Common problems and countermeasures in the development and use of ELISA kits

phenomenon

Possible Causes

Solution

The slats are not colored or have no corresponding values

The order of reagents is disordered, or the procedure is wrong.

Repeat the experiment in strict accordance with the experimental instructions

Different batches of reagents were used

Make sure the reagents are from the same kit

The slats are seriously wet

The slats should be sealed, and the desiccant should be replaced in time.

low

Suck

Light

degree

OD

value

Reagent expired, or mixed in different batches

Verify expired reagents and batches

Incorrect preparation of washing solution, long washing and soaking time

Wash according to the instructions and prepare the lotion properly

Incubation time is too short

Strictly calculate the time according to the instructions

Reagent contamination

Make sure to pick up different liquids and replace the new tips and containers

Reagent temperature is too low

According to the volume of the reagent, the temperature recovery time is extended accordingly to ensure that the reagent is completely warmed up.

Use the wrong wavelength reading, or enzyme

Standard failure

Ensure 450 nm wavelength reading and check if the microplate reader is malfunctioning

The kit is in extreme conditions

Please return the used parts to the refrigerator immediately. Do not put them in an overheated environment for too long.

Excessive

Background value or absorbance ( OD value)

Poor quality water preparation reagent

Use double distilled or deionized water to prepare reagents

Improper washing or poor washing of the washing machine

Add 1-2 washes and add at least 250 μL of wash solution per well

The microplate reader fails, such as the OD reading is very high and the color is very light

Check the microplate reader with a calibration microplate and check the light source

The laboratory temperature is too high and the reaction time is too long

Determine if the operating temperature is appropriate and whether the time is calculated correctly

Confused reagents, contaminated or improperly formulated

Ensure that the correct reagents are used, accurately formulated to avoid contamination

Inside the board

difference

Big

Inconsistent time to add standards and samples, etc.

Multi-channel gun loading, the same reagent can not be interrupted during the sample loading

Improper use of multiple guns

Calibrate multiple guns, check if the nozzles are tight, and ensure consistent liquid absorption

The washing system has failed

Check the washing system and keep it running

board

between

difference

different

Sex

Big

The incubation time between the plate and the plate is too different

Independent timing to ensure consistent incubation time

Inconsistent washing process between plates

Ensure the same number of washings to ensure the washing system is working properly

Improper use of a pipette

Check the pipette to make sure you draw the same amount

Reagents and samples at different temperatures

Make sure that the reagents and sample liquid in the cartridge are fully warmed up. If the reagent volume is large, it needs to be warmed up for a long time. Can not use warm water bath to return samples

Mix different batches of reagents, or the kit expires

Do not mix reagents. Usually , the OD value of 0 standard is less than 0.5, indicating that the reagent quality is degraded.

One or

Multiple standard data points are outside the curve range

Standards are added in disorder, or placed in the wrong location

Repeat the experiment as required to ensure that the standard is added correctly.

Standards are contaminated or confused with other standards

Switch to new standards and add standards in order from low to high

Inconsistent washing or failure of the washing system

Follow the washing method as always, check the washing system

Inconsistent time to add standards and reagents

Standards are added sequentially from low to high concentrations, using multiple guns to pipette

Improper use of multiple guns

Calibrate multiple guns, check if the nozzles are tight, and ensure consistent liquid absorption