ELISA Instrument

Quality assurance
Dr. Ali Mirjalili
 Equipment required for ELISA testing
1. Microplate reader.
2. Microplate washer.
3. Liquid dispensing system (multi-channel
pipettes may be used).
4. Incubator to incubate the plates.
 Microplate Reader
wavelength range to that used in ELISA,
generally between
400 to 750 nm (nanometres)

Some readers (ultraviolet range between

340 to 700 nm.

INSTALLATION REQUIREMENTS
1. A clean, dust free environment.
2. A stable work table away from equipment that vibrates
(centrifuges, agitators).
3. An electrical supply source, which complies with the
country’s norms and standards.
 Calibration of the microplate reader

• Executed by a technician or trained engineer

• Following the instructions provided by each manufacturer.
• It is necessary to have a set of grey filters mounted on a plate of equal geometric
size
• Calibration plates are equipped with at least three pre-established
Optic density values within the measurement
ranges; low, medium, and high value.
 Calibration of the microplate reader

1. Place the calibration plate on the equipment. 4. If the instrument does not have a calibration plate, verify
2. Carry out a complete reading with the calibration plate. it by placing a coloured solution in the wells of a plate
Verify if there are differences in the readings obtained and immediately carry out a complete reading. Then
from well to well. If this is the case, invert the plate (180°) invert the plate 180° and read the plate again. If both
and repeat the reading to rule out that differences are readings display identical, average values in each row,
attributed to the plate itself. In general, it is accepted the reader is calibrated.
that the instrument does not need further calibration if 5. Verify that the reader is calibrated, column by column.
the plate results are as expected at two wavelengths. Place a clean, empty plate and carry out a reading. If
3. Verify if the reader requires calibration. If so, proceed there is no difference between each of the average
with the calibration following the routine outlined by reading of the first to the last column, it can be assumed
the manufacturer, verifying that the reading’s linearity that the reader is calibrated.
is maintained as rigorously as possible.

ELISA Check
Basic maintenance
Frequency: Daily
1. Review that optical sensors of each channel are clean.
If dirt is detected, clean the surface of the windows of
the light emitters and the sensors with a small brush.
2. Confirm that the lighting system is clean.
3. Verify that the reader’s calibration is adequate. 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.
If not, invert the plate and repeat the reading in order
to determine if the deviation originated in the plate or
the reader.
4. Examine the automatic drawer sliding system. It must
be smooth and constant.
Preventive maintenance
Frequency: Quarterly
1. 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.
2. Clean the detectors’ optical systems and the lighting
systems.
3. Clean the plate drawer.
4. Verify the alignment of each well with the light emission
and detection systems.
 Microplate Washer
• The microplate washer has been designed to perform washing operations
in the ELISA technique
Comprise:
• Control subsystem.
• Supply subsystem
• Extraction or suction system
• Advance sub-system
 Microplate Washer
• INSTALLATION REQUIREMENTS
1. A clean, dust-free environment.
2. A stable work table located away from equipment
that generates vibrations, (centrifuges, and agitators).
3. An electric outlet in good condition with a ground pole.
 Microplate Washer Calibration
• Washer calibration The microplate volume distributed is as close as possible
washer is critical for guaranteeing to the maximum capacity of the well;
that the ELISA technique performs confirm that all the wells are filled
as expected. uniformly (at the same level). Verify that
the distributing needles are clean (free
• Position of the needles (supply and of obstructions).
aspiration head). •• Vacuum. The suctioning system must
Situated very close to the well’s wall. be calibrated efficiently. If the vacuum is
too strong, the test can be altered. In
The suction needle should be located in fact, it could dry out the wells and
the centre of the well: considerably weaken the enzyme activity
A needle-base distance is maintained in in the wells and completely alter the test
the well, usually between 0.3 to 0.5 mm. result. The majority of washers function
The needles must never be allowed to with a vacuum ranging between 60 and
touch the bottom of the wells to avoid 70% of atmospheric pressure.
mechanical interferences
• Aspiration time.
• Distributed Volume. Check that the
 Microplate Washer Performance
Verification
• Washing process verification To (TMB/H 2 O 2 –
verify that the washing process is Tetramethylbenzidine/Hydrogen
done according to the Peroxide). Whatever conjugate
specifications of ELISA techniques, remains will hydrolyze the enzyme
manufacturers of ELISA tests have and the chromogen will change to
developed procedures to be blue. After stopping the reaction
carried out regularly. One of the with acid, the TMB will turn yellow
controls again. The resulting colour intensity
• is based on using the peroxidase is directly related to the washing
reagent, which is dispensed using a process efficiency.
pipette in the plate wells to be read
at 405, 450 and 492 nm. At once
the wells are washed and a
colourless substrate is added
ELISA Check
 Microplate Washer Maintenance
Basic maintenance Frequency: Daily 6. Verify that the body of the washer
1. Verify the volume distributed. has been cleaned. If necessary, clean
2. Test the filling uniformity. the exterior surfaces with a piece of
cloth, moistened with a mild
3. Verify the aspiration sub-system’s detergent.
efficiency.
4. Confirm the cleaning of the supply
and extraction needles.
5. Clean the washer with distilled
water after use, to remove
every vestige of salt in the supply and
extraction subsystems’ channels. The
needles may be kept submerged in
distilled water.
 Microplate Washer Preventive
Maintenance
• Preventive maintenance the manufacturer’s recommendations.
Frequency: Quarterly 4. Confirm the integrity of the
1. Disassemble and clean the channels electrical connector and the inter-
and connectors. connection cable.
Verify their integrity. If leaks or any 5. Clean the washer with distilled
vestiges of corrosion are detected, water after using it in order to remove
adjust and/or replace. every vestige of salt in the supply and
2. Verify the integrity of the extraction subsystems’ channels.
mechanical components. Lubricate 6. Verify the integrity of the fuse, and
according to the manufacturer’s that its contact points are clean.
instructions.
3. Test the adjustment of each one of
the subsystems. Calibrate according to
Pipettes and Best pipetting practice

Manual Manual Electronic Electronic

single multi- single multi-
channel channel channel channel
 Ways to optimize pipette performance
• Choose the right pipette for the job.
• Check for leaks or any other pipette malfunctions
• Choose the correct pipette tip
– Correct size
– Correct style
• Have pipettes calibrated and serviced regularly.
• Allow all liquids and equipment to equilibrate to ambient temperature
before beginning work.
• Pre-rinse the pipette tip by aspirating and dispensing the sample liquid
at least 3 times before aspirating a sample for delivery.
• Immerse the tip vertically into the sample liquid well clear of the
container walls and bottom and at a depth of approximately 2 – 5mm
below the meniscus.
 Tips to minimize pipetting errors
• Aspirate using a consistent speed, rhythm, and plunger
pressure.
• Hold the tip in the sample for 1 second after aspiration and
withdraw the tip slowly and smoothly without touching the
sides of the container.
• To dispense touch the pipette tip to the sidewall of the
container where sample is to be delivered just above the
liquid the sample is being dispensed into. Use consistent
speed, rhythm, and plunger pressure to dispense.
• Put the pipette in it’s stand between pipetting cycles to avoid
warming the pipette in your hand – this can affect the volume
of liquid dispensed.
 PIPETTING
GUIDE TO PIPETTING
• ONLY USE FIRST STOP !

• DO NOT DRIP

• DO NOT PRESS HARD INTO

WELL

• DO NOT USE TOO ACUTE

AN ANGLE

• MAKE SURE TIP TOUCHES

SIDE OF WELL AND LIQUID
 Pipetting tips

• Forward Pipetting • Reverse Pipetting

technique technique
• •

• Highly viscous fluid

• Avoid foaming
 Pipette preventive maintenance
• As part of Preventive
Maintenance, each Pipette
disassembled according to
manufacturer’s diagrams and
cleaned thoroughly with the
appropriate chemical solutions.
• All parts are carefully inspected,
piston is polished in most cases,
piston seal and o-ring are
replaced as well as all defective
and malfunctioning parts. [see
12Point Inspection
diagram]Thank You
 Pipette calibration
• Gravimetric
• Colourimetric
Thanks for your attention