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Q1: What core reagents are required for double‑antibody sandwich ELISA?

A: Core reagents include ELISA coating solution, wash buffer, blocking solution (5% skim milk powder or 1% BSA), capture antibody, detection primary antibody, enzyme‑linked secondary antibody, TMB chromogenic solution and stop solution. Dilute concentrated stock solutions to 1× working solution before use according to the instructions.

Q2: Can I quickly thaw reagents directly at 37℃ before experiment?

A: No. All reagents need to be equilibrated to room temperature in advance. Do not thaw or dissolve reagents directly at 37℃. Thoroughly mix reagents and samples during dilution, and avoid bubble generation as much as possible to prevent adverse effects on loading and reaction results.

Q3: Is antibody coating and blocking required for commercial ELISA kits?

A: Most commercial ELISA plates are pre‑coated with capture antibodies. In this case, coating, plate‑washing and blocking steps are not required. Please carefully read the kit manual for confirmation before starting the experiment to avoid redundant operations.

Q4: What buffers can be used for capture antibody coating? What incubation options are available?

A: The capture antibody can be diluted with carbonate buffer or phosphate‑buffered saline. Add 100 μL of diluted antibody to each well. Two incubation protocols are available: incubate at 37℃ for 2 hours, or incubate overnight at 4℃.

Q5: How to perform plate washing correctly?

A: Use 10 mM PBST (10 mM PBS + 0.05% Tween‑20) as washing buffer. Add 200‑300 μL working wash solution to each well and soak for 1‑2 minutes. Discard liquid and tap the plate gently on absorbent paper to remove residual liquid in wells. Repeat washing 3‑5 times per round.

Q6: What are the requirements for sample loading?

A: Set blank wells, standard wells and sample wells for each experiment. Add 100 μL sample diluent to blank wells; add 100 μL standard substance or test sample to other wells. Avoid bubbles, add liquid to the bottom of wells without touching well walls. Complete sample loading for one plate within 3 minutes. Cover with plate lid or sealing film and incubate at 37℃. Prepare fresh standard solution for each independent experiment to guarantee reliable data.

Q7: What are the incubation conditions for detection antibody and enzyme‑linked secondary antibody?

A: Dilute detection antibody and secondary antibody with antibody diluent according to experimental requirements. Add 100 μL per well and incubate separately at 37℃ for 1 hour. Wash the plate 3‑5 times after each incubation step.

Q8: How to control TMB color‑development and termination reaction?

A: Add 100 μL TMB substrate solution to each well and incubate in dark at 37℃ for 15 minutes. Adjust incubation time according to practical conditions but avoid over‑color‑development. When obvious color gradient appears in standard wells, add 50 μL stop solution to each well, and the solution will turn from blue to yellow.

Q9: When and how to read OD values?

A: Complete detection within 5 minutes after adding stop solution. Set 630 nm as reference wavelength, and measure absorbance (OD value) of each well at 450 nm with an ELISA reader.

Q10: How to process raw OD data?

A: Subtract the OD value of blank wells from OD readings of standards and samples. If technical replicates are set, calculate the average OD value for each group.

Q11: How to calculate sample concentration based on standard curve?

A: Take standard concentration as X‑axis and processed OD value as Y‑axis. Use professional software such as Origin or ELISACalc for fitting. Obtain the fitted concentration corresponding to sample OD from the standard curve, then multiply by sample dilution factor to get the final measured concentration of samples.

Q12: High background signal, obvious blue‑yellow color appears in all wells

A: Possible causes: insufficient plate washing, incomplete blocking, excessive antibody concentration or reagent contamination. Extend soaking time during plate washing; verify blocking‑solution concentration and prolong blocking time; re‑optimize antibody dilution ratio; replace contaminated reagents and use new pipette tips for loading.

Q13: Weak or no color signal for both standards and samples

A: Check whether reagents are expired or inactivated; verify incubation temperature and duration of each step; avoid excessive washing which may strip bound complexes; make sure no key steps are omitted; equilibrate all reagents to room temperature beforehand; double‑check wavelength setting of microplate reader.

Q14: High CV value among replicate wells, poor repeatability

A: Common triggers include bubbles during loading, inconsistent liquid volume, residual liquid after plate tapping, and uneven incubation temperature. Avoid bubble formation while loading; calibrate pipettes; thoroughly tap out residual liquid after each wash; seal tightly with sealing film during incubation for uniform temperature distribution across the plate.

Q15: Poor fitting performance of standard curve

A: Prepare fresh standard solution for each run; strictly follow dilution procedures and prevent repeated freeze‑thaw of standard stock solution; finish loading within 3 minutes for one plate; maintain stable incubation temperature; adopt four‑parameter logistic fitting instead of simple linear fitting.

Q16: Edge effect occurs in outer wells of microplate

A: It is mainly caused by liquid evaporation. Use brand‑new sealing film during incubation; do not stack multiple ELISA plates inside incubator; keep stable temperature inside incubator; shorten sample‑loading time for whole plate as far as possible.