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Standard Operating Procedure

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
-------------------------------------------------------------------------------------------------------------PREPARED BY: TP LIM

1. Introduction
The ELISA is an enzyme immunoassay of substances such as drugs, hormones and proteins in
body liquids. It is a biochemical technique used mainly in immunology to detect the presence of an
antibody or an antigen in a sample.
Used widely as a diagnostic tool in medicine, 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.
This SOP incorporates safety protocols into the regular procedures of the ELISA

2. Scope
This generic SOP is intended to provide staff and students in the Department of Medicine with
general guidance on how to safely perform the ELISA in the laboratory.

3. Responsibility and Accountability

The laboratory worker performing the ELISA should be adequately trained in safe BSL2 laboratory
techniques. It is the responsibility of the worker to use ELISA diagnostic kits in accordance with the
principles of good laboratory practice. Each ELISA kit is provided with specific Instructions for Use
pamphlets which the laboratory worker should read carefully prior to performing the assay. It
remains the responsibility of the worker to ensure that any biological and chemical wastes so
generated be disposed of appropriately according to department safety guidelines. The worker
should report accidents if any to the department Safety Officer and biological/chemical safety
committee member.

4. Potential Hazards
a) Human specimens used in ELISA such as plasma, serum, saliva and other human secretions
may potentially carry infections. The main blood borne infections of concern are Hepatitis B,
Hepatitis C and Human Immunodeficiency virus (HIV).
b) Exercise caution as ELISA kits contain reagents (e.g. Standards and controls) produced from
human blood components.
c) All chemicals included in the kit should be considered potentially hazardous. Some ELISA
protocols, for example, use chemicals such as dimethylsulphoxide, tetramethylbenzidene (in
the substrate solution), sulphuric acid (as stopping solution), etc which are harmful and
corrosive. Refer the manufacturers MSDS relating to the safe handling, use and disposal of
these chemicals.

d) Because repetitive pipetting is involved, the worker risk developing repetitive strain injuries
(RSI)

5. Personal Protective Equipment

Wear protective clothing such as laboratory coat, safety goggles, face shield and disposable gloves
where appropriate.

6. Precautions
a) To minimize the risk of spread of infection, all blood and human materials used in ELISA should
be treated as potentially infectious.
b) Exercise caution as ELISA kits contain reagents produced from human blood components.
Observe ALL recommended precautions for the handling of blood derivatives
c) Standard precautions include hand hygiene, wearing of gloves and use of other appropriate
protective clothing and equipment as well as safe disposal of waste.
d) All workers with a significant risk of exposure to human blood should be immunized against
Hepatitis B before starting work.
e) 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 specific handling precautions.
f) Some reagents contain preservatives such as sodium azide which may react with lead and
copper plumbing to form explosive metallic azides. Exercise caution during disposal.
g) Never pipette by mouth - always use safety pipetting devices for all pipetting.
h) To reduce the risk of developing repetitive strain injuries (RSI) to the operator, use pipettors
that are of ergonomic design. Pipettors must also be fully autoclavable
i) Do not eat, drink, smoke or apply cosmetics in the laboratory
j) Wash hands thoroughly with soap and water after work is completed

7. Materials and Methods

a)
b)
c)
d)
e)

Read the kit Instructions for Use carefully before starting the assay.
All assay procedure should be followed as recommended.
Do not use kit reagents after the expiry date.
It is not advisable to use reagents from different kit batches.
At the end of assay: Swap work surfaces with 10% bleach or 70% alcohol and wipe dry with
disposable paper towels.
Wash hands thoroughly after performing the tests.

f)

The basic components in an ELISA kit are:

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)
b)
c)
d)

Incubate diluted serum/plasma, standards and controls in the wells of the microtiter plate
Wash the plate in an automated plate washer
Add conjugate and incubate
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

8. Accidental Spills and Contamination

In case of kit component contamination:

o 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 30
minutes. Seek medical help.
o If in contact with skin, wash immediately with plenty of water. If symptoms occur, get
medical attention.
In case of biological fluid contamination:
o Immediately remove contaminated clothing.
o If blood or body fluids get in contact with skin, wash thoroughly with soap and water
o If eyes are splashed, rinse the affected area (with eyes opened) gently and
thoroughly with water
o If biological fluids get into the mouth, spit out and rinse mouth with water. Repeat
several times.
If blood or biological fluids are spilled on surfaces:
o Wear rubber gloves, eye protection and shoe covers if necessary
o Mop up as much of the spill as possible with absorbent paper towels
o Swap the contaminated area with 10% bleach disinfectant
o Put contaminated paper towels, used gloves and shoe covers in a sealed biohazard
bag and dispose as biohazard waste
o Wash hands well with soap and water.

9. Handling of Waste
a) Refer to kit instructions/MSDS for specific disposal procedures for any unused or leftover kit
components
b) If kit components contain preservatives such as sodium azide, flush with large amounts of
water during disposal.
c) Collect wash waste during microtiter plate washing in a stoppered bottle.
d) Decontaminate wash waste with 10% bleach before flushing down the sewage system with
plenty of water.
e) Decontaminate disposables with 10% bleach before disposal.
f) Disposed of solid materials used to clean spills and work area swaps, including gloves as
biohazard waste in a biohazard bag.

10. Equipment Safety Maintenance

1. A Decontamination Kit should consist of the following items:

70% alcohol
Spray bottle
Deionized/distilled water
Autoclave bags
Absorbent paper towels
Yellow biohazard trash bags

Lab coat and disposable gloves

Surgical masks
Safety glasses

2. Laboratory worker performing the equipment 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, drink, smoke or apply cosmetics 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:
i.
ii.
iii.

Put the entire pipettor into a sterilization bag

Place bag 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:
i.
ii.
iii.

Wipe down the plate carrier and all exposed reader surfaces with 70%
alcohol using absorbent paper towels.
Discard used gloves and paper towels in a biohazard bag and dispose as
biohazard waste
Wash hands thoroughly with soap and water.

C. Microtiter plate washer

Decontamination:
(i)
(ii)

(iii)

(iv)
(v)
(vi)

Wipe down the carrier and all exposed washer surfaces with 70%
alcohol using absorbent paper towels.
Remove the washers 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
Reassemble the washer unit
Discard used gloves and paper towels in a biohazard bag
Wash hands thoroughly with soap and water.