A brief outline of the technique ENZYME LINKED IMMUNOSORBENT ASSAY aka ELISA.

1. ENZYME LINKED
IMMUNOSORBENT ASSAY
E L I S A
P R E S E N T E D B Y
S H A R O N V I J A YA N A N D
B . P H A R M 3 R D Y E A R
C . L . B A I D M E T H A C O L L E G E O F P H A R M A C Y

2. CONTENTS
• HISTORY
• DEFINITION AND PRINCIPLE
• ENZYMES AND SUBSTRATES
• EQUIPMENT
• REAGENTS
• GENERAL PROCEDURE
• TYPES OF ELISA
• INTERPRETATION
• APPLICATION
• ADVANTAGES & DISADVANTAGES

3. HISTORY
1971, Peter Perlmann and Eva Engvall at Stockholm university in
Sweden and Anton Schuurs and Bauke Van Weeman in Netherlands
independently published papers on EIA/ ELISA
ELISA is also known as EIA – ENZYME IMMUNO ASSAY .

4. DEFINITION
ELISA is a highly sensitive and specific immunochemical technique
which is employed to assess the presence of specific proteins usually
AG/AB in the sample and its quantification .
PRINCIPLE : based on the specific AG and AB reaction . EIA uses an
enzyme linked AB as a marker for detecting specific proteins . The
enzyme linked AB binds to the specific protein to be detected .
Substrate/ chromogen specific for the enzyme is added which gives a
coloured end product .
ENZYME-AB-AG + colourless substrate coloured product

5. ENZYMES AND SUBSTRATE
ENZYMES – alkaline phosphatase
horseradish peroxidase
β-galactosidase
SUBSTRATE FOR HORSERADISH PEROXIDASE
 ABTS – (2,2’ azinobis [3-ethylbenzothiazoline-6-sulfonic acid] yields water soluble
green end point – absorbance 410nm and 650nm used
 OPD – (o-phenylenediamine dihydrochloride) yields water soluble yellow orange end
point- absorbacne 492nm
 TMB –(3, 3’,5 ,5’ – tetramethyl benzidine) yields blue colour –absorbance 370nm and
652nm –very sensitive – colour develops faster .
SUBSTRATE FOR ALKALINE PHOSPHATASE
 PNPP –( p-nitophenyl phosphate)

6. EQUIPMENT
Microtiter plate
Multi pipette
Microplate washer

7. REAGENTS
 Enzyme
 Substrate
 Coating buffer – 0.01M phosphate buffer + 0.15M NaCl
 Washing buffer – 0.01M phosphate buffer + 0.50M NaCl + 0.1%
tween20
 Blocking buffer – bovine serum albumin (BSA)
 Stop solution – o.5M sulphuric acid
SAMPLE – cell culture , biological fluids like plasma , serum , urine etc.
can be tested .

8. GENERAL PROCEDURE

9. GENERAL PROCEDURE
1. Add 50-100 µL of prepared standards and sample to wells. Cover plate and
incubate at room temperature for 2 hours.(COATING)
2. Thoroughly decant solution from wells . Wash wells 4 times using a squirt wash
bottle or an automated 96-well plate washer.(WASHING)
3. Add blocking agent to wells. Allow to react for some time .(BLOCKING)
4. Decant and wash .(WASHING)
5. Add 100 µL of diluted enzyme-AB conjugate to each well. Cover plate and incubate
at room temperature for 30 minutes.(DETECTION)
6. Decant and wash (WASHING)
7. Add 100 µL of chromogenic substrate to each well. Develop plate at room
temperature in the dark for 30 minutes.(OBSERVE COLOUR CHANGE )
8. Add 100 µL of stop solution to each well. The solution in the wells should change
colour.(STOPPING REACTION)
9. The plate must be evaluated within 30 minutes of stopping the reaction. Read the
absorbance of each well and plot graph against a standard diluted antigen

10. TYPES OF ELISA
 Direct ELISA
Indirect ELISA
Sandwich ELISA
Competitive ELISA

11. DIRECT ELISA

12. INDIRECT ELISA

13. SANDWICH ELISA

14. COMPETITIVE ELISA

15. INTERPRETATION
The ELISA assay yields three different types of data output:
1) Quantitative: ELISA data can be interpreted in comparison to a standard
curve (a serial dilution of a known purified antigen) in order to precisely
calculate the concentrations of antigen in various samples . The optical density is
plotted against log concentration to produce sigmoid curve .
2) Qualitative: ELISAs can also be used to achieve a yes or no answer indicating
whether a particular antigen is present in a sample, as compared to a blank well
containing no antigen or an unrelated control antigen.
3) Semi-Quantitative: ELISAs can be used to compare the relative levels of
antigen in assay samples, since the intensity of signal will vary directly/indirectly
with antigen concentration.

16. GRAPH

17. APPLICATIONS
• Determining serum AB concentration (HIV screening , west Nile
virus)
• Detection of presence of mycobacterium AB in TB infection
• Detecting food allergens such as milk, peanut, walnuts , almonds,
eggs etc.
• Screening of donated blood for viral contamination (HIV , Hepatitis
C, hepatitis B)
• Measuring hormone levels (ex. Human chorionic gonadotropin
hormone in pregnancy test )

18. ADVANTAGES &
DISADVANTAGES
ADVANTAGES
Reagents –cheap- longer shelf life
Highly specific and sensitive
No radiation hazards as in RIA
Easy to perform
Equipment is widely available
DISADVANTAGES
Enzyme activity may be affected by plasma constituents
False +ve / -ve results are also possible

19. THANK YOU.