Detailed explanation on Serological detection techniques of plant viruses

1. Serological detection
techniques of plant viruses
N. H. SHANKAR REDDY
Ph.D., Plant Pathology
Annamalai University

2. • Serology is a branch of science which deals with immunology and reaction
pertaining to preparation and use of antiserum.
• In agriculture, Horticulture and Forestry, immunological techniques are used
extensively for quick an accurate detection of those pathogens which cause
diseases with variable or latent symptoms on the host plant

3. Serology
Based on antigen - antibody reaction
• Antigen and antibody reaction results in the formation of visible substrate, which
is perhaps due to the formation of bridges between the two and thus form an
aggregate which precipitate when big enough

4. Antigen
• Any substance which evokes the production of antibodies is called an antigens and
includes proteins, polysaccharides, lipids, carbohydrates, nucleic aids, enzymes, toxins
etc.
• Each antigen is made up of distinct sub-regions which have definite spatial and
electronic configuration.
• These restricted regions of an antigen stimulate the antigenic response and are the
regions to which antibodies are/get attached. These regions are called antigenic
determinants or Epitopes
Note – An epitope, also known as antigenic determinant, is the part of an antigen that is
recognized, specifically by antibodies
The part of an antibody that recognizes the epitope is called a paratope.

5. Antibody
• The antibody is a "Y"-shaped molecule that consists of
four polypeptide chains
• A specific protein formed in the blood of warm-blooded
animals in response to injection of an antigen (protein
or polysaccharide)
• Responsible for specific recognition and elimination
(neutralization) of antigens

7. TYPES OF ANTIGEN-ANTIBODY REACTIONS
Two broad categories of antigen-antibody reactions
1. Homologous reactions Homologous reaction is the reaction between an
antiserum and the antigen that caused its production resembles the fit between a
lock and key.
2. Heterologous reaction But heterologous reaction is the reaction between an
antiserum and an antigen closely resembling but not identical to the one causing
the production of the antibody

8. SEROLOGICAL TESTS
• The serological tests can be performed by combining antigen (virus) and its antibody
(antiserum) in several ways for the detection and identification of antigenic substances
and the organism that carry them
• Protein Based Techniques – These techniques detect the virus coat protein by
agglutination reaction or antigen-antibody reaction. Based on the phase in which these
reactions occur, these are further classified as
1. Liquid phase – precipitation test,
agglutination test,
Ouchterlony Double Immunodiffusion Test

9. 2. Solid Phase tests - Enzyme Linked Immunosorbent Assay (ELISA)
Sodium Dodecyl Sulphate – Poly Acrilamide Gel
Electrophoresis (SDS-PAGE)
Immunosorbent Electron Microscopy (ISEM)
Western Blot
Dot Immuno Binding Assay (DIBA)
Tissue Immuno Binding Assay (TIBA)

10. 1. Liquid phase
i. Precipitation test
• When antigen and antibody are mixed they combine and form a precipitate.
This precipitation or precipitin reaction is widely used in Plant Virology
• The extent of precipitate formed is dependent on a number of factors, e.g., salt
concentrations, pH, temperature and presence of interfering compounds and
the ratio of concentration of antibody and antigen is important

11. 1. Liquid phase
ii. Ouchterlony double diffusion test
• In the double diffusion test, high grade agar (0.9%
agarose or ion agar No.2) is prepared in buffer (pH 7.6)
containing 0.85% NaCl in a preservative such as 0.02%
sodium azide
• Various patterns of precipitation lines can be obtained
depending on the relationship between the antigen and
antibody. Because elongated viruses diffuse rather poorly
through agar, the techniques maybe modified either to
use lower concentration of agar (0.5%)

12. 1. Liquid phase
iii. Chloroplast agglutination test :
• This test can be performed on crude sap extracts of suspected virus-infected plants.
• Plant material is ground to produce a sap suspension of chloroplasts and cell
debris.
• Antiserum to the suspected virus is added and the mixture left for a few minutes on
the slide.
• If virus is present, cloudy chloroplast agglutination is visible in the liquid where
the antibody and the matching virus meet and adhere together.

13. iv. Latex agglutination test
• Antiserum (Ab) is adsorbed on to polystyrene
latex beads and then mixed with crude sap
extracts of suspected virus-infected plants
(antigen) in a round bottom micro titer plates
and left for a few minutes.
• Flocculation of the latex indicates positive
reaction of antibody-antigen combination. This
allows the detection of 100-1000 fold smaller
quantities of virus present in the crude extract
within 5-10 minutes.
• The technique is more sensitive than other
precipitation tests.
• When antigen concentration is low precipitation
does not become visible and in such cases
indirect tests are performed to find out the
combination between antigen and antibody.

14. 2. Solid Phase tests
i) ELISA
• Precipitin and agglutination require enough antibody and virus to be present ,but in ELISA
can detect viruses even very low concentration.
Types
1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA

15. 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 of EM
• 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

16. 2. Solid Phase tests
ii) SDS-PAGE
• Aim – Separation of proteins based on their
size and shape. Isolation, separation and
purification of proteins is required to study
their various functions in the organism.
• When proteins are separated by
electrophoresis through a gel matrix, smaller
proteins migrate faster due to less resistance
from the gel matrix. In SDS-PAGE, the use of
sodium dodecyl sulfate (SDS, also known as
sodium lauryl sulfate) and polyacrylamide gel
largely eliminates the influence of the
structure and charge, and proteins are
separated solely based on polypeptide chain
length.

17. 2. Solid Phase tests
iii) Immunosorbent electron microscopy (ISEM)
• In 1973 K.S. Derrick published his "serologically specific electron
microscopy" method, which was later simplified, improved, and renamed more
appropriately as immunosorbent electron microscopy (ISEM).
• The principle of immunosorbent electron microscopy (ISEM) is the selective
trapping of plant viruses on to electron microscope grids precoated with a
specific antiserum.
• The consensus is that ISEM is highly reliable (there are virtually no false
positives), as sensitive as ELISA, fast (results can often be obtained within one
or two hours) and operationally simple.

18. 2. Solid Phase tests
iv) Western blot
• Western blot is usually used for characterization of virus proteins rather than
for detection since it has the advantage of determining the serological and
molecular properties of the virus protein.
• In this method the virus protein antigens are transferred from polyacrylamide
gels in which they were previously separated by electrophoresis to
nitrocellulose or nylon membranes.

19. 2. Solid Phase tests
v) Dot immune binding assay (DIBA)
• The dot immunoblotting assay (DIBA) requires a simple and easier method to
prepare and apply the samples on nitrocellulose or nylon membranes.
• The samples containing the virus antigens are prepared by grinding tissues in
Tris-buffered saline and the extracts are applied directly on the membrane.

20. 2. Solid Phase tests
vi) Tissue immune binding assay (TIBA)
• The tissue immunoblotting assay (TIBA) can be used to detect virus antigens
in plant tissues such as leaf, stem, bulb, tuber, root and fruit or insect vectors
of plant viruses.

21. Video available at you tube
Channel – Geeky Researcher