GOOD

1. Enzyme-Linked
Immunosorbent Assay

2. INTRODUCTION
 Is a biochemical technique used mainly in
immunology to detect the presence of an
antibody or an antigen in a sample.
 ELISA is so named because the technique
involves the use of an immunosorbent, an
absorbing material specific for one of the
components of the reaction, the antigen or
antibody.
 ELISA is usually done using 96-well microtitre
plate suitable for automation

4. What is ELISA?
• Technique used to detect (assay) specific
molecules (e.g. proteins & carbohydrates) in
samples.
• Immunological technique: uses antibodies.
• Quantitative.
• Very sensitive.
• Commonly used in medicine and scientific
research.

5. Substrate
Primary
antibody
Enzyme
Secondary
antibody
Different antigens in sample
Coloured
product
PRINCIPLE

6.  The technique is divided into :
1- Competitive ELISA
2- Sandwich ELISA (also called direct ELISA)
3- Indirect ELISA

7. COMPETITIVE ELISA
 The labelled antigen competes for primary antibody
binding sites with the sample antigen (unlabeled).
 The more antigen in the sample, the less labelled
antigen is retained in the well and the weaker the
signal.
 Advantage:
 The ability to use crude or impure samples and still
selectively bind any antigen that may be present.
 Application:
 Used for the detection of human T-cell leukemia-
lymphoma virus type III (HTLV-III).

9. INDIRECT ELISA

10. COLOR OF SOLUTION &
COLOR OF SOLUTION ARE
COMPLEMENTARYCOLOR OF FILTER WAVELENGTH COLOR OF
SOLUTION
VIOLET 420 BROWN
BLUE 470 YELLOWISH BROWN
GREEN 520 PINK
YELLOW 580 PURPLE
RED 680 GREEN/BLUE

11. INDIRECT METHOD
 This method is largely used to measure
antibodies in almost all human infections
 Eg : HIV antibody detection
 In patients with AIDS, the human immuno
deficiency virus(HIV) produces specific
antibody.
 To detect the HIV antibody, indirect ELISA
method is used

13. SANDWICH METHOD

14. Eg : Assay of thyroid hormone (T4)

15. Reaction :
HRP
 H2O2 H2O + [O]
Diamino
oxidized
Benzidine
DAB
(colorless)
(brown color)

16. MULTIPLE & PORTABLE ELISA
 This new technique uses a solid phase made
up of an immunosorbent polystyrene rod with
8-12 protruding ogives.
 The entire device is immersed in a test tube
containing the collected sample and the
following steps (washing, incubation in
conjugate and incubation in chromogenous)
are carried out by dipping the ogives in
microwells of standard microplates pre-filled
with reagents.

17.  The advantages of this technique are as follows:
 The ogives can each be sensitized to a different
reagent, allowing the simultaneous detection of
different antibodies and/or different antigens for multi-
target assays
 The sample volume can be increased to improve the
test sensitivity in clinical (saliva, urine), food (bulk milk,
pooled eggs) and environmental (water) samples
 One ogive is left unsensitized to measure the non-
specific reactions of the sample
 The use of laboratory supplies for dispensing sample
aliquots, washing solution and reagents in microwells
is not required

19. APPLICATIONS OF ELISA
 Serum Antibody Concentrations
 Detecting potential food allergens
(milk, peanuts, walnuts, almonds and eggs)
 Disease outbreaks- tracking the spread
of disease
e.g. HIV, bird flu, common, colds, cholera,
STD etc
19

20.  Detections of antigens
e.g. pregnancy hormones, drug
allergen,
, mad cow disease

•human chorionic gonadotropin (HCG), the
commonly measured protein which indicates
pregnancy.
•A mixture of purified HCG linked (coupled) to
an enzyme and the test sample (blood, urine,
etc) are added to the test system.
•If no HCG is present in the test sample, then
only HCG with linked enzyme will bind.

21.  The more HCG which is present in
the test sample, the less enzyme
linked HCG will bind.
 The substrate the enzyme acts on is
then added, and the amount of
product measured, such as a change
in color of the solution.

22.  Detection of antibodies in blood sample
for past exposure to disease
e.g. Lyme Disease, trichinosis, HIV, bird
flu
 ELISA can also be used in toxicology as
a rapid presumptive screen for certain
classes of drugs.

23.  To monitor diabetes, glucose concentrations will be
checked.
 Bacterial left-over in milk can be determined with an
ELISA test.
 ELISA assays are as well employed in foodstuff
protection through signifying the occurrence of
salmonella
 ELISA assays can also be utilised to diagnose
several cancers (eg: bladder cancer)

24. ADVANTAGES OF ELISA
 ELISA tests are generally relatively accurate
tests.
 Highly sensitive and specific
 Antigens of very low or unknown concentration
can be detected since capture antibody only
grabs specific antigen
 Generally safe: do not require radioactive
substances, contains diluted sulfuric acid
 Used in wide variety of tests

25. DISADVANTAGES
 Only monoclonal antibodies can be used as
matched pairs
 Monoclonal antibodies can cost more than
polyclonal antibodies
 Monoclonal antibodies are more difficult to find
 Negative controls may indicate positive results if
blocking solution is ineffective [secondary Ab or
antigen can bind to open sites in well]
 Enzyme/substrate reaction is short term so
microwells must be read as soon as possible