Low signal/absorbance in all wells

Plate reader lamp

The intensity of ELISA plate reader lamp is too low.

Check lamp efficiency and plate alignment. This can be tested by calibration of the plate reader, see Microplate reader test for absorbance.

Absorbance

Absorbance was read at 405 nm instead of 450 nm.

Check that the filter is set to 450 nm, (Plates read at 405 nm produces very low levels). Also check that the right filter is in the right slot.

Plate shaker

Plate shaker amplitude is too low.

Plate shakers should have a speed of approximately 700-900 cycles per minute (rpm). Low frequency results in poor calibration curve.

Preparation of enzyme conjugate solution

Over dilution of enzyme conjugate solution.

Dilute enzyme conjugate solution according to Directions for Use.

Pipettes

Pipettes are not calibrated, resulting in low sample/calibrator volume.

Pipettes should be calibrated and cleaned 3-4 times a year.

Incubation time

Incubation time is too short.

Do not prolong or shorten incubations. 

Incorrect wash

Soak was included in the wash procedure.

Program the plate washer to aspirate immediately after the dispensing of Wash Buffer.

If washing manually hold the plate vertically. When washing manually use a laboratory wash bottle, see Instruction for manual washing.