Sandwich ELISA Protocol
Generic protocol for Solarbio sandwich ELISA kits. This protocol is suitable for all Solarbio pre-coated ELISA kits (cytokines, hormones, biomarkers, and immunoglobulins). For kits requiring specific modifications, refer to the product-specific datasheet.
Equipment and Reagents Checklist
| Item |
Recommended Specification |
Purpose |
| Microplate reader |
Capable of 450 nm and 540/570 nm |
Absorbance measurement |
| Incubator |
37°C ± 1°C |
Assay incubation steps |
| Multi-channel pipette |
8- or 12-channel, 20–200 μL |
Sample/antibody/reagent addition |
| Single-channel pipettes |
10 μL, 100 μL, 1000 μL |
Standard dilution, reconstitution |
| Reagent reservoirs |
25–50 mL |
Multi-channel pipetting |
| Wash bottle or auto-washer |
Adjustable volume |
Washing steps |
| Distilled/deionized water |
18 MΩ·cm resistivity |
Wash buffer dilution |
| Vortex mixer |
— |
Reagent mixing |
| Microcentrifuge |
— |
Brief spin for reconstituted reagents |
| Sealing film |
Adhesive, provided in kit |
Cover plate during incubation |
| Paper towels |
Lint-free |
Blotting plate after wash |
Reagent Preparation
| Component |
Preparation |
Stability |
| Wash Buffer (1×) |
Dilute 20× concentrate with distilled water (e.g., 50 mL concentrate + 950 mL water) |
2 weeks at RT |
| Standard |
Reconstitute with the volume indicated on vial label; let stand 15 min at RT; no vortexing |
1 h at RT; 2 weeks at -20°C |
| Standard serial dilutions |
Prepare 7 serially diluted tubes (7-point curve) plus 1 zero standard (diluent only) |
Prepare fresh, use within 1 h |
| Detection antibody |
Dilute in antibody diluent as indicated on vial label |
30 min at RT (do not store diluted) |
| HRP-Streptavidin |
Dilute 1:100–1:200 in diluent (or as per kit instruction) |
30 min at RT (do not store diluted) |
| TMB Substrate |
Ready to use; bring to room temperature (20–25°C) |
Stable at 2–8°C (dark); warm to RT before use |
| Stop Solution |
2N H₂SO₄, ready to use; contains strong acid |
Stable at RT (handle with gloves) |
Standard Serial Dilution Protocol (7-point + Zero)
- Label 8 microcentrifuge tubes: S1 (top standard) through S7, plus S0 (zero/blank)
- Reconstitute lyophilized standard as per vial label to obtain the top standard concentration
- Add 200 μL of Assay Diluent to tubes S2–S7
- Transfer 200 μL from S1 to S2; mix by pipetting (do not vortex)
- Continue 2-fold serial dilutions: 200 μL from S2 → S3, S3 → S4, etc.
- For S0 (zero standard), use 200 μL Assay Diluent only
- Dispense 100 μL per well in duplicate for each standard
S1 ──200 μL──→ S2 ──200 μL──→ S3 ──200 μL──→ S4 ──200 μL──→ S5 ──200 μL──→ S6 ──200 μL──→ S7
│ │ │ │ │ │ │
Top conc 1:2 1:4 1:8 1:16 1:32 1:64
Step 1: Equilibrate and Prepare Plate
| Detail |
Time |
Notes |
| Remove plate from sealed pouch |
— |
Bring to room temperature (20–25°C) before opening |
| Equilibrate to RT |
20 min |
Condensation on plate = not fully equilibrated |
| Remove unused strips |
— |
Reseal with desiccant; store at 2–8°C |
Step 2: Standard and Sample Incubation
| Detail |
Time |
Notes |
| Add 100 μL standard or sample to designated wells |
— |
Run all standards and samples in duplicate |
| Seal plate with adhesive film |
— |
Ensure complete seal to prevent evaporation |
| Incubate at 37°C |
2 h |
Alternatively, overnight at 4°C for low-abundance targets (increases sensitivity ~2×) |
Quality check: After incubation, inspect wells for uniform liquid volume; evaporation indicates poor seal.
Step 3: Wash
| Detail |
Volume |
Cycles |
| Aspirate contents |
— |
Blot plate inverted on paper towel |
| Add Wash Buffer |
300 μL/well |
Do not overflow wells |
| Let soak |
30 s |
Critical for complete removal of unbound material |
| Aspirate |
— |
Complete removal; blot between cycles |
| Repeat |
— |
5 total wash cycles |
| Final blot |
— |
Firm blot on clean paper towel; no residual buffer |
Step 4: Detection Antibody
| Detail |
Time |
Notes |
| Add 100 μL biotinylated detection antibody |
— |
Dilute immediately before use |
| Seal plate |
— |
— |
| Incubate at 37°C |
1 h |
— |
Step 5: Wash
Repeat Step 3 — 5 wash cycles with 30 s soak each.
Step 6: HRP-Streptavidin
| Detail |
Time |
Notes |
| Add 100 μL HRP-Streptavidin working solution |
— |
Protect from light; cover plate with foil or black lid |
| Incubate at 37°C in dark |
30 min |
— |
Step 7: Wash (Critical Step)
| Detail |
Volume |
Cycles |
| Wash as before, but increase to 7 cycles |
300 μL/well |
This thorough wash is critical to remove unbound HRP-streptavidin |
| Increase soak time for cycles 5–7 |
45–60 s |
Lower background |
Step 8: TMB Substrate
| Detail |
Time |
Notes |
| Add 90 μL TMB substrate to each well |
— |
Pre-warmed to RT; protect from light |
| Incubate at 37°C in dark |
15–30 min |
Blue color develops |
| Monitor visually |
— |
High standard should show deep blue (A₄₅₀ ~2.0) |
| Do NOT shake plate during incubation |
— |
Shaking accelerates non-specific color development |
Step 9: Stop and Read
| Detail |
Time |
Notes |
| Add 50 μL Stop Solution |
— |
In same order as TMB addition; color changes blue → yellow |
| Gently tap plate |
— |
Ensure complete mixing |
| Read at 450 nm (reference 540 or 570 nm) |
Within 15 min |
Signal decays ~1% per 5 min after stopping |
Data Analysis
Standard Curve Generation
| Fit Method |
Recommendation |
Formula |
| 4-Parameter Logistic (4PL) |
Preferred; best for sigmoidal ELISA curves |
y = A + (D−A) / (1 + (x/C)^B) |
| Cubic spline |
Acceptable for most targets |
— |
| Linear (log-log) |
Only for very narrow linear range |
Not recommended for full curve |
4PL parameters:
- A = Bottom asymptote (zero standard signal)
- B = Hill slope (curve steepness; typical 0.8–1.5)
- C = EC₅₀ (inflection point concentration)
- D = Top asymptote (saturating signal)
Calculation Example (Mouse IL-6, EM0042)
| Well |
Standard (pg/mL) |
A₄₅₀ |
Corrected (Blank-Subtracted) |
| Blank |
0 |
0.055 |
— |
| S1 |
500 |
2.345 |
2.290 |
| S2 |
250 |
1.532 |
1.477 |
| S3 |
125 |
0.891 |
0.836 |
| S4 |
62.5 |
0.482 |
0.427 |
| S5 |
31.2 |
0.265 |
0.210 |
| S6 |
15.6 |
0.154 |
0.099 |
| S7 |
7.8 |
0.098 |
0.043 |
Acceptance criteria:
- R² (4PL fit) > 0.98
- Blank CV < 10%
- Duplicate CV < 10%
- Recovery of quality control sample within 80–120%
Optimization Notes
| Parameter |
Standard Protocol |
Optimized Protocol |
When to Use |
| Sample incubation |
2 h at 37°C |
Overnight at 4°C |
Low-abundance targets (<10 pg/mL expected) |
| TMB incubation |
15 min at 37°C |
20–30 min at 37°C |
When signal is weak after 15 min |
| Wash cycles (HRP step) |
7 cycles |
10 cycles with 60 s soak |
High-background samples (serum, plasma) |
| Standard reconstitution |
15 min at RT |
30 min at RT with gentle agitation for high-MW targets |
TGF-β1, large cytokines |
| Sample pre-treatment |
— |
Heat or acid activation |
TGF-β1 (acid activation), complement proteins |
Troubleshooting
| Problem |
Likely Cause |
Solution |
| Low signal (all wells) |
Kit expired; TMB degraded; HRP inactive |
Check expiry date; test TMB with 1 μL HRP (should turn blue in 1 min) |
| High background (blank ≥ 0.1 A₄₅₀) |
Insufficient washing; TMB contaminated |
Increase wash to 7× with 60 s soak; verify TMB is not green before use |
| High duplicate variation (CV > 15%) |
Inconsistent pipetting; edge effects |
Calibrate pipettes; use multi-channel; pre-warm plate to RT; seal thoroughly |
| Non-linear standard curve |
4PL fit not applied; pipetting error in serial dilution |
Use 4PL regression; verify serial dilution accuracy (2-fold ± 5%) |
| Low sensitivity (cannot detect low standard) |
Insufficient amplification; too short TMB time |
Extend TMB to 30 min; verify antibody and HRP dilutions |
| Hook effect (high sample reads lower than expected) |
Antigen concentration exceeds antibody capacity |
Dilute sample 1:5 in Assay Diluent and re-test |
| Edge effect (outer wells different from inner) |
Temperature gradient during incubation |
Use pre-warmed plate; avoid stacking plates; use sealing film |
Cross-References
For product procurement: solarbio.store