Microplate reader is a device which used to read the result of ELISA test utilizing theory of spectrophotometer.

1. Microplate Reader
 It is a device which used to read the result of ELISA test
utilizing theory of spectrophotometer.
 ELISA-(Enzyme-Linked Immunosorbent Assay)
Biochemical technique used
mainly in Immunology to
detect the presence of an
antibody or an antigen in a
sample.
 Also called Photometric
Microplate reader,
ELISA reader

2. Theory of operation
 Microplate reader is specialized spectrophotometer which facilitates
readings on a wide range of wavelengths, generally between 400 to 750 nm.
 Beer Lambert law,
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A = absorption
ε = absorption coefficient
l = path length
c = concentration
I0 = reference light intensity
I = light intensity
c
l
 A light beam, passing through the sample has a diameter ranging between
1 to 3 mm.
 Then detection system detects the light coming from the sample, amplifies
the signal and determines the sample’s absorbance.

3. Theory of operation

4. Internal Parts
Light Source
ELISA Plate
Power supply

5. Installation
 A clean, dust free environment.
 A stable work table away from equipment that vibrates
(centrifuges, agitators).

6. calibration
 Calibration plates are equipped with at least three
predetermined optic density values within the
measurement ranges; low, medium, and high value.
 Place the calibration plate on the equipment and carry out
a complete reading.
 Verify if there are differences in the readings obtained from
well to well. If this is the case, invert the plate (180°) and
repeat the reading to rule out that differences are
attributed to the plate itself.
 Identify the reader requires calibration. If so, proceed with
the calibration according to manufacturer.

7. Maintenance
Basic : Daily
Preventive : Quarterly
Review that optical sensors of each
channel are clean.
2. If dirt is detected, clean the light
emitters and the sensors with a
small brush.
3. When the daily operations begin,
let the reader warm up for 30
minutes. Next, do a blank reading
and then read a full plate of
substrate. The readings must be
identical.
4. Examine the automatic drawer
sliding system. It must be smooth
and constant.
1.
1.
2.
3.
4.
Verify the stability of the lamp.
Use the calibration
plate conducting readings with
intervals of 30 minutes with
the same plate. Compare
readings. There must be no
differences.
Clean the detectors’ optical
systems and the lighting
systems.
Clean the plate drawer.
Verify the alignment of each
well with the light emission
and detection systems.

8. Basic fault Identification
The reader gives a reading that does not
make sense
2. The reader’s readings vary from row to
row.
3. The reader displays unexpected
variation in the optical density
readings.
1.
4. The reader displays a gradual increase
or decrease from column to column.
5. Low reproducibility.
6. Misaligned light beam.

9. 1.0 The reader gives a reading that does not make sense …?
The illumination
lamp is out of
service.
 Replace the lamp with one with the
same characteristics as the original.

10. 2.0 The reader’s readings vary from row to row.
• Dirty optical
sensors.
• The illumination
system’s lenses or
parts are dirty.
• Lack of
calibration in one
or more channels.
 Clean the sensors.
 Clean the lighting system’s lenses.
 Verify the calibration of each one of
the channels.

11. 3.0 The reader displays unexpected variation with
different wavelengths.
The reader’s lamp is
unstable.
 Replace the lamp with one that has
similar characteristics as the
original.

12. 4.0 The reader displays a gradual increase or decrease from
column to column.
Inappropriate
calibration of the
plate’s controlling
motor.
 Calibrate the plate’s controlling
system so that at each step the
wells remain exactly aligned with
the lighting system.

13. 5.0 Low reproducibility.
• Sample homogeneity.
 Mix the reagents before use. Allow these to
equilibrate to room temperature.
•Incorrect pipetting procedure.  Ensure pipette’s tips are changed between
samples and that excessive liquid inside is
removed
 Check the calibration.
•Reader not calibrated.
•Reading without sufficient
warm up time.
•Expired reagents.
•Insufficient or Inefficient
washing
 Wait until the reader has warmed up to its
operating temperature.

14. 6.0 Misaligned light beam.
•The reader was transferred
or moved without using
 Call the specialized service technician.
the necessary precautions.
•The light source has been
changed and the
replacement has not been
installed or aligned
incorrectly
 Align according to manufacturer instructions

15.  Thank you.