Possible Causes

What You Can Do?

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Improper standard dilution

Use appropriate diluent as blank. Make sure that the dilution is performed as according to protocol

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Standard improperly reconstituted

Briefly spin standard vial before opening. Make sure that there is no undissolved material after reconstituting

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Standard degraded

Store standards as according to protocol

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Curve doesn’t fit the scale

Try plotting log-log or 5 parameter logistic curve fit

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Pipetting error

Calibrate pipettes to make sure that the correct volume is dispensed

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Incomplete washing

Increase washing cycles

Possible Causes

What You Can Do?

►

Improper washing

Make sure that the washing is done as according to protocol

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Poor mixing of samples

Mix samples gently and evenly

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Dirty plate

Make sure that the bottom of plate is clean

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Reagents too old

Make sure that the reagents are not expired. Use freshly prepared reagents

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Bubbles in wells

Make sure that there is no bubble in wells before reading

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Inconsistent pipetting

Calibrate pipettes to make sure that the correct volume is dispensed

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Edge effects

Make sure that the plate and reagents are equilibrated to room temperature before starting assay

Possible Causes

What You Can Do?

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Too much antibodies was used

Reduce the concentration of primary or secondary antibodies

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Antibodies bind nonspecifically

Use blocking buffer or choose another affinity-purified antibody

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Too much substrate reagent used

Use substrate with higher dilution

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Insufficient washing

Increase washing cycles

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Wrong concentration of blocking reagent

Check the recommended concentration of blocking buffer

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Reaction not stopped

Stop reactions with STOP buffer before reading

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Plate left too long before reading

Take measurements shortly after addition of substrate and STOP buffer

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Insufficient Tween in wash buffer

Use PBS+0.05% Tween as wash buffer

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Incubation temperature too high

Optimize incubation temperature for each experiment

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Plate stacking during incubation lead to uneven temperature throughout the plate

Avoid stacking plates together during incubation

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Pipetting error

Calibrate pipettes to make sure that the correct volume is dispensed

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Reagents not mixed properly

Make sure that all reagents are mixed properly and equilibrated to room temperature before assay

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Salt concentration of incubation and wash buffer

Increase salt concentration to reduce nonspecific interaction

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Substrate incubation carried out in light

Perform substrate incubation in dark

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Dirty plate

Make sure that the bottom of plate is clean

Possible Causes

What You Can Do?

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Insufficient coating

Use more antigens or antibodies for coating

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Substrate reagents have expired or prepared at a wrong pH

Use fresh substrate reagents

Possible Causes

What You Can Do?

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Assay set up incorrectly

Make sure that the instructions in the protocol is followed carefully

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Incorrect secondary antibody used

Check if the correct secondary antibody is used

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Insufficient antibodies used

Increase concentration of primary or secondary antibody

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Substrate reagents not fresh

Use fresh substrate reagents

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Wrong settings of plate reader

Check the settings (wavelength, filters, gain etc) of plate reader

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Insufficient incubation

Follow the incubation time as indicated in the protocol booklet

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Sample concentration falls below detection limits of kit

Decrease dilution factor or concentrate samples

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Plate washing too vigorous

Check the setting of plate washer. Pipette wash buffer into wells gently

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Wells dried out

Cover plate with adhesive film or incubate in humidified chamber throughout experiment

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Enzyme inhibitor present in buffers or reagents

Inhibitors such as Sodium Azide can affect enzyme and assay performance. Ensure that there is no enzyme inhibitor in any buffers