● Always verify reagent labels and lot numbers before the assay; only use reagents from the same ExKits kit.
● Maintain consistent wash buffer volume per well and tap the plate dry on absorbent paper after washing.
● Strictly follow the kit protocol for incubation and color development time.
● Change tips between reagents and use separate vessels for different solutions.
● Check dilution calculations and dilute detection antibody or HRP-avidin further if needed.
● Keep TMB substrate protected from light until use.● Double-check reagent labels during preparation and handling.
● Ensure all wash buffer containers are clean and contamination-free.
● Carefully read the operating protocol and strictly follow the reaction sequence specified in the instructions.● Confirm validity and store ExKits kits under recommended conditions to avoid contamination.
● Equilibrate all reagents and samples at room temperature before use.
● Use calibrated pipettes with proper, single-use tips.
● Use a timer; typical color development is 15–30 minutes.
● Dilute concentrate as directed and control washing volume and frequency.
● Use pure, neutral water for buffer preparation.A: Poor well-to-well consistency may arise from improper standard dilution, premature preparation of working reagents, insufficient mixing after sample addition, inconsistent incubation, washing, or coloration conditions, unstable incubation temperature, cross-contamination or reused consumables, scratches, dirt, or fingerprints on the plate bottom, clogged washer heads or insufficient sample centrifugation, or sample degradation or contamination.
Solutions:
● Use only the recommended diluent and follow ExKits guidelines.
● Prepare working solutions 10 minutes before use.
● Mix thoroughly and handle plates gently to prevent splashing.
● Keep parameters uniform across experiments.
● Maintain a constant 37°C environment.
● Replace tips and use separate reservoirs for each reagent.
● Avoid touching the bottom and wipe gently before reading.
● Clear washer nozzles and centrifuge samples fully.
● Use fresh samples or store at low temperatures.
● When pipetting samples, take care to avoid generating air bubbles.
● Weather conditions vary by region. Be sure to eliminate static electricity before operation to prevent enlarged differences in reaction liquid levels between wells.
| Problem Description | Possible Cause | Solution |
|---|---|---|
| Whole plate yellow or high background | Wrong reagents or mixed batches | Use same ExKits kit, check lot numbers |
| No color in any wells | Enzyme conjugate inactivation | Keep containers clean, avoid contamination |
| Weak color in all wells | Reagents not equilibrated to room temperature | Equilibrate 30min at 18-25°C |
| Poor repeatability | Inconsistent washing or incubation | Keep conditions uniform each test |
| Low reader reading but normal color | Wrong wavelength filter | Use 450nm |