ELISA Troubleshooting

Problem

Possible Causes

Solutions

Light color,

low sensitivity

1. Long transportation time with high temperature.

Shorter transportation time, pack the kit with foam box and ice bags.

2. Without fully equilibration of the kit to room temperature before use.

Bring all reagents to room temperature (25°C) before use.

3. Incubator temperature less than 37°C.

Make sure the steady Incubator temperature.

4. Insufficient incubation time.

Accurate timing.

5. Incorrect washing process.

See washing procedure in the manual, The wash procedure is critical.

6. Insufficient fluid absorption of pipette, too much water or uncleanness inside of suction nozzle.

Adjust pipette, use clean suction nozzle matching with pipette tightly, dispose suction nozzle after use.

7. Distilled Water contamination.

Use fresh qualified distilled water.

8. Insufficient substrate working time.

Accurate timing.

9. low concentration in HRP or detection antibody.

Raise content of Avidin-HRP and detection antibody.

High background

1. Insufficient washing.

Prepare Wash Concentrate accurately. See washing procedure in the manual, The wash procedure is critical.

2. Sample contamination.

Collect fresh sample or store at low temperature to avoid pollution.

3. Incubator temperature higher than 37°C or too long reaction time.

Adjust Incubator temperature, Accurate timing.

4. Reuse suction nozzle, Insufficient washing or disinfecting.

Dispose suction nozzle after use.

5. Distilled Water contamination.

Use fresh distilled water.

6. Mix reagents like enzyme.

Do not mix reagents from different batches.

7. Too many samples cause long addition time and reaction time.

Control of reaction time to avoid adding too many samples once.

8. Insufficient sealing cause unspecific adsorption.

Extend sealing time.

9. High concentration in HRP or detection antibody, Insufficient washing.

Reduce content of Avidin-HRP and detection antibody.

Poor reproducibility

1. Variations in samples quantity and addition time.

Controlling of addition time.

2. Variations in Incubation time, washing methods and operators.

Make the same experiment condition.

3. Variations in Sample addition.

Samples should be mix well before diluting, use the same transferpettor and set suction nozzle tightly.

Whiteboard

1. Substrate deteriorated.

Replace new substrate.

2. Wash Solution error.

See Dilution ratio in the manual.

3. Miss Enzyme conjugate.

Do not miss Enzyme conjugate.

4. Mistake stop solution for wash buffer or substrate.

Check the labels of each reagent carefully.

5. Process sequence error.

Make sure the correct process sequence in the manual.