Biotin ELISA Kit

ELISA (Enzyme-Linked Immunosorbent Assay) is a plate-based assay used to detect and quantify specific proteins, antibodies, or hormones in a sample. CUSABIO kits use three main formats: sandwich, competitive, and indirect ELISA. The general workflow follows these steps:

• Storage & Reagent Preparation – Bring all kit components to room temperature; prepare wash buffer at the correct dilution.
• ELISA Plate Setup – Add standards, controls, and samples to pre-coated wells.
• Incubation – Incubate at 37 °C (optimal for antigen–antibody binding) for the time specified in the kit manual.
• Washing – Remove unbound material; ensure wells are filled completely and blot dry after the final wash.
• Detection & Color Development – Add TMB substrate; allow 15–30 minutes (20 min recommended) for color to develop at room temperature.
• Stop & Read – Add stop solution (2N H₂SO₄); read OD within 10 minutes using a dual-wavelength setting (450 nm / 570 nm or 450 nm / 630 nm).

Every CUSABIO ELISA kit is evaluated under strict QC standards before release. Parameters assessed include:

• Linearity & Linearity Range – Serial dilution linearity of spiked samples
• Sensitivity (LLOQ) – Lowest detectable concentration above background
• Intra- & Inter-Assay Precision (CV%) – Repeatability within and across plate runs
• Recovery Rate – Spike-recovery in representative matrices (e.g., serum, plasma)
• Stability – Kit stability under recommended storage conditions
• Specificity – Cross-reactivity with related proteins and isoforms tested
• Natural Sample Detection – Validation in authentic biological samples

• Quantitative kits include a set of calibration standards matched to the entire reagent system. Running the standard curve confirms system integrity and serves as a built-in quality control.
• Qualitative kits include positive and negative controls.
• CUSABIO recommends running duplicate wells for all standards and samples to assess precision and catch pipetting errors.
• For best curve fit, use a 4-parameter logistic (4-PL) or 5-PL model as recommended in the kit insert.

CUSABIO ELISA kits are validated and quality-controlled for the species explicitly listed on the product datasheet. Use with unlisted species is not recommended without prior cross-reactivity data, because antibody epitope conservation varies significantly even among closely related species. If you need to test a non-listed species, please contact our technical support team for guidance before ordering.

No. Reagents from ELISA kits with different lot numbers must not be mixed. Antibody concentrations, standard concentrations, and reagent formulations are lot-matched and co-validated as a complete system. Mixing lots will invalidate your standard curve and may produce unreliable quantification. CUSABIO does not sell kit components (standards, antibodies, substrate) separately.

• BSA: Some components (typically diluents and conjugate buffers) contain BSA as a carrier protein and non-specific binding blocker.
• Preservative: Commercial Proclin 300 (a broad-spectrum antimicrobial) is used in most reagents at concentrations consistent with industry practice.
• Stop Solution: 2N Sulfuric Acid (H₂SO₄) is used in most kits. It terminates the HRP-TMB reaction by denaturing the enzyme and shifts TMB color from blue (OD 650 nm) to yellow (OD 450 nm). Read the plate within 10 minutes of adding stop solution.

TMB (3,3′,5,5′-Tetramethylbenzidine) is a chromogenic HRP substrate widely used in ELISA. In the presence of HRP and H₂O₂, TMB is oxidized and turns blue (λmax ≈ 650 nm). After addition of sulfuric acid stop solution, it converts to a yellow diimine form (λmax ≈ 450 nm).

Dual-wavelength reading (450 nm − 570 nm or 450 nm − 630 nm) is recommended because subtracting the reference wavelength OD from the signal wavelength OD corrects for:

• Optical imperfections and scratches in the well bottom
• Non-specific light scattering
• Plate reader lamp fluctuations

This improves assay precision and reduces well-to-well variation (CV%).

Uniform color across all wells (including blanks) indicates non-specific signal. Common causes and fixes:

• Wrong reagents added – Verify kit component labels and lot numbers before starting.
• Insufficient plate washing – Ensure wells are completely filled during each wash step; blot plate firmly on absorbent paper after the final wash to remove residual buffer.
• Cross-contamination – Use dedicated pipette tips for each reagent; do not allow enzyme conjugate to contact TMB substrate container or positive control wells to contact blank wells.
• Reagent contamination – Use fresh, clean reservoirs and single-use pipette tips.
• Unqualified distilled water – Verify wash buffer pH is neutral (pH 7.2–7.4) after dilution.

Complete absence of color (zero signal in all wells) points to a failure in the enzyme-substrate reaction chain. Check the following:

• TMB substrate expired or degraded – Substrate solution should be clear and colorless before use; blue-tinted substrate indicates oxidation and must be discarded.
• Stop solution added before color development – Ensure TMB has been incubated the full required time (15–20 min) before stopping.
• HRP conjugate omitted or inactivated – Confirm conjugate was added and stored correctly (2–8 °C, avoid freeze–thaw).
• Over-washing / sodium azide contamination – Azide irreversibly inhibits HRP; ensure buffers and reagents are azide-free.
• Incorrect incubation temperature – All CUSABIO sandwich ELISA kits require 37 °C incubation; room temperature incubation significantly reduces signal.

Low signal across the entire plate (including high standards) suggests a systemic assay problem rather than a true low-concentration result. Troubleshoot in this order:

• Insufficient incubation time – Use a timer and adhere strictly to protocol timing at each step.
• Insufficient color development time – Allow 15–20 minutes in the dark at room temperature; avoid stopping the reaction early.
• Incorrect color development reagent addition order – Follow kit protocol exactly; TMB Component A and B (if two-component) must be combined correctly.
• Over-washing or excessive wash force – Reduce manifold pressure on plate washers; dilute concentrated wash buffer accurately; do not exceed the specified number of wash cycles.
• Pipetting errors – Use calibrated pipettes with well-fitted tips; pipette slowly to avoid air bubbles and uneven volumes.
• Reagent storage issue – Check that all components were stored at 2–8 °C and not frozen (unless specified); degraded detection antibody conjugate is a common cause of signal loss.

A poor standard curve undermines all sample concentration calculations. Common causes:

• Improper standard reconstitution – Dissolve the lyophilized standard completely in the specified diluent; briefly vortex or centrifuge to ensure homogeneity.
• Serial dilution error – Change pipette tips between every dilution step; mix each dilution thoroughly before transferring to the next tube.
• Wrong curve-fitting model – Use the manufacturer-recommended 4-PL or 5-PL logistic model; avoid linear or log-log fits unless specified.
• Pipetting inconsistency – Calibrate pipettes; pipette at a consistent angle and speed; dispense against the well wall rather than dropping into the liquid.
• Edge effects – Use a plate sealer and a calibrated incubator; do not stack plates; allow all reagents to reach room temperature before adding.

• Reading too high (above upper standard) – Dilute the sample further in the kit's assay diluent and re-run. Apply the dilution factor to back-calculate the true concentration.
• Reading too low (below detection limit) – Concentrate the sample (e.g., by lyophilization, ultrafiltration), or use a more sensitive kit format. Confirm the analyte is present and stable in your sample matrix.
• Matrix interference – Run a spike-recovery test to confirm acceptable recovery (80–120%) in your specific sample type. If recovery is poor, optimize sample dilution or switch to a compatible matrix diluent.