Sandwich ELISA — General Protocol

Enzyme-Linked Immunosorbent Assay (ELISA) is a plate-based immunoassay that quantifies a specific analyte (protein, hormone, cytokine) in a biological sample through antibody–antigen binding coupled to an enzyme-mediated colorimetric readout. This protocol describes the **sandwich (two-antibody) format**, in which a capture antibody immobilized on a microplate binds the target antigen; a biotinylated detection antibody then binds a second epitope on the same target; HRP-conjugated streptavidin amplifies the signal; and TMB substrate produces a color proportional to analyte concentration. Absorbance at 450 nm is compared to a standard curve to derive concentration. Covers both standard-curve quantification and quality-control acceptance criteria. Positive control (top standard) and blank (zero standard) are required.

biochemistry Intermediate 3–4 hours 10 steps

Stage 01 / 05Preparation

Equilibrate reagents and prepare standards and samples

Bring kit components to room temperature, then prepare the serial standard and sample inputs for consistent binding.

Done
STEP 1Equilibrate all reagents to room temperature30 min
Remove kit components from 4°C storage and allow to equilibrate at room temperature (20–25°C) for a minimum of 30 minutes. Prepare 1× Wash Buffer: dilute the 10× concentrate 1:10 with ddH₂O. Mix thoroughly. Calculate volume needed: 300 µL/well × 4 washes × number of wells used (add 10–20% overage).

WhyCold reagents reduce antibody-binding kinetics and enzyme activity, leading to underestimated signals and a compressed standard curve. Uniform temperature across all wells is critical for intra-plate reproducibility (Tighe et al., 2015).
Done
STEP 2Reconstitute and serially dilute the standard30 min
Reconstitute lyophilized standard: add the volume of Sample Diluent specified by the kit. Allow to sit undisturbed for 15 minutes; mix by gentle inversion — do not vortex. Label 7 microcentrifuge tubes (S6 through S1) plus one tube for the blank (S0). Pipette Sample Diluent into tubes S6–S1 (volume per kit; commonly 300 µL each). Transfer the volume specified by the kit from the reconstituted top standard (S7) into tube S6; mix by pipetting up and down 10 times. Change tips. Perform 2-fold serial dilution: transfer from S6 → S5 → S4 → S3 → S2 → S1, changing tips between each transfer. S0 = Sample Diluent only (0 pg/mL blank). Do not add standard. Run all standards in duplicate.

WhySerial dilution accuracy is the single largest source of standard curve error. Changing tips between transfers prevents carryover; consistent pipetting motion (aspirate and dispense 10×) ensures complete mixing at each step. A 2-fold dilution series is standard for most cytokine ELISA kits and produces a 7-point calibration range spanning 2–3 orders of magnitude.
CautionDo not vortex the reconstituted standard — protein aggregation can cause non-linearity at the high end of the curve.
Done
STEP 3Prepare samples20 min
Cell culture supernatant: centrifuge at 1,000 × g for 10 min at 4°C to pellet debris. Collect the clarified supernatant. Use immediately or aliquot and store at −80°C; avoid more than 2 freeze–thaw cycles. Serum: allow blood to clot at room temperature for 30 min, then centrifuge at 2,000 × g for 10 min. Collect serum layer (top). Plasma: collect blood into EDTA or heparin tube; centrifuge at 2,000 × g for 10 min. Collect the plasma layer (top). Dilute samples in Sample Diluent as required (start with a 2-fold dilution if analyte concentration is unknown; dilutions must fall within the linear range of the standard curve).

WhyCellular debris and lipid in undiluted samples can cause matrix interference (hook effect at high analyte concentrations, non-specific binding). Serum is preferred over plasma for cytokine ELISA when anticoagulants may interfere with the assay (Lequin, 2005).

Stage 02 / 05Capture

Load the well and capture the target analyte

The pre-coated capture antibody retains its target while washing removes unbound material.

Done
STEP 4Load standards and samples; incubate2 hr or overnight
Add 100 µL per well of each standard (S0–S7) and each sample. Assign duplicates; include a blank (S0) in ≥ 2 wells. Seal the plate with adhesive film. Incubate at room temperature for 2 hours on a plate shaker at gentle agitation (~150 rpm). Alternatively, incubate at 4°C overnight (16–18 h) for higher sensitivity. After incubation, aspirate or flick out well contents. Tap plate firmly on absorbent paper to remove residual liquid.

WhyExtended incubation at 4°C increases sensitivity by allowing analyte–capture-antibody equilibrium to shift toward complete binding. However, it introduces more room for timing variability; for routine assays, 2 h at room temperature is sufficient and more reproducible.
Done
STEP 5Wash the plate (1st wash cycle)5 min
Add 300 µL Wash Buffer to each well. Soak for 1–2 minutes. Aspirate or flick out wash buffer. Tap plate on absorbent paper. Repeat for a total of 4 wash cycles. After the final wash, invert and tap the plate firmly on a fresh sheet of absorbent paper to remove all residual buffer.

WhyIncomplete washing leaves unbound sample proteins and antibodies in the well, raising background signal (OD of blank > 0.1). Four wash cycles are the standard minimum for pre-coated sandwich ELISA kits.
CautionDo not allow the plate to dry between washes. Dry wells can lead to non-specific antibody binding to exposed plastic. Proceed immediately to the next step.

Stage 03 / 05Detection

Build the enzyme-linked detection complex

A second antibody binds a distinct epitope, then streptavidin binds its biotin tag and carries HRP.

Done
STEP 6Add detection antibody; incubate1 hr
Dilute the biotinylated detection antibody to working concentration per kit instructions (commonly 1:100 in Sample Diluent or Assay Buffer). Add 100 µL per well. Seal the plate. Incubate at room temperature for 1 hour on a plate shaker. Wash the plate 4 times (same procedure as Step 5).

WhyThe detection antibody binds a second, distinct epitope on the captured analyte. Biotin conjugation enables signal amplification via streptavidin-HRP binding (K d ≈ 10 −15 M), which is orders of magnitude tighter than most antibody–antigen interactions (Engvall & Perlmann, 1971).
Done
STEP 7Add HRP-Streptavidin; incubate45 min
Dilute HRP-Streptavidin to working concentration per kit instructions (commonly 1:40–1:100 in Assay Buffer). Prepare fresh immediately before use. Add 100 µL per well. Seal the plate. Protect from light (cover with aluminum foil or place in dark). Incubate at room temperature for 45 minutes on a plate shaker. Wash the plate 4–5 times (increase to 5 washes to minimize HRP background at this step).

WhyStreptavidin–biotin binding is near-irreversible, ensuring quantitative HRP localization at each captured analyte. Thorough washing at this step is especially important because residual HRP causes false-positive TMB oxidation in all wells.
CautionHRP-Streptavidin is sensitive to denaturation. Do not freeze the diluted working solution; use within the session.

Stage 04 / 05Color

Develop the blue TMB signal under light protection

HRP oxidizes TMB; the developing blue signal reflects the amount of bound analyte.

Done
STEP 8Add TMB substrate; develop color15–30 min
Add 100 µL TMB Substrate Solution per well. Do not allow tip to contact the well surface. Do not seal the plate. Protect from light. Incubate at room temperature. Monitor color development: wells with high analyte should turn blue within 5–10 minutes. Development is complete when the top standard shows a deep blue color and the blank remains colorless or very faintly blue. Target incubation: 15–30 minutes total; stop before the blank well shows any visible color.

WhyTMB is oxidized by HRP to a blue-colored diimine product. The rate of color development is proportional to HRP (hence analyte) concentration. Stopping when the blank shows color indicates non-specific oxidation is occurring; this indicates excessive development time, contaminated TMB, or insufficient washing.
CautionTMB is light-sensitive. Store in the dark; use a colorless (not blue-tinted) solution only. A blue-tinted TMB stock has been partially oxidized and will produce elevated background across all wells.

Stage 05 / 05Readout

Stop the reaction, read absorbance, and fit the curve

Stop solution converts blue TMB to yellow; corrected absorbance is then mapped to concentration with a 4PL fit.

Done
STEP 9Stop the reaction; read absorbance15 min
Add Stop Solution per kit instructions (typically 50–100 µL per well) to each well in the same order and at the same pace as TMB was added. Mix gently by tapping the plate sides. Color should change from blue to yellow uniformly. Any wells that remain green or mixed-color indicate incomplete mixing — tap again. Read absorbance at 450 nm (primary wavelength) and 570 nm (reference wavelength) within 15 minutes of adding Stop Solution. Record: Final OD = OD₄₅₀ − OD₅₇₀.

WhySulfuric acid (Stop Solution) converts the blue TMB diimine to the stable yellow sulfonated form with a sharp 450 nm peak. Reference subtraction at 570 nm eliminates optical artifacts (plate well scratches, fingerprints, bubbles) that affect raw OD 450 . Reading must occur within 15 minutes because the yellow product is not indefinitely stable (Lequin, 2005).
CautionStop Solution is 2 N H₂SO₄ (strong acid). Wear gloves and eye protection. Pipette carefully; acidic aerosols can contaminate neighboring wells.
Done
STEP 10Fit standard curve and calculate concentrations
Using ELISA reader software or a data analysis tool, plot standard concentration (X-axis, log scale) vs. corrected OD (Y-axis). Fit a 4-Parameter Logistic (4PL) model. Verify R² > 0.99 before accepting the curve. For each sample well: interpolate OD on the standard curve to obtain concentration. Multiply interpolated concentration by the dilution factor to obtain the concentration in the original sample. Average duplicates; report the mean ± SD (or CV%).

WhyThe 4PL model accurately describes the sigmoidal relationship between OD and analyte concentration across the full dynamic range. Linear regression is acceptable only for a narrow, linear sub-region of the curve and will introduce systematic error at the extremes (Tighe et al., 2015).
CautionSamples with OD above the top standard (S7) must be re-assayed at a higher dilution. Report values below the lowest non-zero standard (S1) as "< LLOD" (Lower Limit of Detection); do not extrapolate outside the curve.

Sandwich ELISA — General Protocol

biochemistry · Intermediate · 3–4 hours

https://labgrimoire.com/protocols/sandwich-elisa-general/

Materials

☐ ELISA Kit (pre-coated 96-well plate included): 1 kit (Kit-specific; this protocol follows the sandwich ELISA format common to most commercial cytokine/growth-factor kits)
☐ Lyophilized or liquid standard: Per kit (Store as instructed; reconstitute lyophilized powder in Sample Diluent; do not vortex)
☐ Biotinylated detection antibody: Per kit (Dilute to working concentration per kit instructions (commonly 1:100))
☐ HRP-Streptavidin concentrate: Per kit (Dilute to working concentration per kit instructions (commonly 1:40–1:100); prepare fresh immediately before use)
☐ TMB Substrate Solution: Per kit (Store protected from light; solution should be colorless before use; discard if blue-tinted)
☐ Stop Solution (2 N H₂SO₄): Per kit (Corrosive; wear gloves and eye protection)
☐ Wash Buffer (10× PBS-T concentrate): Per kit (Dilute 1:10 with deionized water to prepare 1× working solution)
☐ Sample Diluent / Assay Buffer: Per kit (Used for standard reconstitution, serial dilution, and sample dilution)
☐ Deionized water (ddH₂O): As needed (For Wash Buffer preparation)
☐ Plate sealer film: As needed (Prevents evaporation during incubation steps)

Equipment

☐ Microplate spectrophotometer (ELISA reader) (Must read at 450 nm; dual-wavelength correction at 570 nm or 630 nm recommended)
☐ Multichannel pipette (8-channel, 50–300 µL) (Reduces pipetting CV; use for all liquid additions to the plate)
☐ Plate washer (manual or automated) (Automated preferred; manual: use squirt bottle, 300 µL/well/wash)
☐ Plate shaker / orbital rocking platform (Set to gentle agitation (~150–200 rpm) during incubation steps)
☐ 37°C incubator or water bath (For standard room-temperature incubations; alternatively, ambient (20–25°C) bench incubation is acceptable if room temperature is stable)
☐ Refrigerator (4°C) (For overnight incubation option (Step 4))
☐ Microcentrifuge (For clearing cell culture supernatant before assay (1,000 × g, 10 min))
☐ 1.5 mL microcentrifuge tubes (For serial dilution of standards)
☐ Absorbent paper / lint-free tissue (For plate blotting after wash steps)

Procedure

☐ Equilibrate all reagents to room temperature (30 min)
Remove kit components from 4°C storage and allow to equilibrate at room temperature (20–25°C) for a minimum of 30 minutes. Prepare 1× Wash Buffer: dilute the 10× concentrate 1:10 with ddH₂O. Mix thoroughly. Calculate volume needed: 300 µL/well × 4 washes × number of wells used (add 10–20% overage).
☐ Reconstitute and serially dilute the standard (30 min)
Reconstitute lyophilized standard: add the volume of Sample Diluent specified by the kit. Allow to sit undisturbed for 15 minutes; mix by gentle inversion — do not vortex. Label 7 microcentrifuge tubes (S6 through S1) plus one tube for the blank (S0). Pipette Sample Diluent into tubes S6–S1 (volume per kit; commonly 300 µL each). Transfer the volume specified by the kit from the reconstituted top standard (S7) into tube S6; mix by pipetting up and down 10 times. Change tips. Perform 2-fold serial dilution: transfer from S6 → S5 → S4 → S3 → S2 → S1, changing tips between each transfer. S0 = Sample Diluent only (0 pg/mL blank). Do not add standard. Run all standards in duplicate.
☐ Prepare samples (20 min)
Cell culture supernatant: centrifuge at 1,000 × g for 10 min at 4°C to pellet debris. Collect the clarified supernatant. Use immediately or aliquot and store at −80°C; avoid more than 2 freeze–thaw cycles. Serum: allow blood to clot at room temperature for 30 min, then centrifuge at 2,000 × g for 10 min. Collect serum layer (top). Plasma: collect blood into EDTA or heparin tube; centrifuge at 2,000 × g for 10 min. Collect the plasma layer (top). Dilute samples in Sample Diluent as required (start with a 2-fold dilution if analyte concentration is unknown; dilutions must fall within the linear range of the standard curve).
☐ Load standards and samples; incubate (2 hr or overnight)
Add 100 µL per well of each standard (S0–S7) and each sample. Assign duplicates; include a blank (S0) in ≥ 2 wells. Seal the plate with adhesive film. Incubate at room temperature for 2 hours on a plate shaker at gentle agitation (~150 rpm). Alternatively, incubate at 4°C overnight (16–18 h) for higher sensitivity. After incubation, aspirate or flick out well contents. Tap plate firmly on absorbent paper to remove residual liquid.
☐ Wash the plate (1st wash cycle) (5 min)
Add 300 µL Wash Buffer to each well. Soak for 1–2 minutes. Aspirate or flick out wash buffer. Tap plate on absorbent paper. Repeat for a total of 4 wash cycles. After the final wash, invert and tap the plate firmly on a fresh sheet of absorbent paper to remove all residual buffer.
☐ Add detection antibody; incubate (1 hr)
Dilute the biotinylated detection antibody to working concentration per kit instructions (commonly 1:100 in Sample Diluent or Assay Buffer). Add 100 µL per well. Seal the plate. Incubate at room temperature for 1 hour on a plate shaker. Wash the plate 4 times (same procedure as Step 5).
☐ Add HRP-Streptavidin; incubate (45 min)
Dilute HRP-Streptavidin to working concentration per kit instructions (commonly 1:40–1:100 in Assay Buffer). Prepare fresh immediately before use. Add 100 µL per well. Seal the plate. Protect from light (cover with aluminum foil or place in dark). Incubate at room temperature for 45 minutes on a plate shaker. Wash the plate 4–5 times (increase to 5 washes to minimize HRP background at this step).
☐ Add TMB substrate; develop color (15–30 min)
Add 100 µL TMB Substrate Solution per well. Do not allow tip to contact the well surface. Do not seal the plate. Protect from light. Incubate at room temperature. Monitor color development: wells with high analyte should turn blue within 5–10 minutes. Development is complete when the top standard shows a deep blue color and the blank remains colorless or very faintly blue. Target incubation: 15–30 minutes total; stop before the blank well shows any visible color.
☐ Stop the reaction; read absorbance (15 min)
Add Stop Solution per kit instructions (typically 50–100 µL per well) to each well in the same order and at the same pace as TMB was added. Mix gently by tapping the plate sides. Color should change from blue to yellow uniformly. Any wells that remain green or mixed-color indicate incomplete mixing — tap again. Read absorbance at 450 nm (primary wavelength) and 570 nm (reference wavelength) within 15 minutes of adding Stop Solution. Record: Final OD = OD₄₅₀ − OD₅₇₀.
☐ Fit standard curve and calculate concentrations
Using ELISA reader software or a data analysis tool, plot standard concentration (X-axis, log scale) vs. corrected OD (Y-axis). Fit a 4-Parameter Logistic (4PL) model. Verify R² > 0.99 before accepting the curve. For each sample well: interpolate OD on the standard curve to obtain concentration. Multiply interpolated concentration by the dilution factor to obtain the concentration in the original sample. Average duplicates; report the mean ± SD (or CV%).

Sandwich ELISA — General Protocol

Materials

Equipment

Why it works, and how it goes wrong