Serological tests involve diagnostic procedures for identifying antibodies and antigens in a patient's blood sample. Serology definition tells that Serological tests could be used to diagnose infections and autoimmune disorders, as well as to see whether a person is resistant to these kinds of diseases and for a variety of other purposes, including assessing a person's blood type.

1. UNIVERSITY OF AGRICULTURAL SCIENCES, BANGALORE
COLLEGE OFAGRICULTURE V. C. FARM MANDYA
Reddy Kumar A V
PAMM3005
Dept. of Plant Pathology
Immuno/ serological assays(Slide agglutination tests, tube
precipitation test, double agar diffusion test, ELISA and Dot
immuno binding assay) for detection of plant viruses.
PRESENTATION

2. INTRODUCTION

3.  Any substance which evokes the production of antibodies is called an
antigens and includes proteins, polysaccharides, lipids, carbohydrates,
nucleic acids, enzymes, toxins etc. Each antigen is made up of distinct
sub-regions which have definite spatial and electronic configuration.
 The regions to which antibodies are/get attached. These regions are
called antigenic determinants or Epitopes.
 Epitopes: 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.
 A single epitope gives rise to a monoclonal antibody (Mab) and many
epitopes of different types produces polyclonal antibodies.
ANTIGEN

4. Antibody
Antibodies are a large family of glycoproteins.
Antibodies present in the antiserum are globulin
and two types are relevant in serological
reactions.
The main one is IgG with a molecular weight of
around 150000 and the other IgM is a bigger
molecule (800000). The antibody is a “Y”-
shaped molecule that consists of four
polypeptide chains.
IgG antibody is made up of heavy and light
chains linked together by disulphide bonds or
bridges (-S-S-). IgG molecules have three
protein domains, forming the arms of the Y, two
are identical and are called the Fab domain.

5. ANTIGEN-ANTIBODY

6. ANTIGEN-ANTIBODY BINDING
Hydrogen bonding :Results from the formation of
hydrogen bridges between appropriate atoms
 Electrostatic forces : Are due to the attraction of
oppositely charged groups located on two protein side
chains
Van der Waals bonds : Are generated by the
interaction between electron clouds (oscillating dipoles)
 Hydrophobic bonds

7. 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.
 There are many monoclonal antibody determinants in the virus coat
protein. With TMV 600-700 antibody determinants completely cover
one virus particle. These sites depend on particular configurations of
amino acids in the protein molecules.
 Antibodies act by forming bridges between virus particles when mixed
in optimum proportion. The large antigen complexes formed appear as
precipitate.
 The highest dilution of the antiserum reacting with antigen (virus) is
called the antiserum titre and the highest dilution of virus which gives a
visible precipitate is called as virus end point.

8. These techniques detect the virus coat protein by agglutination
reaction or antibody reaction. Based on the phase in which
these reaction occur, these are further classified as:
 Tube Precipitation test
 Agglutination test
 Micro-precipitin test
 Double agar diffusion test
 ELISA
 Dot immune binding assay

9.  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.
Tube Precipitation test

10. Principle
 A precipitation reaction is based on the principle of
“antigen-antibody reaction”, which occurs at the
equivalence zone. At the equivalence region, the ratio of
both antigen and antibody is equal, which brings out the
formation of lattice or cross-linked structure.
 In the equivalence zone, cross-linkage occurs between the
reactants that result in the formation of the antigen-
antibody complex as a visible ring or line of a precipitate.
 The least soluble antigens and antibodies form a complex at
the point of equivalence, whereas the free antigens and
antibodies remain as the supernatant.

12. Agglutination test:
 Its also known as chloroplast agglutination test, a few drops of
crude freshly expressed plant leaf sap from diseased plant
containing high concentration of virus is mixed with double
amount of diluted antiserum on a microscope slide.
 Due to combination of virus present in crude sap and antiserum,
chloroplasts and chloroplast fragments along with small particles
of host material clump or co-precipitate together.
 With crude sap from healthy plants there is no clustering of
chloroplast.

13. Principle
agglutination reaction is based on the “Clumping of antigen and
antibody”. Like precipitation reaction, it also involves the binding
of antigen and antibody at the equivalence zone, where the
concentration of both are at equilibrium. Antibodies possess a y-
shape with two Fab sites made of the hypervariable region, which
target the specific antigenic determinants or epitopes of an antigen.
The binding of antigen and antibody is similar to the “Lock-key
model”. Therefore, we can take a reference by considering the
epitopes of an antigen as a key and the Fab sites of an antibody as a
lock. Thus, the specific epitope will fit into the cleft of an
antibody’s Fab sites.

15. Micro-precipitin test
 This test is done on a micro-scale to economize on
antiserum. Drops of series of dilution mixtures (antiserum
and clarified virus suspension) are mixed at the bottom of a
petri-dish.
 The precipitates produced are observed with a microscope
with dark-ground illumination.
 The precipitation varies, depending on the ratio of
concentrations of antigen and antibodies. This test is a
miniatured version of the precipitation test.

16. Double diffusion test
In this test the antigen and antibody diffuse towards one another through
an agar gel whenever they meet in suitable concentration they react
with each other forming a whitish line or zone .

17. The double immunodiffusion test in agar gel also known as the
Ouchterlony test (Ouchterlony, 1962) is based in the fact that the antigens
and the antibodies deposited into wells opened in agar gels diffuse in all
directions through the medium.
This test is preferentially developed in Petri dishes but it can also be
accomplished on microscope slides. Reactant wells are opened in the agar
gel with cork borers or adjustable gel cutting device and the agar plugs
are removed with glass tubing connected to a vacuum pump.
A useful gel pattern consists of up to six peripheral antigen wells of 3 to 7
mm in diameter, surrounding a central serum well. Each peripheral well is
4 to 5 mm from the central well at the closest point.

18.  The antigens are pipetted into the peripheral wells and the
antiserum into the central well. Reactions usually appear within 12
h and are complete within 24 -48 h after the addition of the
reactants.
 The results can be viewed and recorded photographically by dark-
field illumination (Purcifull & Batchelor, 1977).
 The standard double immunodiffusion technique recommended to
define the serological relationship among virus species or strains.
 It is still used to define the relationship among virus species and
isolates from the genera Potyvirus and Comovirus.

19. Enzyme linked immunosorbent assay (ELISA)
 ELISA is a very specific and sensitive serological technique introduced
to the study and identification of plant viruses in the 1970s (Clark &
Adams, 1977; Voller et al., 1976). This technique is able to detect virus
particles in very low concentrations and can be used with viruses of
different particle morphology.
 Because of its adaptability, high sensitivity, and economy in the use of
reagents, ELISA is used in a wide range of situations, especially for
indexing a large number of samples in a relatively short period of time.
 The ELISA technique is based on the basic principle in which the virus
antigens are recognized by their specific antibodies (IgG) in association
with colorimetric properties. The ELISA method is commonly
accomplished in a 96-well polystyrene plate by adding the antigens and
antibodies into the wells in an established sequence, involving several
stages.

20. In the final stage, the positive reactions are detected when a
colorless substrate, usually p-nitrophenyl phosphate, undergoes
a chemical change resulting in a yellow colored product as the
result of exposure to the enzyme alkaline phosphate linked to the
antibody. The degree of color change indicates the degree of
reactivity that is read by an ELISA plate reader apparatus.

21.  The principle of ELISA techniques consists of detecting the
antigen-antibody interactions by enzyme induced color
reaction rather than by observing their precipitation.
 It is always recommended to include a homologous antigen
(positive control) for the specific virus antibody and extracts
from healthy plants (negative control) to compare the
absorption readings and to obtain a correct interpretation of
the results

22. TYPES OF ELISA
• Direct ELISA
• Indirect ELISA
• Sandwich ELISA

23.  The direct ELISA, also called double antibody sandwich (DAS-
ELISA), is highly strain-specific and requires each detecting
antibody to be conjugated to an enzyme. Typically, the enzyme is
alkaline phosphatase. The first step in the test is the adsorption of
virus-specific antibodies to the wells of ELISA plates.
 Unbound antibody is removed by washing, and the samples to be
tested for virus antigen are added. Controls include extracts from
known infected plants (positive control), and extracts from healthy
plants (negative control).
 After incubation and washing, the enzyme-antibody conjugate is
added. If virus attached to the coating antibody is present, the
enzyme-antibody conjugate will combine with the virus. Plates are
washed, and the colorless substrate (p-nitrophenyl phosphate) is
added.
 Positive wells will show a yellow reaction, due to the action of the
conjugated enzyme (alkaline phosphatase) on the substrate.
Direct ELISA

24.  Negative wells will remain colorless. The colorimetric changes
are measured in an ELISA reader, using a filter for 405 nm
wave length. The washing procedures can be accomplished by
the use of a plate washing apparatus, according to a
programmed schedule.
 The quality of the antiserum is critical in achieving certain
objectives, but a good, broad spectrum polyclonal antiserum
will give satisfactory results in most virus indexing programs.
 On the other hand, monoclonal antibodies could be useful for
identification and characterization of specific plant virus
strains.

26.  Although the direct ELISA technique has high sensitivity and
specificity, a method called indirect ELISA or plate-trapped
antigen (PTA- ELISA) was developed to avoid the
inconveniences and the difficulties of conjugating the enzyme
with the IgG specific for each virus species to be used in the
second layer of antibodies in direct ELISA.
 For this reason, the indirect ELISA or PTA-ELISA requires
antibodies produced in two different animal species and the
virus particles are trapped in the wells of the ELISA plate.
 The indirect ELISA also requires the use of a universal IgG
enzyme conjugate which can be used with the antibodies of
all virus species.
Indirect ELISA

27.  The complete indirect ELISA protocol consists of, initially, covering
the plate wells with extracts from infected and healthy plant tissues
prepared in the proportion of 1:10 in carbonate buffer, pH 9.6 and the
plates are incubated at 37 0C for 1 h.
 The plates are washed three times with PBS Tween buffer and 100
microlitre of the virus polyclonal antiserum produced in rabbit
previously absorbed by extracts from healthy plants, diluted to 2,000
to 6,000 are added into the wells.
 The plates are incubated again at 37 0C for 1 h, after which they are
washed three times with PBS-Tween.
PROTOCOL

28.  The washing procedures can be accomplished by the use of a
washing ELISA plates apparatus, according to programmed
schedules. Finally, 100 microliter of a substrate of p-nitrophenyl
phosphate in the concentration of 0.5 mg/ml dissolved in a buffer
containing 12% of diethanolamine and 0.25% of sodium azide, pH
9.8 are added into the wells. After 20, 40 and 60 min the plates are
analyzed in the ELISA plate reader apparatus, using a filter for 405
nm wave length.
 After drying, 100 microltre of anti-rabbit IgG produced in goat or
mouse conjugated to alkaline phosphatase, diluted in the
proportion of 1:2,000 to 1:6,000 in a buffer contain 2% of
polyvinylpyrolidone, 0.2% of albumin and 0.02% of sodium azide
are added into the wells.
 The plates are incubated once more at 37 0C for 1 h and washed
again three times with PBS-Tween.

29. Another widely used ELISA variation is the triple antibody
sandwich (TAS- ELISA), which is similar to the direct ELISA
(DAS- ELISA), except that an additional antibody produced in
another animal is used.
First, the bottom of the ELISA plate wells are coated with a virus
antibody produced in a species of animal (e.g., rabbit) and the virus
antigen is linked in the trapped antibodies. The virus antigen is
covered with a second layer of virus specific antibody produced in
another animal species (e.g., mouse or goat) and the presence of this
antibody is detected by adding an enzyme-conjugated specific
antibody (e.g., rabbit anti-mouse IgG), that does not react with the
plate well trapped antibody, followed by colorimetric changes of a
specific substrate that is added into the wells.
3 Triple Antibody Sandwich (TAS-
ELISA)

30. Considering that virus specific monoclonal antibodies are usually
used in the second layer of antibodies this procedure is an effective
method of combining the broad reactivity of polyclonal antibodies
in the virus trapping phase with the specificities of the monoclonal
antibodies (Purcifull et al., 2001).

31.  This ELISA variation is based on the property of protein
A combining specifically with the Fc portion of the IgG.
The protein A is obtained from the cell wall of
Staphylococcus aureus and has a molecular weight of
approximately 42 – 56 Kd (Almeida, 2001).
 This protein is very stable at a broad pH range and it is
produced commercially, including a protein A-enzyme
conjugate to be used in plant virology.
 It is prepared by direct dilution in pure water (1 mg/ml)
and diluted in ELISA buffer to determine its adequate
concentration for good results in PAS-ELISA. In the PAS-
ELISA the antibody–virus–antibody layers that occur in
the direct ELISA are sandwiched between two layers of
protein A.
4 Protein A-Sandwich (PAS- ELISA)

32.  The method consists of coating the bottom of the ELISA plate wells with
a layer of protein A before the addition of the trapped virus antibody.
 Since the Fc region from the antibodies (IgG) has affinity to protein A,
the added antibodies link specifically with the protein A trapped at the
bottom of the a wells keeping the virus antibodies in a specific
orientation so that the F(ab´)2 portion of the antibodies will be free to
trap the virus particles.
 The F(ab´)2 portion of the virus antibody orientation will increase the
sensitivity of the PAS- ELISA by increasing the proportion of
appropriately aligned antibody molecules. The exposed virus particles
will link to the F(ab´)2 portion of a second added layer of the same
antibodies which will be detected by an enzyme-conjugated protein A
followed by colorimetric changes of a specific substrate that is added into
the wells.

34. Sandwich ELISA
Sandwich ELISA (or sandwich immunoassay) is the most
commonly used format. This format requires two antibodies
specific for different epitopes of the antigen. These two
antibodies are normally referred to as matched antibody pairs.
One of the antibodies is coated on the surface of the multi-well
plate and used as a capture antibody to facilitate the
immobilization of the antigen. The other antibody is conjugated
and facilitates the detection of the antigen.

36. Competitive ELISA
 Competitive ELISAs are commonly used for small
molecules when the protein of interest is too small to
efficiently sandwich with two antibodies.
 Similar to a sandwich ELISA, a capture antibody is coated
on a microplate. Instead of using a conjugated detection
antibody, a conjugated antigen is used to complete binding
with the antigen present in the sample.
 The more antigen present in the sample, the less conjugated
antigen will bind to the capture antibody. Substrate is added
and the signal produced is inversely proportional to the
amount of protein present in the sample.

38. Advantages of ELISA
 Tests are extremely sensitive.
 Large number of samples can be tested
simultaneously .
 Only small amount of antiserum is required.
 Results are quantitative.
 Procedure can be semi automated.

39.  Serological solid support matrix methods similar to ELISA techniques
were developed in which the virus antigens are trapped onto a membrane
rather than in a microtitre plate.
 Similar to indirect ELISA, virus particles or their proteins are
immobilized on nitrocellulose or nylon membranes (Almeida, 2001;
Purcifull et al., 2001).
 As distinguished from indirect ELISA, it is not necessary to use an
ELISA reader for detecting the virus antibodies interactions and for this
reason it is not possible to quantify the results by numerical absorbance
values (Almeida, 2001; Astier et al., 2007).
 According to the process by which the virus antigens are applied in the
membranes these methods can be divided into three categories: a)
Western blot; b) Dot blot or dot immuno binding assay (DIBA) and c)
Tissue blot immuno assay (TIBA).
Immunoblotting methods

40.  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. Several
methods can be used to transfer the virus protein and the electro-
blotting is the most used system.
 Similar to ELISA techniques, the proteins are detected in the
membrane by the use of specific enzyme labeled antibodies
(Almeida, 2001; Purcifull et al., 2001). Different antibody labeling
systems, including biotin-avidin and chemiluminescent systems
are sometimes used to increase sensitivity.
 The 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.
Western blot

41. Dot blot immunobinding assay (DBIA)
 Blotting technique has become widely used for specific
identification of nucleic acid and proteins. This dot assay was
modified to detect protein by spotting the antigen on a nitrocellulose
membrane and incubating the membrane in test antibody followed
by incubation in peroxidase-conjugated second antibody to the first
antibody, and by development in 4-chloro-1-naphthol.
 Specific monoclonal antibodies are bound to strips (dip-sticks) of
nitrocellulose which can be carried into the field, allowing the
ELISA reactions to be carried out in situ. Sap or juice from plants
under examination is placed on the coated surface. Any pathogen
cells or particles with the specific epitope are trapped, the dip-stick
is washed and treated with the second antibody-enzyrne conjugate
before washing and developing the reaction.

42.  Although based on the Western blot principle, the dot
immunoblotting assay (DIBA) requires a simple and easier method to
prepare and apply the samples on nitrocellulose or nylon membranes
(Almeida, 2001; Astier et al., 2007; Purcifull et al., 2001).
 The samples containing the virus antigens are prepared by grinding
tissues in Tris-buffered saline and the extracts are applied directly on
the membrane.
 The sample application on the membrane is usually accomplished
through the use of a plastic mold with 96 wells which presses the
membrane marking the places where the samples should be applied.

43.  Usually, the spaces not occupied by the antigens on the
membrane are blocked with neutral protein solution.
 The addition of virus IgG produced in rabbit and the anti-
rabbit IgG produced in mouse follow protocols similar to
indirect ELISA or PTA-ELISA, except that the positive
reactions in DIBA are recorded as colored dots on the
membrane.
 Considering that DIBA is a simple, less laborious and
quick test, it can be used routinely for plant virus
indexing and survey programs.

44. Tissue Blot Immune Assay (TIBA)
 This is the simplest immunoblotting assay technique developed for virus
antigen detection in different types of plant and insect tissues. It is a
variation of DIBA in which the samples consist of preparation of infected
plant tissues. 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.
 The tissues are cut with razor blades and pressed on the membrane to
transfer the virus particles or protein. The detection of the virus antigens
applied on the membrane is accomplished by protocols similar to those
used for indirect ELISA or PTA-ELISA and for DIBA. As with DIBA,
sometimes the sap components can interfere with the diagnostic results and
the color of the sap interferes with the observation of weak virus antigen
antibody reactions.

45.  The TIBA technique has been demonstrated to be sensitive enough to
evaluate the in situ distribution of plant virus species from different
families and genera (Astier et al., 2007; Makkouk et. al., 1993). As with
DIBA the virus-antibody interactions are not quantified since the results
are not presented by numerical forms by absorbance values as in the
ELISA variations (Almeida, 2001; Astier et al., 2007).
 The low cost and simplicity of these immunoblotting assay techniques
(DIBA and TIBA) make them useful for laboratories with limited
facilities (Astier et. al., 2007; Makkouk & Kumari, 2002).
 Another advantage of these methods is that the samples can be blotted
onto the membranes right in the field or in simple laboratories and
shipped for further processing at a more equipped laboratory (Naidu &
Hughes, 2001).

46. CONCLUSION
 Diagnosis using serological methods has many advantages. Although
antibodies may take several weeks to produce, they are generally stable
for long periods if stored correctly and produce results quickly.
 There are some limitations to the use of antibodies in pathogen
diagnosis. Firstly, the nature of the cross reactions between
heterologous antibody-antigen complexes are not well understood so
the degree of relatedness between cross reacting isolates cannot be
estimated.
 Secondly, diagnosis is based on only part of the organism's structure
such as the coat protein of a virus which represents only a small
proportion of the information about the virus.
 Thirdly, serology is only useful when the antiserum has been prepared
or when an antigen is available for producing an antiserum.

47. REFERENCES
 Ken Goulter and John Randles,1997, Serological And Molecular
Techniques To Detect And Identify Plant Pathogen, Plant Pathogens and
Plant Diseases,174-178
 E. VAN SLOGTEREN and D. H. M. VAN SLOGTEREN, 1957,
Serological Identification Of Plant Viruses And Serological Diagnosis Of
Virus Diseases Of Plants, Annual Review Microbiology,149-164.
 Chu et al,1989,New Approaches to the detection of microbial plant
pathogens, Biotechnology and Genetic Engineering Reviews,45-55
 Bhat K.A et al,2010,Serodiagnosis in plant pathology : Present status and
future prospects, Journal of ecobiotechnology,21-28
 Abd El-Aziz et al, 2019,Three modern serological methods to detect plant
viruses, Journal Of Plant science and phytopathology,101-105

48. THANK
YOU