1. TEST FOR DETECTION OF
PLANT VIRUS
BY
R.LOKESH
I -M.sc.Agriculture
Plant pathology
Annamalai university

2. ANTIBODY
 An antibody, also known as an immunoglobulin, is a large,Y-shaped protein used
by the immune system to identify and neutralize foreign objects such as
pathogenic bacteria and viruses.
 The antibody recognizes a unique molecule of the pathogen, called an antigen
 What are antibodies made of?
 A typical antibody molecule is composed of four polypeptide chains, two identical
heavy chains and two identical light chains.
 Parts of both the heavy and light chains usually combine to form the antigen-
binding sites.

3. di-sulfide bond
• Each chain is linked by di-sulfide bond

5. ANTIGEN
 Any substance that causes the body to make an immune response against that
substance.
 Antigens include toxins, chemicals, bacteria, viruses, or other substances that
come from outside the body.

6. Enzyme-linked immunosorbent
assay (ELISA)
 ELISA stands for enzyme-linked immunoassay. It is a commonly used laboratory
test to detect antibodies in the blood.
 ELISA gives qualitative and quantitative information about the presence of a
antigen or antibody.
 ELISA is widely used as a diagnostic test for many viral disease including AIDS.
 ELISA is a colorimetric test that uses antibodies and colour changes to identify a
substances.

7. Advantages of ELISA
 ⚫ It is a simple technique, easy to learn and perform
 Fast and easy method compared to other techniques
 ELISA is 1000 times sensitive for the detection of viruses, than any
conventional methods .
 Large amount of samples can be tested in relatively very short time (2-3 hr)
 Detection kits available commercially
 Very small volumes (0.1 µl) of virus extract may be sufficient.

8. TYPES OF ELISA
Direct ELISA
Indirect ELISA
Sandwich ELISA
Competitive ELISA

9.  INVENTED BY –PETER PERLMANN AND EVA ENGVALL
 ON - 1971

10. MICROTITER PLATE
 There are 96 well plate each well is microtiter well.

11. Solutions used
 COATING BUFFER – for antigen coating
 WASHING BUFFER – to remove unbound material
 SULFURIC ACID – to stop reaction

12. WE GOING TO DISCUSS ABOUT
INDIRECT ELISA
SANDWICH ELISA

13. INDIRECT ELISA

14. STEP : 1
 Surface of the well is
coated with a specific
antigen

15. STEP : 2
 Mixture of antibodies are
added to the well .
 If the antibody of interest is
present , it will bind to the
antigen
 Washing steps removes the
unbounded antibodies

16. STEP : 3
 Enzyme-linked antibodies that can
bind to the antibody of the
interest are added into the well.
 If the antibody of interest is bound
to the antigen, the enzyme –linked
antibody will bind to the antibody-
antigen complex.
 Washing steps removes the
unbounded antibodies

17. STEP : 4
 Unbound antibodies are removed by
washing .
 The substrate specific to the enzyme is
then added .
 If the enzyme is present the substrate
will react and cause the colour
change .
 This signifies the presence of antibody
of interest.
 This can be investigated and
quantified using the
spectrophotometry.
 For ex : horseradish peroxidase
enzyme reacts with the substrate TMB
gives blue colour product.

19. SANDWICH ELISA

20. SANDWICH ELISA
 It is generally used to detect the presence of
antigen rather than the antibody

21. STEP : 1
 The antibody specific to the
antigen is attached to the bottom
of the well.

22. STEP : 2
 Some sample containing the
antigen, such as prepared plant
samples, is placed into the well.
 The antigen then binds to the
antibody.

23. STEP : 3
 Now an enxyme linked
monoclonal antibody is added .
 It binds to the antigen attached to
the adsorbed antibody.

24. STEP : 4
 The well is then washed to remove anything
unbound .
 The substrate added to react with the
enzyme, producing the coloured product.
 This can be investigated and quantified using
the spectrophotometry.
 (3,3′,5,5′-Tetramethylbenzidine or TMB is a
chromogenic substrate used in staining
procedures in immunohistochemistry as well
as being a visualising reagent used in
enzyme-linked immunosorbent assays
(ELISA)).
4

27. How long does ELISA test results take?
How long does it take to get ELISA test results?
Depending on what the test is being used for, you may
get results as quickly as about 24 hours if the test is done
locally. However, there are some tests that may take days
to weeks.

28. THANK YOU