1. ENZYME–LINKED IMMUNO
SORBENT ASSAY (ELISA)
PRESENTED BY PRANZLY

2. CONTENT
• INTRODUCTION
• PRINCIPLE
• MATERIAL REQUIRED
• REAGENTS
• TYPES
 NON-COMPETITIVE ELISA
o DIRECT ELISA
o INDIRECT ELISA
o SANDWICH ELISA
 COMPETITIVE ELISA
• ELISA RESULT
o QUALITATIVE
o QUANTITATIVE
o SEMI-QUANTITATIVE
• PRECAUTIONS

3. INTRODUCTION
• The term ELISA was first used by Engvall & Perlma
in 1971.
• high sensitivity
• useful & powerful method in estimating ng/mL to
pg/mL ordered materials in the solution.
• Similar To RIA, Except No Radio-labelling.
• Alkaline phosphatase, horseradish peroxidase and
beta-galactosidase are the enzymes used in the EIA
tests.

4. Why known as Enzyme Linked
Immunosorbent Assay
1.Antigen of interest is absorbed on to plastic surface
(sorbent).
2. Antigen is recognized by specific antibody (immuno).
3. This antibody is recognized by second antibody
(immuno) which has enzyme attached (enzyme- linked).
4. Substrate reacts with enzyme to produce product,
usually coloured

5. PRINCIPLE
• Lock and key concept
• Antigen key
• Antibody lock
• Key fits into the lock
• Enzyme conjugate substrates
• Bound to a secondary antibody that bind with the
Ag-Ab complex

6. EQUIPMENTS
MICROWELL PLATE
•FLAT BOTTOM
POLYSTYRENE
CONTAIN 96 WELLS
HOLDING 350µL
EACH
MULTIPIPETTE
8-CHANNEL 100µL
PIPETTE IS A GOOD
HELP FOR EVEN
SMALL SCALE
WORK
WASHING DEVICE
MANUALLY
OPERATED
PREVENT
CONTAMINATION
MICROPLATE WASHER
VERY EFFICIENT

7. REAGENTS
COATING
BUFFER
0.01M
phosphate
buffer
+
0.15M NaCl
WASHING
BUFFER
0.01M
phosphate
buffer
+
0.50M NaCl
BLOCKING
BUFFER
BOVINE
SERUM
ALBUMIN
CHROMOGENIC
SUBSTRATE
TRIMETHYL
BENZIDINE
STOP
SOLUTION
0.5M H2SO4
ENZYME
HORSERADISH
PEROXIDASE
ALKALINE
PHOSPHATASE

8. TYPES
NON-COMPETITIVE ELISA
DIRECT ELISA
INDIRECT ELISA
SANDWICH ELISA
COMPETITIVE
ELISA

9. DIRECT ELISA
PROCEDURE
1. Apply a sample of known antigen to a surface
2. Enzyme HRPO (Horseradish peroxidase) linked primary antibody is
applied to the plate
3. Washed after this only antigen antibody complexes remain attach
4. Apply a substrate which is converted by the enzyme to elicit a
chromogenic signal
ADVANTAGE
 Quick methodology since only primary antibody is used
 Cross reactivity of secondary antibody is eliminated
DISADVANTAGE
 Labelling of every primary antibody is time consuming and expensive
• little signal amplification

11. INDIRECT ELISA
PROCEDURE
1. Antigen is added to the plate
2. Added blocking buffer
3. Suitable primary antibodies added
4. Secondary antibody-HRPO(Horseradish peroxidase) then added which recognizes and
binds to primary antibodies
5. Tmb substrate is added and is converted to detectable form
ADVANTAGE
 With variety of labelled secondary antibody are available
 Versatile since many primary antibody can be made in primary Species and the same
labelled secondary antibody can be used for detection
DISADVANTAGE
 Cross reactivity mein occur with secondary antibody resulting in non specific signal
 and extra incubation step is required in the procedure

13. SANDWICH ELISA
1. Plate is coated with suitable antibody
2. Blocking buffer is added
3. Sample is added to plate so antigen is bounded by capture antibody
4. A suitable biotin labelled detection antibody is added to the plate
5. Enzyme HRPO(Horseradish Peroxidase) is added and binds the biotin
labelled detection antibody
6. TMB(trimethyl benzidine) substrate is added and converted by HRPO to
coloured product
ADVANTAGE
• High Specificity
• Suitable for complex sample since the antigen does not require purification
prior to measurement

15. COMPETITIVE ELISA
1. Solid face coated with antibody
2. Add unknown amount of unlabeled antigen and known amount of labelled
antigen
3. Free and labelled antigen are present
4. Colour formation by oxidation of substrate into a coloured compound
ADVANTAGE
• Suitable for complex (crude/impure) sample, since the antigen does not
require purification prior to measurement.
DISADVANTAGE
• Each antigen may require a different a different method to couple it to the
enzyme.

16. oELISA RESULT
oQUALITATIVE
o Determines antigen or
antibody is present or
absent in a sample as
compared to a blank
well containing no
antigen or unrelated
control antibody
oQUANTITATIVE
oELISA data can be
interpreted in comparison
to a standard curve in order
to precisely calculate the
concentration of antigen in
various sample.
o
oSEMI-QUANTITATIVE
oCan be used to compare
the relative levels of
antigen in assay samples
since the intensity of
signal will vary directly
with antigen
concentration.

17. PRECAUTIONS
 Negative Control with strong signal
 Positive control with no signal
 Microwell plates not coated properly
 Reagents applied in wrong order/ step omitted
 Elisa with weak signal
 Use exchange type pipette
 Do not use same reservoir for multiple reagents
 Improper coating gives false positive result