Incorrect configuration of standard solution

Confirm if the dilution is done correctly.

Improper reconstitution of standard samples

Centrifuge before opening the lid; Check for insoluble substances after reconstitution.

The standard product has degraded

Save and process standard samples according to the recommended method.

The scale of the curve is not suitable

Attempt to plot curves using different scales, such as double logarithmic and 5-parameter fitting.

Sample addition error of pipette

Correct use of calibrated pipettes

The incubation time is too short

Incubate the sample overnight at 4 ℃, or follow the experimental protocol of the reagents.

The target content is below the detection range

Reduce the dilution factor of the sample or concentrate the sample.

Sample type not applicable

For unverified sample types, the detection signal may be weakened or unverified sample types may not be used as positive controls for simultaneous detection.

The antigen epitope is adsorbed by the well plate and cannot be recognized

Enhance the ability to detect peptides using direct or indirect ELISA methods by conjugating peptides onto large carrier proteins and then coating them onto microtiter plates.

Test the compatibility of the buffer solution

Ensure that the detection buffer is compatible with the target (e.g. preserving enzyme activity, preserving protein interactions).

Insufficient testing reagents

Follow the experimental protocol of the reagents and increase the concentration or dosage of the detection reagents.

Incorrect sample preparation

Ensure proper sample preparation/dilution. The sample may not be compatible with the determination form of the microtiter plate.

Insufficient antibodies

Try different antibody concentrations/dilutions.

Incubation temperature too low

Ensure incubation is carried out at the correct temperature. All reagents (including well plates) should be at room temperature or the temperature recommended by the experimental protocol before conducting the experiment.

Incorrect wavelength

Confirm the wavelength and read the board again.

The orifice plate is strongly washed

Check and ensure that the pressure of the automatic washing system is correct. If washing manually, gently absorb the rinsing buffer.

Kong Biangan

After the measurement begins, do not let the hole dry out. Seal the orifice plate with sealing film or tape for all incubation steps.

Slow color development rate of enzyme reaction

Prepare substrate solution before use. Ensure that the mother liquor is not expired or contaminated. Extend the incubation time.

There are bubbles in the hole

Before reading the board, ensure that there are no bubbles present.

Uneven/insufficient washing of holes

Check if all the pipe openings of the washing machine are unobstructed. Use the recommended method for washing.

Insufficient mixing of reagents

Ensure that all reagents are thoroughly mixed.

Inconsistent transfer volume

Correct use of calibrated pipettes

Edge effect

Ensure that the orifice plate and all reagents are at room temperature.

Inconsistent sample preparation or storage conditions

Ensure consistent sample preparation and use optimal sample storage conditions (such as minimizing repeated freeze-thaw cycles).

Insufficient hole washing

Wash according to the experimental protocol recommendations.

Wash buffer contamination

Prepare fresh washing buffer.

Too many detection reagents

Ensure that the reagents are diluted correctly or reduce the recommended concentration of the detection reagents.

Invalid blocking buffer (e.g. detection reagent binding to blocking agent; wells not completely blocked)

Try different sealants and/or add sealants to the washing buffer.

Salt concentration of incubation/washing buffer

Increasing salt concentration may reduce non-specific and/or weaken off target interactions.

Too long time after adding termination solution before reading the board

Read the board immediately after adding the termination solution.

Non specific binding of antibodies

Use appropriate blocking buffer, such as BSA or 5-10% normal serum. If it is a direct primary antibody, use the same serum as the primary antibody species. If it is a non direct primary antibody, use the same serum as the secondary antibody species. Ensure that the holes have been pre treated to prevent non-specific adhesion.

High antibody concentration

Try different dilutions to achieve optimal results.

Substrate incubation is carried out under light

Substrate incubation should be carried out in the absence of light or according to the experimental protocol of the reagents.

After the substrate is added, precipitation is generated in the well

Increase the dilution factor of the sample or decrease the substrate concentration.

Dirty orifice plate

Clean the bottom of the orifice plate.

Improper storage of ELISA kit

Save all reagents according to the recommended method. Please note that the storage conditions for each reagent may vary.

Insufficient target

Concentrate the sample or reduce the sample dilution.

Detection reagent inactivation

Ensure that the reported enzyme/fluorescein has the expected activity.

The enzyme-linked immunosorbent assay (ELISA) reader is not set correctly

Ensure that the ELISA reader is set to the correct absorption wavelength or excitation/emission wavelength during testing.

The measurement method is not sensitive enough

Replace with a more sensitive detection system (such as transitioning from colorimetric detection to chemiluminescence/fluorescence detection). Replace with more sensitive measurement methods (such as switching from direct ELISA method to sandwich ELISA method). Extend the incubation time or increase the temperature.

The adsorption effect of the target on the microtiter plate is poor

Covalently bind the target to a microtiter plate.

Insufficient substrate

Add more substrates.

Sample type incompatibility (e.g. serum and cell extract)

For unverified sample species, the detection signal may be weakened or absent. Use validated sample types as positive controls for simultaneous testing.

Interference from buffer solution or sample components

Confirm whether there are interfering compounds in the reagent. For example, sodium azide in antibodies can inhibit HRP enzyme, while EDTA used as an anticoagulant in plasma can inhibit enzyme reactions.

Mixing or blending reagents from different reagent kits

Avoid mixing reagents from different test kits.