000 × g
Add 100 μL sample or standard per well. Incubate 1–2 h at RT or overnight at 4°C. Wash 3–5× with PBS-T.
4. Detection Antibody
Add the detection antibody (biotinylated or HRP-conjugated) diluted in blocking buffer. The detection antibody must recognize a different epitope on the target antigen than the capture antibody. For monoclonal vs polyclonal antibody selection, see our guide.
Protocol: Add 100 μL/well. Incubate 1 h at RT. Wash 3–5× with PBS-T.
If using a biotinylated detection antibody, add streptavidin-HRP (typically 1:200–1:1,000 in blocking buffer), incubate 20–30 min at RT, and wash 3–5×.
5. Substrate & Detection
| Enzyme |
Substrate |
Read At |
Stop Solution |
| HRP |
TMB (3,3′,5,5′-tetramethylbenzidine) |
450 nm (after stop) or 650 nm (kinetic) |
2N H₂SO₄ (turns blue → yellow) |
| AP |
pNPP (p-nitrophenyl phosphate) |
405 nm |
3N NaOH (optional) |
Protocol: Add 100 μL TMB substrate per well. Incubate 10–30 min at RT in the dark. When the highest standard develops a deep blue color, add 50 μL stop solution (2N H₂SO₄) to all wells. Read absorbance at 450 nm within 30 min of stopping.
Tip: Add stop solution to all wells in the same order and at the same pace as substrate to ensure equal development time across the plate. An 8-channel multichannel pipette makes this reproducible.
6. Data Analysis
Plot the standard curve (OD₄₅₀ vs. concentration) using a 4-parameter logistic (4-PL) curve fit — not linear regression. The 4-PL model accounts for the sigmoidal shape of immunoassay dose-response curves and provides more accurate interpolation, especially at the extremes of the dynamic range.
Key quality checks:
R² of standard curve ≥ 0.99
Blank OD < 0.1 (indicates low background)
Duplicate CV < 10% (inter-well precision)
Sample OD within the linear range of the standard curve (not above the highest standard or below the lowest)
Common ELISA Problems & Quick Fixes
| Problem |
Likely Cause |
Solution |
| High background (blank OD >0.2) |
Insufficient blocking or washing |
Increase blocking time; increase washes to 5×; check wash buffer has 0.05% Tween-20 |
| No signal (all wells clear) |
Detection antibody mismatch; substrate expired; wrong plate type |
Confirm antibody pair compatibility; use fresh TMB; use high-binding plates |
| Poor replicate precision (CV >15%) |
Pipetting error; plate edge effect |
Use calibrated multichannel pipette; avoid outermost wells or fill them with buffer |
| Standard curve plateau too early |
Capture antibody saturated; substrate depleted |
Increase capture antibody coating concentration; stop the reaction earlier |
| Samples above standard curve |
Analyte concentration too high |
Dilute samples further and re-run; multiply result by dilution factor |
| Matrix interference |
Serum components interfere with antibody binding |
Perform spike-and-recovery test; increase sample dilution; use sample diluent matched to standard diluent |
Ready-to-Use ELISA Kits from abinScience
Adalimumab ELISA Kit — Cat# DF879018 | Sandwich PK format, serum/plasma
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Frequently Asked Questions
Q: What is the difference between a sandwich ELISA and a competitive ELISA?
In a sandwich ELISA, the target antigen is captured between two antibodies (capture + detection), and signal increases with analyte concentration. In a competitive ELISA, the sample antigen competes with labeled reference antigen for antibody binding, so signal decreases as analyte concentration increases. Sandwich ELISA is more sensitive for large proteins; competitive ELISA is used for small molecules and haptens. See our ELISA Formats Guide for a detailed comparison.
Q: Can I reuse an ELISA plate?
No. ELISA plates are single-use. Stripping and re-coating is unreliable because residual bound protein and blocking reagent interfere with the next coating cycle. The cost of a new plate is negligible compared to the cost of unreliable data from a reused plate.
Q: Why should I use 4-PL curve fitting instead of linear regression?
Immunoassay dose-response curves are sigmoidal (S-shaped), not linear. Linear regression only fits the middle portion of the curve and underestimates concentrations at the high and low ends. The 4-PL model fits the entire sigmoidal curve, providing accurate interpolation across the full dynamic range. Most ELISA analysis software (SoftMax Pro, Gen5, Prism) includes 4-PL fitting.
Q: Do I need a kit, or can I build my own ELISA?
Kits include pre-optimized capture antibody, detection antibody, standards, and buffers — saving weeks of optimization. For common targets (cytokines, drug levels, ADA), a validated kit is strongly recommended. For novel or custom targets where no kit exists, you can build a sandwich ELISA using individual antibodies, but expect significant optimization of coating concentration, antibody pair selection, and buffer conditions. abinScience offers both ready-to-use ELISA kits and individual antibodies for custom assay development.
References
1. Crowther JR. The ELISA Guidebook. Methods in Molecular Biology. 2nd ed. Humana Press; 2009. doi: 10.1007/978-1-60327-254-4
2. Aydin S. A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides. 2015;72:4-15. doi: 10.1016/j.peptides.2015.04.012
3. Lequin RM. Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clin Chem. 2005;51(12):2415-2418. doi: 10.1373/clinchem.2005.051532
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This article is provided for educational purposes only. For technical support, contact info@abinscience.com.
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