Why These Two Kits Require Different Protocols, Mechanistic Comparison
🔵 QuickTest Format, FineTest QT-EH1581
The QuickTest plate is coated with an anti-tag secondary antibody, not the capture antibody directly. The Cap/Det Ab reagent contains both the tagged capture antibody and the biotin-detection antibody in a single pre-mixed solution.
Step 1: Cap/Det Ab + Sample added simultaneously → sandwich complex forms in solution
↓ faster kinetics in liquid phase
Complex captured onto plate via anti-tag interaction
Wash → HRP-Streptavidin binds biotin-detection Ab
Wash → TMB → Stop → Read 450 nm
⚡ Total active incubation: 60 + 30 min = 90 min
Faster because antibody–antigen kinetics are faster in solution than at solid phase.
Faster because antibody–antigen kinetics are faster in solution than at solid phase.
🟠 Standard Sandwich Format, CUSABIO CSB-EL007460HU
Capture antibody is directly pre-coated on the microplate. Sample antigen binds the immobilized capture antibody in solid-phase. Detection antibody and HRP conjugate are added in sequential separate steps.
Sample added to pre-coated plate → antigen captured (solid-phase, 2 h)
Remove liquid (no wash) → add Biotin-antibody (1 h)
↓ sequential steps, each requiring full incubation
Wash × 3 → HRP-Avidin (1 h)
Wash × 5 → TMB → Stop → Read 450 nm
🎯 Lower LOD: <0.039 ng/ml vs 0.094 ng/ml
More sensitive because each step reaches equilibrium independently. Trade-off: ~4 h total time.
More sensitive because each step reaches equilibrium independently. Trade-off: ~4 h total time.
Supplier: FineTest®
Intra-CV: 3.62–4.19%
Inter-CV: 4.25–4.77%
Inter-CV: 4.25–4.77%
Before You Begin, Reagent Preparation
QuickTest-specific requirement: The Cap/Det Ab reagent contains both the affinity-tagged capture antibody and the biotin-detection antibody in a single ready-to-use solution. It must be added simultaneously with, not before or after, the sample. Do not pre-incubate Cap/Det Ab in empty wells.
P1
Equilibrate Kit to Room Temperature
⏱ 20 min before start
→
Remove sealed kit from 2–8°C storage. Allow all components to equilibrate to 18–25°C for at least 20 minutes before use.
→
Remove only the number of strips and standards required for the current assay. Return unused strips to the sealed foil bag with desiccant and store at 2–8°C.
Storage reminder: Kit must be stored at 2–8°C in sealed box. Do not freeze. TMB substrate should also equilibrate to room temperature during this period.
P2
Prepare Wash Buffer (1×)
→
96T: Dilute 30 ml concentrated Wash Buffer (25×) into 720 ml deionized water to prepare 750 ml of 1× Wash Buffer. Resistivity ≥ 18 MΩ recommended.
→
48T: Dilute 15 ml concentrated buffer into 360 ml deionized water (375 ml total).
Crystal formation: If crystals are present in concentrate, warm at 40°C (water bath) until fully dissolved. Do not exceed 50°C. Use prepared 1× buffer within 48 h; store remainder at 2–8°C.
P3
Reconstitute Lyophilized Standard (E002)
1
Centrifuge the lyophilized standard vial at 10,000 × g for 1 min before opening. Label this vial as the Zero Tube.
2
Add exactly 0.5 ml Sample Dilution Buffer (E039) to the vial. Cap tightly. Let stand 2 min at room temperature, then invert several times to mix. Do not vortex vigorously.
3
Centrifuge at 1,000 × g for 1 min to settle liquid to tube bottom and remove bubbles.
Zero tube stock concentration: 20 ng/ml (2× the top standard). Store reconstituted Zero Tube at 2–8°C and use within 24 h. Do not freeze reconstituted standard.
P4
Prepare 8-Point Standard Dilution Series
→
Label 7 EP tubes: 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, and Blank. Add 150 µl Sample Dilution Buffer to each tube.
→
Transfer 150 µl from the Zero Tube into the 1/2 tube. Mix thoroughly. Continue serial 1:2 transfers (150 µl each) through to the 1/64 tube. Blank contains buffer only.
→
Use fresh tips for each transfer. Mix completely at each step before proceeding. Do not foam.
| Tube | Zero | 1/2 | 1/4 | 1/8 | 1/16 | 1/32 | 1/64 | Blank |
|---|---|---|---|---|---|---|---|---|
| ng/ml | 20* | 10 | 5 | 2.5 | 1.25 | 0.625 | 0.312 | 0 |
| Loaded as | S8 | S7 | S6 | S5 | S4 | S3 | 0 ng/ml |
* Zero Tube stock (20 ng/ml) is not loaded directly. Load the 1/2 tube as the highest standard (10 ng/ml). Diluted standards must be used within 2 h of preparation.
Typical Standard Curve Data (QC-validated batch, 18–25°C, 60% humidity):
| STD (ng/ml) | OD-1 | OD-2 | Average OD | Corrected OD* |
|---|---|---|---|---|
| 0 | 0.119 | 0.123 | 0.121 | 0 |
| 0.156 | 0.180 | 0.178 | 0.179 | 0.058 |
| 0.312 | 0.208 | 0.204 | 0.206 | 0.085 |
| 0.625 | 0.310 | 0.307 | 0.296 | 0.175 |
| 1.25 | 0.495 | 0.491 | 0.469 | 0.348 |
| 2.5 | 0.695 | 0.699 | 0.715 | 0.594 |
| 5 | 1.444 | 1.457 | 1.419 | 1.298 |
| 10 | 2.286 | 2.232 | 2.247 | 2.126 |
* Corrected OD = Average OD − OD of blank. Reference data provided for QC purposes; establish your own standard curve per run. Fit using 4-parameter logistic (4-PL) model. Online calculator: elisa.fn-test.com
P5
Sample Preparation & Dilution
Pre-experiment dilution scouting is strongly recommended for samples without prior reference data. Test 3–4 dilution gradients (10-fold steps) before the formal assay. Do not assume the listed recommended dilutions are appropriate for disease-model or non-standard samples.
Serum
1
Allow whole blood to clot at RT for 2 h or at 2–8°C overnight.
2
Centrifuge at 1,000 × g for 20 min. Collect supernatant.
3
Assay immediately or store at −20°C / −80°C in aliquots.
!
Avoid hemolyzed or lipemia samples. Use non-endotoxin collection tubes.
Plasma (EDTA or Heparin)
1
EDTA-Na₂/K₂ recommended. Collect and centrifuge within 30 min.
2
Centrifuge at 1,000 × g for 15 min at 2–8°C.
3
Collect supernatant. Assay immediately or store at −20°C / −80°C.
Cell Culture Supernatant
1
Centrifuge at 2,500 rpm, 2–8°C, 5 min.
2
Collect clarified supernatant.
3
Assay immediately or store at −80°C.
Cell Lysate / Tissue Homogenate
1
Lyse on ice with 0.5–1 ml lysis buffer + 1 mM PMSF (30–60 min or ultrasonic disruption).
2
Centrifuge at 10,000 rpm, 2–8°C, 10 min. Collect supernatant.
3
Measure total protein by BCA. Target 1–3 mg/ml for assay.
!
Do not use NP-40, Triton X-100, or DTT-containing lysis buffers, inhibit assay. Use PBS or 50 mM Tris + 0.9% NaCl + 0.1% SDS, pH 7.3.
Assay Procedure, 4 Steps, 120 Minutes
Critical continuity requirement: Once the assay is started, each step must proceed without interruption. Never allow wells to dry between steps. After each wash, add the next reagent immediately.
1
Cap/Det Ab + Sample Co-Incubation (QuickTest-Specific)
⏱ 60 min @ 37°C, simultaneous addition
→
Designate wells for standards (duplicate recommended), samples, and blank. Record plate layout.
1
Add 50 µl Cap/Det Ab (E054, ready-to-use) to each well first.
2
Immediately add 50 µl standard or sample to the corresponding wells. Disposable tip should lightly touch the liquid surface, do not touch well walls. Use fresh tips for each standard and sample.
3
Gently tap the plate for 10 seconds to ensure thorough mixing. Seal plate with plate sealer.
4
Static incubate for 60 minutes at 37°C (water bath: ensure internal air temp 35–38°C; CO₂ incubator: use sealed bag).
QuickTest critical: Both Cap/Det Ab and sample/standard must be added in the same step. The sandwich complex forms in solution during this incubation. Do not pre-incubate Cap/Det Ab alone, and do not skip mixing. Total volume per well at this point: 100 µl.
Wash × 2
Remove plate sealer. Discard liquid by inverting plate over waste. Add 300–350 µl wash buffer per well (no immersion). Discard. Tap plate on absorbent paper. Repeat once more (total: 2 washes).
300–350 µl/well
× 2 cycles
× 2 cycles
2
HRP-Streptavidin Incubation
⏱ 30 min @ 37°C, begin equilibrating TMB now
1
Place the whole bottle of TMB substrate (E024) at room temperature (18–25°C) to equilibrate for ≥ 30 min during this incubation.
2
Add 100 µl HRP-Streptavidin (E053, ready-to-use, orange) to each well. Seal plate.
3
Static incubate for 30 minutes at 37°C.
Light protection: HRP-Streptavidin is light-sensitive. Work in subdued light. Do not expose to direct sunlight during addition or incubation.
Wash × 5
Using same method as Wash × 2 above. Repeat complete wash cycle (add 300–350 µl, discard, tap on absorbent paper) five times total. After final wash, firmly tap plate to remove all residual buffer. Insufficient washing causes high background.
300–350 µl/well
× 5 cycles
× 5 cycles
3
TMB Substrate Development
⏱ 10–20 min @ 37°C, light-protected, monitor color gradient
→
Do not use the reagent reservoirs previously used for HRP conjugates, even trace HRP contamination will cause TMB false-color development.
1
Add 90 µl TMB Substrate (E024) to each well in the same order as HRP-Streptavidin was added.
2
Seal plate and incubate at 37°C in the dark. Wells should begin turning blue. Monitor every 5 min.
3
Terminate when a clear color gradient is visible across all standard wells (lightest at low concentrations, darkest at high). Weak or oversaturated color both impair curve quality.
Timing limit: Do not exceed 30 minutes. Add Stop Solution as soon as visible gradient appears. Preheat microplate reader now, requires 15 min warm-up.
4
Stop Reaction & Read
⏱ Read immediately after stop, do not delay
1
Add 50 µl Stop Solution (E026) to each well in the same order as TMB. Wells will turn from blue to yellow immediately.
2
Read absorbance at 450 nm in microplate reader immediately after adding Stop Solution.
3
Optional dual-wavelength correction: If reader supports 570 nm or 630 nm reference wavelength, subtract those readings from OD450 to correct for optical plate imperfections.
Formula reminder: If samples were loaded undiluted, multiply calculated concentration by ×2 (accounts for 50 µl sample + 50 µl Cap/Det Ab in-well dilution). For pre-diluted samples: multiply by (dilution factor × 2). Example: 1:100 pre-diluted → multiply by 200.
Performance Validation Data, QT-EH1581 (FineTest)
Precision (n=20 per level)
| Sample | Mean ng/ml | Intra-CV% | Inter-CV% |
|---|---|---|---|
| Low | 0.29 / 0.31 | 4.19% | 4.77% |
| Mid | 1.16 / 1.25 | 3.97% | 4.32% |
| High | 5.07 / 4.99 | 3.62% | 4.25% |
Recovery (spiked samples, n=5)
| Matrix | Range (%) | Average (%) |
|---|---|---|
| Serum | 87–97% | 92% |
| EDTA Plasma | 86–98% | 91% |
| Heparin Plasma | 90–101% | 94% |
Linearity (serial dilution, n=5)
| Matrix | 1:2 | 1:4 | 1:8 |
|---|---|---|---|
| Serum | 86–105% | 84–98% | 82–93% |
| EDTA Plasma | 86–98% | 82–101% | 80–98% |
| Heparin Plasma | 89–104% | 84–97% | 82–100% |
Supplier: CUSABIO
Intra-CV: <8%
Inter-CV: <10%
LLD: <0.039 ng/ml
Inter-CV: <10%
LLD: <0.039 ng/ml
Before You Begin, Reagent Preparation
Key difference from QuickTest format: In this kit, Biotin-antibody and HRP-Avidin are supplied as 100× concentrates requiring a 1:100 dilution before use. Do not use them directly from the vial. Prepare fresh dilutions immediately before use.
P1
Equilibrate All Reagents to Room Temperature
⏱ 30 min minimum
→
Bring all kit components to 18–25°C for at least 30 min before use. Includes Biotin-Ab, HRP-Avidin, TMB Substrate, and Stop Solution.
→
Centrifuge any vials if liquid is visible in cap before opening.
P2
Prepare Wash Buffer (1×)
→
Dilute 20 ml Wash Buffer Concentrate (25×) into deionized or distilled water to make 500 ml of 1× Wash Buffer.
If crystals are present in concentrate, warm to room temperature with gentle mixing until fully dissolved before diluting.
P3 + P4
Dilute Biotin-Antibody and HRP-Avidin (1:100 each)
1
Centrifuge the Biotin-antibody (100×) vial before opening. Prepare 1× working solution: 10 µl Biotin-antibody + 990 µl Biotin-antibody Diluent (per 10 wells; scale accordingly).
2
Centrifuge the HRP-Avidin (100×) vial before opening. Prepare 1× working solution: 10 µl HRP-Avidin + 990 µl HRP-Avidin Diluent.
Prepare fresh for each assay. Use diluted working solutions within 4 hours. Do not use undiluted concentrate directly in wells. If Biotin-Ab working solution appears cloudy, warm to room temperature and mix gently until uniform before use.
P5
Reconstitute Standard
1
Centrifuge the lyophilized Standard vial at 6,000–10,000 rpm for 30 s.
2
Add 1.0 ml Sample Diluent to the vial. Stock concentration = 10 ng/ml. Do not use any other diluent.
3
Mix until fully dissolved. Allow to sit with gentle agitation for at least 15 minutes before making dilutions.
Prepare fresh for each assay. Use within 4 hours. Do not freeze reconstituted standard.
P6
8-Point Standard Dilution Series
→
Label 7 tubes S6–S1 and one S0 (zero standard). Add 250 µl Sample Diluent to each tube S0–S6. Use the 10 ng/ml stock as S7 (undiluted).
→
Transfer 250 µl from S7 into S6. Mix thoroughly. Continue serial transfers (250 µl each) through S1. S0 contains Sample Diluent only (0 ng/ml).
| Tube | S7 | S6 | S5 | S4 | S3 | S2 | S1 | S0 |
|---|---|---|---|---|---|---|---|---|
| ng/ml | 10 | 5 | 2.5 | 1.25 | 0.625 | 0.312 | 0.156 | 0 |
Minimum pipetting volume: 10 µl. Use calibrated pipettors. Do not make serial dilutions directly in the assay plate wells. Use within 4 hours.
Assay Procedure, 5 Steps, ~4 Hours
1
Sample / Standard Capture on Pre-Coated Plate
⏱ 2 hours @ 37°C, solid-phase capture
→
Designate wells for standards (duplicate recommended), samples, and blank. Record plate layout using the Assay Layout Sheet.
1
Add 100 µl of standard or sample to each designated well. Cover with adhesive strip.
2
Incubate for 2 hours at 37°C ± 0.5°C.
Do not wash after this step. After incubation, simply remove and discard the liquid from each well by inversion or aspiration. Proceed immediately to Step 2.
2
Biotin-Antibody Incubation
⏱ 1 hour @ 37°C
1
Add 100 µl Biotin-antibody (1×, diluted in P3) to each well. Cover with a new adhesive strip.
2
Incubate for 1 hour at 37°C.
Wash × 3
Fill each well with 200 µl Wash Buffer. Let stand 2 minutes. Aspirate or decant completely. Repeat for a total of 3 cycles. After final wash, invert plate and blot firmly on clean absorbent paper. Incomplete washing elevates background.
200 µl/well
2 min soak
× 3 cycles
2 min soak
× 3 cycles
3
HRP-Avidin Incubation
⏱ 1 hour @ 37°C
1
Add 100 µl HRP-Avidin (1×, diluted in P4) to each well. Cover with a new adhesive strip.
2
Incubate for 1 hour at 37°C.
Wash × 5
Repeat aspiration/wash as in Step 2 wash, for 5 complete cycles. After final wash, aspirate all residual buffer. Invert plate and blot firmly on absorbent paper. Critical for low-background results.
200 µl/well
2 min soak
× 5 cycles
2 min soak
× 5 cycles
4
TMB Substrate Development
⏱ 15–30 min @ 37°C, protect from light
1
Add 90 µl TMB Substrate to each well. Cover. Incubate at 37°C for 15–30 minutes in the dark.
2
Observe color development every 10 min. TMB should progress from colorless/light blue to distinct blue gradient across standard wells.
Timing: If color develops rapidly, add Stop Solution before 15 min to prevent OD saturation. If too deep, accuracy is compromised. TMB substrate must remain colorless or light blue before adding to plate; store protected from light.
5
Stop Reaction & Measure
⏱ Read within 5 minutes of Stop Solution addition
1
Add 50 µl Stop Solution to each well in the same order as TMB. Wells should turn from blue to yellow. Any green color indicates incomplete mixing, tap plate gently to resolve.
2
Read absorbance at 450 nm within 5 minutes. If dual-wavelength correction is available, subtract OD at 540 nm or 570 nm from OD450 for higher accuracy.
Concentration calculation: Average duplicate ODs for each standard. Subtract zero standard OD. Fit 4-PL logistic curve (concentration on y-axis, OD on x-axis). Back-calculate sample concentrations from curve. Multiply by dilution factor if applicable.
Performance Specifications, CSB-EL007460HU (CUSABIO)
Precision
| Metric | CV Specification |
|---|---|
| Intra-assay | <8% |
| Inter-assay | <10% |
Sensitivity
| Parameter | Value |
|---|---|
| Lower Limit of Detection (LLD) | <0.039 ng/ml |
| Method | Mean + 3 SD of 20× zero standard replicates |
Head-to-Head Comparison: FineTest QT-EH1581 vs. CUSABIO CSB-EL007460HU
| Parameter | 🔵 FineTest QT-EH1581 (QuickTest) | 🟠 CUSABIO CSB-EL007460HU (Standard) |
|---|---|---|
| Format / Mechanism | Solution-phase sandwich capture (QuickTest) Cap+Det Ab co-incubated with sample; complex captured onto plate via anti-tag
|
Classical solid-phase sandwich ELISA Capture Ab pre-coated; antigen captured in solid phase; detection Ab added sequentially
|
| Total Run Time | 120 min ✓ Faster | ~240 min (4 h) |
| Detection Range | 0.156 – 10 ng/ml | 0.156 – 10 ng/ml |
| Sensitivity (LOD/LLD) | 0.094 ng/ml | <0.039 ng/ml ✓ More sensitive |
| Sample Volume/Well | 50 µl | 100 µl |
| Sample Types Validated | Serum, plasma, cell culture supernatant, cell lysate, tissue lysate, other biological fluids | Serum, plasma, tissue homogenates, cell lysates |
| Standard Reconstitution | 0.5 ml Sample Diluent → 20 ng/ml stock Serial 1:2, 150 µl transfers; 8 points
|
1.0 ml Sample Diluent → 10 ng/ml stock Serial 1:2, 250 µl transfers; 8 points
|
| Step 1 Incubation | 60 min @ 37°C Cap/Det Ab + Sample simultaneously
|
2 h @ 37°C Sample only; no wash after this step
|
| Step 2 Incubation | 30 min @ 37°C HRP-Streptavidin (ready-to-use)
|
1 h @ 37°C Biotin-antibody (100× → dilute to 1×)
|
| Step 3 Incubation |
Only 2 incubation steps in assay
|
1 h @ 37°C HRP-Avidin (100× → dilute to 1×)
|
| Wash Protocol | Wash × 2 after Step 1 Wash × 5 after Step 2 300–350 µl/well, no immersion
|
No wash after Step 1 capture Wash × 3 after Step 2 (200 µl, 2-min soak) Wash × 5 after Step 3 |
| TMB Development | 90 µl, 10–20 min @ 37°C, dark | 90 µl, 15–30 min @ 37°C, dark |
| Stop + Read | 50 µl Stop → 450 nm (read immediately) Optional correction: 570 or 630 nm
|
50 µl Stop → 450 nm within 5 min Optional correction: 540 or 570 nm
|
| Intra-assay CV | 3.62–4.19% (measured) ✓ | <8% (specification) |
| Inter-assay CV | 4.25–4.77% (measured) ✓ | <10% (specification) |
| Recovery | Serum: 87–97% (avg 92%) EDTA Plasma: 86–98% (avg 91%) Heparin Plasma: 90–101% (avg 94%) |
Not reported in datasheet |
| Curve Fitting | 4-PL logistic (recommended) Free online tool: elisa.fn-test.com
|
4-PL logistic (recommended) CurveExpert or equivalent software
|
| Dilution Correction Note | Undiluted samples: multiply result × 2 (on-plate 1:1 dilution by Cap/Det Ab). Pre-diluted at 1:100: multiply × 200. | Multiply by dilution factor only (no inherent in-well dilution). |
| Best Use Case | High throughput, time-constrained runs. Strong precision data, validated recovery across 3 matrices. Suitable when sensitivity >0.094 ng/ml is sufficient. | Low-abundance targets requiring maximum sensitivity. <0.039 ng/ml LLD. Appropriate when EFNA1 levels expected near or below 0.1 ng/ml range. |
ELISA Troubleshooting Guide, Applicable to Both Kits
| Problem | Likely Cause | Resolution |
|---|---|---|
| No signal (standard + samples) | Incorrect reagent addition order; missing step; mixed kit components | Verify each step was completed in sequence. Use only components from the same kit lot. Repeat assay with fresh reagents. |
| Overflow OD (all wells saturated) | Cross-contaminated kit components; over-concentrated working solutions | Confirm reagent dilutions (particularly for CUSABIO: 100× concentrate must be diluted 1:100). Use only same-kit components. |
| No signal from samples (standards OK) | Sample concentration below detection range; incompatible lysis buffer; excessive freeze-thaw | Decrease dilution ratio or concentrate sample. For lysates: avoid NP-40, Triton X-100, DTT. Aliquot samples to avoid freeze-thaw cycles. |
| High CV% (poor reproducibility) | Uneven washing; foaming in wells; inconsistent pipetting; precipitate in wells | Use calibrated multichannel pipette. Increase dilution to reduce precipitate. Ensure washer tubing is unobstructed. Check pipettor calibration. |
| Low standard curve signal | Improperly reconstituted standard; degraded standard; over-dried plate after washing | Centrifuge lyophilized standard before opening. Follow storage conditions strictly. Never allow wells to dry, add reagents immediately after each wash. |
| High background | Insufficient washing; contaminated wash buffer; TMB exposed to light; reading delayed after Stop Solution | Complete all prescribed wash cycles. Prepare fresh wash buffer. Incubate TMB step in dark. Read within 5 minutes of adding Stop Solution. |
| Poor standard curve fit | Inappropriate curve fitting model; standard preparation error | Use 4-PL logistic fitting. Try alternative models if 4-PL fit is poor. Verify serial dilution volumes and mixing at each step. Exclude outlier replicates if technically justified. |
| QuickTest only: low signal despite good standards | Cap/Det Ab not added simultaneously with sample; insufficient mixing after loading | Both Cap/Det Ab and sample must be added to wells and mixed immediately by plate tapping (10 s). Do not pre-incubate Cap/Det Ab in empty wells before adding sample. |