STANDARD OPERATING PROCEDURE
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
Prepared by TP Lim
BACKGROUND
The ELISA is an immunoassay of substances such as drugs, hormones and proteins in
body liquids.
Commercial ELISA kits certified to meet international health and safety standards
(such as the IVD CE for in-vitro diagnostic medical devices manufactured in the EU),
are available for a wide range of assays. Each of these kits is provided with specific
‘Instructions for Use’ pamphlets which the laboratory worker should read carefully
prior to performing the assay.
MATERIALS AND METHODS
The basic components in an ELISA kit are:
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Wash buffer
Sample diluent buffer
Reference standards and Controls
Conjugate
Substrate
Stop Solution
Coated 96-well microtiter plate
The basic steps in ELISA are:
a. Incubate diluted serum/plasma, standards and controls in the wells of the
microtiter plate
b. Wash the plate in an automated plate washer
c. Add conjugate and incubate
d. Wash the plate again in an automated plate washer
e. Add substrate and incubate
f. Add stopping solution
g. Read absorbance in a microtiter plate reader
Warnings and Precautions (to be observed by the laboratory worker when
performing the ELISA at Biosafety Level 2):
1) Wear protective disposable gloves, safety glasses and a laboratory coat.
2) All patient samples should be regarded as potentially contaminated and
treated as if capable of transmitting disease.
3) Do not smoke, eat or drink in areas where samples or kit reagents are
handled.
4) Never pipette by mouth, always use safety pipetting devices for all pipetting.
Pipettors should be (a) of ergonomic design, to reduce the risk of developing
repetitive strain injuries (RSI) to the operator, and (b) fully autoclavable.
5) ELISA kit components:
(i)
contain reagents manufactured from human blood
components. Although source materials provided
have been tested for the presence of antibodies to
Hepatitis B and C as well as antibodies to HIV, and
have been found to be negative, and since no test
method can offer absolute assurance that these
pathogens
are
not
present,
observe
ALL
recommended precautions for the handling of blood
derivatives.
(ii)
should be handled with care since some reagents
may be harmful by inhalation, if swallowed, or in
contact with eyes and skin. Refer to the MSDS for
these
chemical
components
for
handling
precautions.
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If inhaled, remove to fresh air. If not
breathing,
get
immediate
medical
attention.
If swallowed and if person is conscious,
wash out mouth with water. Get immediate
medical attention. Do not induce vomiting
unless directed by the medical personnel.
If in contact with eyes, hold eyelids apart
and flush eyes with plenty of water. After
initial flushings, remove any contact lenses
and continue flushing for at least 20
minutes. Seek medical help.
If in contact with skin, wash immediately
with plenty of water. If symptoms occur,
get medical attention.
6) Decontaminate wash waste (collected in a stoppered bottle during the
microtiter plate washing steps) with 10% bleach before flushing down the
sewage system.
7) Decontaminate disposables with 10% bleach before disposal.
8) In case of biological spills: Immediately remove biological spillage with
absorbent paper towels and swap the contaminated area with 10% bleach or
70% alcohol.
9) At the end of assay: Swap work surfaces with 10% bleach or 70% alcohol and
wipe dry with disposable paper towels.
10) Disposed off materials used to clean spills and work area swaps, including
gloves as biohazardous waste.
11) Wash hands thoroughly after performing the tests.
EQUIPMENT SAFETY MAINTENANCE
1. A Decontamination Kit should consist of the following items:
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70% isopropyl alcohol
Spray bottle
Deionized/distilled water
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Sterilization bags
Absorbent paper towels
Yellow biohazard trash bags
Lab coat and disposable gloves
Surgical masks
Safety glasses
2. Laboratory worker performing the decontamination process must be familiar
with the basic setup and operation of the equipment.
3. Always wear rubber gloves when decontaminating equipment, keeping
gloved hands away from eyes, mouth and nose.
4. Do not eat nor drink while decontaminating.
5. As mucous membranes are prime entry routes for infectious agents, wear
eye protection and a surgical mask when there is a possibility of aerosol
contaminations.
A.
Pipettor
Decontamination: Put the entire pipettor into a sterilization bag and
place it into the autoclave at 121oC for 20 minutes. After autoclaving,
cool the pipettor down and leave to dry overnight before use.
B.
Microtiter plate reader
Decontamination: Wipe down the plate carrier and all exposed reader
surfaces with 70% isopropyl alcohol using absorbent paper towels.
Discard used gloves and paper towels in a biohazard bag.
C.
Microtiter plate washer
Decontamination:
(i)
(ii)
(iii)
(iv)
Wipe down the carrier and all exposed washer
surfaces with 70% isopropyl alcohol using
absorbent paper towels.
Remove the washer’s aerosol cover and wipe down
the inside of the cover, the plate carrier and the top
surface of the base.
Run the decontaminate program by following the
washer menu path:
Maintenance

Decontaminate

Connect
disinfectant and press <START> key  Running:
Decontamination/Disinfecting  Connect rinse
bottle and press <START> key  Running:
Decontamination/Rinsing
Discard used gloves and paper towels in a
biohazard bag after reassembling the washer unit