Serological Techniques in Detecting Plant Virus

1. SEROLOGICALVIRUS
DETECTION
Roselyn G.Andamon
Graduate student
Visayas State University, Baybay City, Leyte

2. • Diagnosis and identification of plant
virus through symptoms is
impossible because symptoms
caused by viruses vary according to
the plant variety involved, the
environmental conditions, the strain
of the virus, and sometimes the
disease infection cab be caused by
two different viruses
• Symptoms sometimes may be very
slight and symptomless

3. • Over the last decades
laboratory-based has
been developed.
• However, SEROLOGY
is the most specific and
easiest methods to
obtain a rapid and
precise
identification/detection

4. SEROLOGY DEFINITION:
 The science that studies the reaction between antigens and
antibodies in serum
 Serologic testing is used to identify unknown antigens such as
viruses. It uses known antibodies, called ANTISERUM, to identify
an unknown antigen (virus)
 The great value of serological methods for plant virus
identification is based on the specific reaction between the viral
antigens and their specific antibodies
 Antigen-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

5. PRINCIPLE:
The principles of serology are simple
If an antigen is parenterally introduced into the body of a
rabbit, it may stimulate the formation of specific
antibodies.The blood serum of the rabbit contains those
antibodies which may specifically react in some
observable way with the antigen that stimulated their
formation
Plant viruses can be used as antigens and, in a suspected
plant, their presence can be demonstrated with
homologous antisera.

6. ANTIGEN
• An antigen (Ag) is a molecule that has the
ability to induce an immune response (to
produce an antibody) in the host organism
• Antigens are typically proteins, peptides, or
polysaccharides. Lipids and nucleic acids can
combine with those molecules to form more
complex antigens. Parts of bacteria, viruses,
and other microorganisms can be an antigen.
• each antigen is made up of distinct sub-
regions which have definite spatial and
electronic configuration and it stimulate the
antigenic response and are the regions to
which antibodies are/get attached. These
regions are called antigenic
determinants or EPITOPES
EPITOPES- are the actual stimulus for the
production of particular antibody and are the
combining sites of antibody.
Video presentation about
epitope and paratope

7. ANTIBODY
• a protective protein produced by the
immune system in response to the
presence of a foreign substance, called an
antigen
• Antibodies have combining sites on their
surfaces depending upon shape, charge
and hydrophobicity to the antigenic sites
and number depends upon size and
complexity of antigen
• E.g.TMV have 2100 similar protein sub-
units, each active have the same antigenic
determinants

8. CHEMICAL NATURE OF AN ANTIBODY
From the chemical perspective, an antibody (Ab) is a protein
composed of four polypeptide chains, including two light chains and
two heavy chains.The four chains jointly form a 'Y' shaped structure

9. ANTIBODY-ANTIGEN INTERACTION
• When a foreign antigen enters the
body, it stimulates the immune
system to produce antibodies. The
protective antibody molecules help
the body fight against the antigen.
• Antibodies recognize antigens
(usually proteins) based on their
structure as well as content, and
bind to only a small part of an
antigen, known as the EPITOPE,
OR ANTIGENIC
DETERMINANT *
• Each type of antibody binds to one unique epitope,
because of the unique antigen-binding site of an
antibody. In a word, an antibody binds to a particular
Antigen-Antibody specific binding

10. Biological Functions
• From the biological perspective, an antibody is a protective protein
produced by the immune system in response to the presence of an
antigen.
• More specifically, antibodies are produced by plasma cells that are
developed from B cells, or B lymphocytes, which are a type of white
blood cells.
• When a B cell becomes activated due to a particular antigen, it
differentiates into the antibody-secreting cell, plasma cell.
• Plasma cells release antibodies that specifically bind to the
corresponding antigen.

11. CLASSFICATION OF ANTIBODY
• An antibody is also known as an immunoglobulin (Ig).
• In humans, there are five primary classes of antibodies, with each class playing a distinct role in the
immune response.
• These classes are identified as IgM, IgG, IgA, IgE, and IgD, which differ in the constant region of their
heavy chains

12. PRODUCING ANTIBODIES
• The special properties of antibodies is the
reason why they are a useful tool for
scientific research, disease diagnosis
treatment, and other areas.
• There is a growing demand for antibodies
due to their wide applications.
• The Man-made antibodies are primarily
divided into two types:
a. monoclonal antibody (mAb) and
b. polyclonal antibody (pAb)

13. MONOCLONAL ANTIBODIES
• Burnet (1959) put forth the clonal selection hypothesis:“ Each antibody forming
cell (B-lymphocyte) is committed to the production of one type of anitbody
molecule, which have the potential to react with one or at most few structurally
similar epitopes
• But attempts to culture B-lymphocytes failed because these cells can not be
cultured
• Kohler and Milstein (1975) showed somatic hybridization- between B-
lymphocytes (antibody producing cells) and myeloma cells and rising hybrids
called HYBRIDOMA.
• The hybrid cell has the ability to be cultured indefinitely in vitro.
• Antibodies produced by single hybridoma are identical and specific for a single
epitope
• This technique is called HYBRIDOMA TECHNOLOGY- which have the potential
for producing an unlimited quantity of monospecific or monoclonal antibodies

14. HYBRIDOMATECHNOLOGY

19. Video Presentation: HYBRIDOMA TECH
Video presentation of the actual equipment in producing monoclonal antibodies

20. POLYCLONAL ANTIBODIES
• POLY- many
• Describe a collection of antibodies which
are grown from different B cells
• This makes them capable of recognizing
multiple epitopes on the same antigen

21. Video presentation: Polyclonal Antibody

22. Polyclonal Antibody
Where can we buy Antisera ofViruses:
• Specific plant virus antisera have
been produced in many different
PlantVirus Research Laboratories
throughout the world.
• Often, these collections have served
as a major source of antisera for
commercial companies that sell
plant virus antisera and diagnosis
services on an international basis
• The PVLab/UFC in the State of
Caera, Brazil has been given an
improtant collaboration in the
production of antisera for tropical
plant viruses and in the
development and adaptation of
serological techniques for plant
virus identification since 1970-
decade

25. Importance of Virus purification
• It is necessary to purify viruses to study virus
STRUCTURE,VIRUS REPRODUCTION and
others
• Due to the virus features such as larger size
than proteins, more stable than cell
components and presence of surface proteins,
techniques used for virus purification are
similar to the techniques used for isolation
and purification of proteins and cell organelles
TECHNIQUES INVIRUS
PURIFICATION:
1. Density Gradient Centrifugation
2. Precipitation
3. Denaturation of Contaminants
4. Enzymatic Digestion of host cell constituents

26. Importance of Virus purification
1. DENSITY GRADIENT CENTRIFUGATION
• Involves sedimentation of particles in a density gradient medium
• Separates particles based on their bouyant densities
• This method allows separation of sub-cellular particles,
macromolecules, viruses, etc. and measurement of
sedimentation coefficients
2 METHODS ARE:
A. Rate Zonal Centrifugation
B. Isopycnic or equilibrium gradient
centrifugation

27. Methods ofVirus purification
A. RATE ZONAL CENTRIFUGATION
• Sample is centrifuged in a preformed
gradient
• Density gradient is prepared in a centrifuge
tube before centrifugation
• Various substances are used as gradient
material include sucrose, CsCl, cesium
acetate, glycerol, ficoll and ludoxx (silical
sols)
• The highest density solution is at the bottom
of centrifuge tube whereas lowest density
solution is at the top of centrifuge tube
• The virus sample to be separated is layered
on top of gradient

28. Methods ofVirus purification
B. Isopycnic or Equilibrium
Density Gradient
Centrifugation
• The sample to be separated is
dissolved in a solution of cesium
chloride or cesium sulfate
• This mixture is distributed
uniformly in a centrifuge tube
• Centrifuge is carried out at a high
speed for a long time (48 hrs)
• The cesium salt redistribute
under the influence RCF to form
a continuously increasing gradient
from op to bottom

29. Methods ofVirus purification
Video presentation
on how to purify
virus particle
2. Precipitation
• Although after centrifugation some cell
components still remain in the virus
preparation
• Viruses are separated from cellular
components by adding ammonium sulphate or
polyethylene glycol
3. Denaturation of Contaminants
• The remaining cellular impurities may be
treated with pH or temperature that will
denature them
• Organic solvent like butanol and chloroform
can be added to remove lipids by emulsifying
them ie. Breaking them into tiny pieces

30. 1. Enzyme-Linked Immunosorbent Assay (ELISA)
a. Direct ELISA
b. Indirect ELISA
c. Triple Antibody Sandwich (TAS-ELISA)
d. Protein A-sandwich (PAS-ELISA)
e. Immune Precipitation ELISA (IP-ELISA)
f. Rapid Immunochromatographic tests for field and
laboratory diagnosis
g. Immune Capture Polymerase Chain Reaction (IC-PCR)
2. Immune Precipitation Polymerase Chain Reaction (IP-PCR)
3. Immunoblotting methods
a. Western blot
b. Dot blot or Dot Immuno binding assay (DIBA)
c. Tissue Blot Immune Assay (TIBA)
4. Serologically specific electron microscopy (SSEM)
5. Double Immunodiffusion
SEROLOGICAL METHODS

31. I. ELISA
• Enzyme-Linked Immunosorbent Assay
(ELISA) is a biochemical technique that
uses antibodies and color change to
identify a substance
• It is the most sensitive and reproducible
technologies available.These assays are
rapid, simple to perform and easily
automated
PRINCIPLE
• Virus antigens are recognized by their specific antibodies (IgG) in
association with colorimetric properties
• Consists of detecting the antigen-antibody interactions by
enzyme induced color reaction

33. Equipment

35. GENERAL PROCEDURE**

36. Video presentation on how to prepare samples
Video presentation on how to use ELISA kit

37. A. DIRECT ELISA

38. B. INDIRECT ELISA

39. Video presentation on direct and indirect ELISA

40. C.Triple Antibody Sandwich (TAS-ELISA)
Video presentation on TAS ELISA

41. D. Protein A-sandwich (PAS-ELISA)

42. E. Immune Precipitation ELISA (IP-ELISA)

43. F. Rapid Immunochromatographic tests
for field and laboratory diagnosis
Video presentation

44. G. Immune Capture Polymerase Chain Reaction
(IC-PCR)
Video presentaion on the protocol of PCR

45. 2. Immune Precipitation Polymerase Chain
Reaction (IP-PCR)

46. a. Western blot
b. Dot blot or Dot Immuno binding assay (DIBA)
c. Tissue Blot Immune Assay (TIBA)
3. Immunoblotting methods

47. a. Western blot
Video presentation on actual procedure of western blot

48. b. Dot blot or Dot Immuno binding assay (DIBA)
Video presentation on the actual procedure of DOT blot

49. C. Tissue Blot Immune Assay (TIBA)

52. 4. Serologically specific electron
microscopy (SSEM)
Video presentation on what is Electron microscopy

53. 5. Double Immunodiffusion
Video presentation