TECHNIQUES IN PLANT VIROLOGYCIP Training Manual2.3 DETECTION/Serology

Section 2.3.1An Introduction toSerology

IntroductionThe morphological characteristics of plants and animals are used asclassification criteria by the descriptive natural sciences. However, thedevelopment of serology as an independent science has made it possibleto clarify the differences between living beings based on chemicalconsiderations by determining their structures. These considerationswere arrived at indirectly and not as the consequence of simpleobservation.

The concept of specificity originates in the knowledge that after recoveryfrom an infectious illness there is a certain resistance to that disease(immunity). Jenner used this fact to develop the first practical vaccine.

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The search for an explanation to this phenomenon led to the discovery ofa peculiar group of substances in blood called antibodies. Someantibodies protect the organism against infectious agents (bacteria andviruses) or neutralize toxins.

These substances, which have now been identified as modified globulins,are formed not only as the result of an infection, but also as theconsequence of the administration of certain dangerous substances ofhigh molecular weight—toxins of bacteria, animals, or plants—or of deadbacteria.

Apart from the initial interest in resistance (immunity) to diseases, otherresearch in immunology found that immunity caused by bacteria andtoxins is only a part of a more general principle: the same mechanism istriggered when animals are inoculated with materials, such as cells orproteins derived from foreign species. In this case, antibodies appear inthe blood causing agglomeration, cell destruction, or precipitation ofthose proteins.

All immuno-antibodies are specific. This means, literally, that they reactonly to one antigen (the one used for immunization). Antigens may beproteins, cells from the blood of a different species, bacteria, or viruses.

Modern biological sciences have developed efficient and precisebiochemical and immunological techniques for the diagnosis of variousimportant diseases, thus replacing more conventional methods.

History

Edward Jenner (1749–1823) pioneered the use of vaccines for theprevention of infectious diseases.

Louis Pasteur (1822–1895) studied various methods for attenuatingpathogen virulence, leading to the development of vaccines.

H. Bence-Jones (1847) discovered an abnormal protein in the urine ofmultiple myeloma patients. This protein, produced in excess, is a dimerof light (L) chains.

Paul Ehrlich (1854–1915) discovered that antibodies are synthesized as aresponse to antigens.

George Nuttall (1888) discovered the bactericidal property of blood.

Gruber and Durham (1896) produced the first serological agglutinationreaction of vibrium cholera, and the corresponding antiserum.

Albert Coons (1942) developed histochemical methods for detectingpathogens by utilizing fluorescent antibodies.

O. Ouchterlony, J. Ondin, and S. Elek (1946–1948) described the geldiffusion tests for virus study and detection.

P. Grabar and C. Williams (1953) developed the immuno-electrophoresistechnique.

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Alick Isaacs (1957) produced interferon.

Solomon Berson and Rosalyn Yallw (1959–1960) developedradioimmunology.

Rodney Porter (1962) proposed a basic four-chain model for the structureof immunoglobulin molecules.

G. Edelman et al. (1968) established the complete amino acid sequenceof an IgG antibody from the blood of a multiple myeloma patient.

A. Völler et al. (1976) first used the enzyme linked immunosorbent assay(ELISA) for detecting three plant viruses.

George Köhler and Cesar Miltstein (1981) developed the technique forproducing monoclonal antibodies.

Immunochemistry

Leukocytes are blood cells that protect organisms. There are severalkinds of leukocytes, among which the most important are:

Monocytes: 15 to 18 �m long; are up to 8% of all leukocytes. (Theyare macrophages and along with the phagocytosis are responsible forconsuming large molecules).

Lymphocytes: 8–9 �m long; constitute 30–35% of all leukocytes andtake part in antigen–antibody reactions.

Eosinophiles: 12–13 �m long; are up to 8% of blood components.They take part in antigen–antibody reactions, particularly in allergies.The immune system reacts in either of two ways, through cellimmunity or humoral immunity. Lymphocytes are the primary cellsinvolved in these responses. Two different lymphocyte populationshave been identified: T cells (T lymphocytes) and B cells (Blymphocytes).

T lymphocytes are responsible for cell immunity, including cutaneousreactions, graft rejection, anti-tumor immunity, and cellular defenseagainst fungi and intracellular pathogens.

B lymphocytes develop in the Fabrician sack of birds, but are thoughtto derive from the bone marrow of mammals. They are in charge ofhumoral immunity expressed in the production of specific circulatingplasma proteins called antibodies or immunoglobulins.

Antigens

Substances capable of inducing an immunological response (humoral,cellular, or mixed T and B cells), when introduced in animals are calledantigens or immunogens. Most antigens are macromolecular proteins butalso may be immunogenes, polysaccharides, synthetic polypeptides, aswell as other synthetic polymers.

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Immunogenic molecules have the following characteristics:

• The molecules should be foreign to the host.

• Molecules with a molecular weight higher than 10,000 are weakimmunogens; proteins with a molecular weight higher than100,000 are stronger immunogens.

• The molecule must have a certain degree of complexity to beantigenic. Immunogenicity increases with structural complexity.Moreover, aromatic amino acids contribute to a larger degree thanresidues of non-aromatic amino acids in immunogenicity.

• The capacity to respond to an antigen varies with the animalspecies, and even with its genetic constitution.

Antigenic Determinants

The production of immunoglobulins requires the linkage of the antigen tothe surface of the lymphocyte.

Combination sites on the surface of the lymphocyte that consist ofmolecules similar to antibodies are called antigen receptors. Onlyrestricted portions of the antigenic molecules are related to antibodycombination sites. These areas are called antigenic determinants, andthey determine antigen–antibody reactions. An antigenic determinant cancomprise only 6 to 7 amino acids of a protein's total number. In otherwords, a virus, for example, induces the production of a mixture ofantibodies that react specifically to various antigenic determinantspresent in the particle. Thus, we can define the antisera containing amixture of antibodies as "polyclonal antisera." The number of antigenicdeterminants in a single molecule varies with the size of the molecule andits complexity.

Hapten

This is a small, chemically defined molecule that cannot induce theproduction of specific antibodies against itself. However, when it iscovalently linked to a larger molecule, it can act as an antigenic determinantand induce antibody synthesis.

Adjuvants

Adjuvants are chemical substances used to enhance immunologicalresponse. They not only stimulate the formation of antibodies, but alsolocalize them at the site of injection as deposits from which they are slowlyreleased during the period of antibody synthesis, either through adsorption tosolid particles or through their incorporation in to an oily emulsion. The mostfrequently used adjuvants are calcium phosphate, inorganic gels, aluminumhydroxide, bentonite, methiolated serum albumin, and incomplete Freundadjuvant. This is the most commonly used adjuvant with viral antigens and ismade of 9 parts mineral oil (lanolin) and one part detergent; it allows theformation of a stable emulsion. If the mixture also contains deadMycobacterium sp. cells, it is called a complete Freund adjuvant.

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Antibodies

Antibodies are immunoglobulins produced by an organism as a response tothe invasion of foreign compounds such as proteins, glucosides, or nucleicacid polymers. The antibody molecule associates non-covalently to theforeign substance starting a process for eliminating the foreign substancefrom the organism.

The antibody molecule is basically formed by four polypeptide chainsforming the basic unit: two identical heavy (H) chains (MW 53,000 to75,000), and two light (L) chains (MW 23,000) united by disulfur bonds.There are two regions in the antibody molecule: the common fraction (Fc)and the variable fraction (Fab):

• Fc: constituted of a portion of heavy chain.

• Fab: this fraction chain (MW 50,000) determines the specificity ofthe antibodies and is constituted by the other portion of the heavychains (MW 100,000).

Immunoglobulins made up of more than one basic monomeric unit arecalled polymers. Electrophoretic and ultracentrifugation studies haveallowed the identification of 5 groups of immunoglobulins.

1. IgG. This is the main fraction of antibodies and comprises 80% ofimmunoglobulins having molecular weights of between 150,000 and160,000. It contains 2–4% carbohydrates and shows the lowestelectrophoretic mobility among all immunoglobulins. It is the onlyimmunoglobulin capable of crossing the placenta.

2. IgA. This immunoglobulin has a molecular weight of between180,000 and 400,000. It has a higher carbohydrate content (5–10%)than IgG. IgA is found in high concentrations in blood, in secretionssuch as colostrum, saliva, tears, and bronchial and digestive tubesecretions. IgA cannot cross the placenta.

3. IgM. This immunoglobulin has the most proteins and its amino acidsequence has not yet been determined. It contains 576 amino acidsand has a molecular weight of 950,000. It is the first antibodysynthesized by a newborn animal or human being. The cellsproducing IgM are divided into two daughter cells, which produceIgG. The IgMs can promote phagocytosis of microorganisms bymacrophages and polymorphonuclear leukocytes. IgMs do not crossthe placenta.

4. IgD. No antibody activity is attributed to IgDs. Its role in the generalscheme of immunoglobulins is still unknown.

5. IgE. This appears in serum at very low concentrations. It has amolecular weight of 190,000. Approximately half of the patients withallergic diseases show high IgE concentrations.

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Properties of human inmunoglobulins (Harper, 1980)

LgG lgA lgM lgD lgE

Molecular weight 150,000 160,000 900,000 180,000 190,000

Sedimentation coefficient 6–7 7 19 7–8 8

Concentration in serum (mg %) 1,000 200 120 3 0.05

Placenta transfer yes no no no no

Bacterial lysis + +++ + ? ?

Antiviral activity + +++ + ? ?

Monoclonal Antibodies

Monoclonal antibodies are specific antibodies to only one antigenicdeterminant. Monoclonal antibodies are produced by cloned cells(hybridomes) resulting from the union of a B lymphocyte with acarcinogenic myeloma cell. The lymphocyte confers the monoclonalantibody the capability to produce antibodies, and the myeloma cell givesit the ability to reproduce itself indefinitely. Because all cell clones from ahybridome come from a single B lymphocyte, they produce specificantibodies to only one antigenic determinant.

Antigen–Antibody Reactions

The antigen–antibody union is non-covalent and irreversible under normallaboratory conditions, and it includes hydrogen bonds, Van der Waalsforces, and hydrophobic and coulombic interactions. The resulting chemicalcomplementarity is similar to that between a key and lock.

The union area between the antigen and the antibody is only 1% of the totalglobulin surface and its specificity depends on the sequence of amino acidspresent in that region. The degree of association between the antigen andthe antibody depends on the characteristics of each molecule and isdetermined by the combined effects of the interactions between them. Ifaffinity and avidity are high, the molecules will unite more rapidly anddissociation will be very slow. Antisera with strong precipitation reactionsshow more avidity than those reacting weakly at a similar degree of dilution.

Antiserum affinity depends on:

• The temperature at which the reaction takes place. Highertemperatures cause stronger reactions. However, temperatures over40OC result in protein denaturation.

• Salt concentration. Low salt content favors the union of reactingsubstances.

• The pH level can cause changes in antigen–antibody affinity.

• The concentration of reacting substances for the formation ofprecipitates.

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Preparation of Viral Antigens

1. Animal-infecting viruses

These may be propagated in laboratory animals, embryonic eggs, or invitro cultures. The animal species used for the production of antiserummust be as phylogenetically distant as possible from the usual species tobe infected by the pathogen. The viral preparation must contain a highvirus concentration, and the viruses must be free of host contaminants.For example, in the case of rabies, the inactivated virus must be used inthe first immunizations in a series.

2. Plant-infecting viruses

These can be propagated to obtain high particle-concentration in vivo inhost plants allowing virus propagation in their tissues. Difficulty ofpurification depends on the virus and plant host. For viruses whosemultiplication is restricted to the phloem, the concentration obtained inplant tissue will be low and purification difficult.

Immunization and Production of Antibodies

a) Immunization

Animals commonly used in the production of antisera for researchpurposes include rabbits, guinea pigs, mice, goats, sheep, monkeys,horses, and hens. Selection of species depends on availability andvolume of antiserum required. The mechanism regulating antibodysynthesis and the reactions produced in the cells of the immune systemin the presence of viral particles are extremely complex, and somecharacteristics are still unknown.

The immunization process can be summarized as follows (see Figure):

Some of the viruses entering the organism (1) are ingested by themacrophage cells (2).

Some of the many millions of the helper T lymphocytes (3) normallyflowing in the blood system are activated to identify the new enemy, inthis case a viral particle. The T cell is activated by its union with themacrophage that captures the particle. The union takes place when the Tlymphocyte recognizes in the macrophage one of its own markers andanother corresponding to the antigen.

The activated helper T lymphocyte multiplies itself and thus stimulatesthe multiplication of killer T lymphocytes (4) and of B lymphocytes (5).

Once the B lymphocytes have multiplied (approximately a million differentlines), the T lymphocytes signal them to start the production of a greatnumber of antibodies (6) that will join the blood stream.

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Figure 1. Production of antibodies by organisms.

When a virus infects an organism, some particles penetrate and startmultiplying in the infected cells. Killer T lymphocytes cause chemicaldamage to the membranes of the infected cells, thus interrupting virusreplication.

Some of the synthesized antibodies neutralize the remaining virusparticles.

When the infection stops, the suppressor T lymphocyte (7) stops allactivity of the immune system, thus preventing uncontrolled reactions.

T lymphocytes and memory B lymphocytes (8) store information on theviral particles and remain in the blood and lymphatic systems. Thus, theyare ready to mobilize and start a reaction if the same viral particlesinvade the organism again.

b) Immunization methods

Immunization can be intravenous, intramuscular, subcutaneous,intraperitoneal, intradermal, intra-articular, or intranodular. This increasesthe stimulating effect upon the immune response.

c) Antisera production

Antibody production increases during the first days following the firstimmunization to reach a maximum concentration that can be maintainedfor a few days. If a new dose of antigen is injected, the synthesis ofantibodies increases rapidly to a higher concentration than initially

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produced by the first dose of antigen. Antibodies are found in the bloodserum of immunized animals. This serum is called serum.

To produce antiserum, allow a blood sample to stand to separate serumfrom other blood components. The serum is centrifuged (7840 g x 15min) to remove debris that have not separated. Serum may be storedfrozen (–20 OC to –70 OC) and lyophylized, or as liquid at 4OC mixed withan antiseptic.

Adequately stored, the antisera can remain active for many years.

Immunodiagnostic Techniques

A. Precipitation tests

Antigen and antibody precipitation occurs when the reaction betweenthese substances forms a grid-like structure preventing the passage ofwater molecules (hydrophobic reaction). The formation of the gridstructure requires a proportional concentration of reacting substances.For every antigen molecule, a given number of antibodies are needed.

Antibodies usually act as bivalent molecules and the antigens asmultivalent molecules. The presence of too many antibodies will makethe antibodies act as monovalent molecules only to one particle of theantigen, thus preventing the formation of the grid structure. Too manyantigens also prevent the formation of the grid, because the antigens willthen act only as monovalent molecules. The antigen–antibody reactioncannot be observed in either case.

1. Interface ring tests

In this test, a solution containing both antigens and antibodies is used. Aproportional amount of the sample containing the antigen is placed on agiven volume of antibodies. Precipitation occurs in the interface and theprecipitation plane creates the illusion of a ring.

2. Tube liquid precipitation test

This quantitative test is mainly used to determine the titer of antisera, andthe antigen concentration. In this test, several (double) dilutions of theantiserum and the antigen are mixed and incubated in two small testtubes for the formation of precipitates. Varying degrees of precipitationallow determination of the concentration of the reacting substances.

3. Microprecipitation test

In this test, individual drops of each of the reacting substances are placedon a petri dish. The plaque is stirred to mix the antibody and the antigen.After incubation, the precipitate forms. A stereoscopic microscope isused to observe the reaction.

B. Gel diffusion tests

These precipitation tests are usually done using agar or agarose gels.Their advantage is that the mix of the antigen and its corresponding

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antibody can be physically separated by the difference in the diffusioncoefficients of the components of their gels. Thus, these tests canprovide information on the homogeneity and purity of the reactingsubstances, as well as on their size and relations.

1. Simple immunodiffusion (Oudin 1946)

This is based on the use of antibodies immobilized in an agar or agarosematrix melted in a water bath at 50OC. It is pre-cooled for use and put into10 x 75 ml or smaller tubes. The antigen can diffuse through the agar,thus provoking the appearance of a zone or band migrating along thetube until the concentration of both reacting substances reaches anoptimal stage. Precipitation occurs at this point.

Some factors affecting band migration are antigen concentration anddiffusion coefficient. The latter depends on molecular weight and on thesize of the antigen molecule. Lower molecular weight and size meanhigher diffusion coefficients. The distance migrated by the diffusion bandalso depends on time, as well as on the kind, quality, and concentrationof the antibody.

2. Double immunodiffusion (Ouchterlony 1948)

• Simple dimension system. A neutral agar layer is placed betweenthe antigen and antibody solutions. The same principles apply as inthe Oudin simple diffusion test. However, both reacting substancesare diffusable and a precipitation line appears when they meet.The position and width of the band allow determination of theconcentration of the reacting substances.

! Double diffusion system. Plaques or slab holders are covered withneutral agar at a concentration of 0.7–1.5%. A well pattern isdesigned that suits the purpose of the test, and the antigen and theantibody are placed in separate wells.

Types of Reactions

Identity reaction: The fusion of precipitation lines occurs when bothantigens are identical. A compact barrier, the immunospecific barrier, isthus formed.

Non-identity reaction: In this case, diffusion varies and the precipitationarches do not act as barriers to the antigen, which are not related andtherefore intercross.

Partial double identity reaction: A partial double identity occurs betweenantigens. (Almost identical antigens differ in only one antigenicdeterminant.)

Partial identity reaction: The formation of a "spur" is observed, indicatingpartial identity between antigens (e.g., as in the case of different strainsof the same virus).

3. Immunoelectrophoresis (Grabar and Williams, 1953)

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This is one of the most important analytical tools used in the solution ofcomplex antigen mixes. It is based on electrophoretic mobility andantigenic specificity. The antigen mix is first separated in its componentsthrough electrophoresis in an agar gel. Then the antiserum is placed in aparallel channel or electrophoretic migration pathway to allow theformation of precipitation lines. Alkaline buffers between pH 7.5 and 8.6are generally used because this provides conditions under which theproteins are negatively charged and move toward the anode. Thismethod is used for virus characterization and strain differentiation.

C. Agglutination Test

The main difference between precipitation and agglutination tests is thatin the latter the antigen is very often larger than the antibody. For thisreason, few antibody molecules are necessary to form a visible particlegrouping. The principles governing reactions are similar to those of theprecipitation tests.

1. Passive agglutination (indirect or reverse)

This test is based on the use of inert substances that carry antigens orantibodies. These substances (latex spheres, bentonite) are severaltimes larger than the reacting substances, thus making it possible to usesoluble antigens (viral particles) in agglutination tests.

2. Latex test

This test is based on the use of polysterene spheres (800 nm diameter)covered by immunoglobulin molecules. This technique is 10–100 timesmore sensitive than the traditional microprecipitation tests for detectingplant viruses. A reaction may be seen with the naked eye.

3. Neutralization tests

In some cases, the interaction of biologically active antigens withhomolog antibodies results in loss of the antigen's biological activity. Thisreaction is called neutralization. Since the sensitivity of these testsfundamentally depends on the activity of the antiserum, the antigen'sactivity must be biologically detectable.

D. Immunological tests with markers

These tests use antibodies and antigens marked with independentlyacting substances called markers that increase test power and sensitivity.The higher the marker's level of activity, the faster the antigen–antibodyreaction can be detected.

1. Immunofluorescence (IFA)

This technique uses substances transforming light in the ultraviolet range(200 to 400 nm) into longer wavelength radiation. A modified microscope(a fluorescence microscope) allows you to see the light emitted by thefluorescing substance (fluoresceine isocyanate: FITC).

2. Radioimmunology

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The immunoglobulins are marked with radioactive substances (P32, I128).Their presence is determined by a reaction against photographicmaterial.

3. Immunologic assays with enzymatic conjugates

[Enzyme Linked Immunosorbent Assay (ELISA)]

These tests are based on the property of certain antigens and antibodiesto be absorbed into a solid medium allowing them to construct an orderedsequence of biological material (antibody, antigen, antibody conjugatedwith an enzyme), and which can be seen thanks to the color reactionresulting from the addition of the enzyme-specific substrate conjugated tothe antibody, thus allowing adequate quantifying of the antigen.

Depending on research needs, the following assays of this kind arefrequently used.

� DAS-ELISA (Double Antibody Sandwich)

� NCM-ELISA (Nitro Cellulose Membrane)

E. Immunosorbent Electron Microscopy (ISEM)

This is used for detecting antigens, or in ultrathin sections of virus-infected tissues.

When the virus is suspended, the positive reaction can be identified withthe help of an electron microscope.

1. Aggregation of viral particles

An adequate dilution of specific antiserum is prepared for detecting thesuspected virus in the tissue suspension. As a result of particle addition,complexes appear that can be seen under the electron microscope.

This technique is particularly useful when low virus concentrationprevents direct observation. An excess of antibodies will result in reactioninhibition, as in precipitation tests.

2. Antibody coating

This is based on the immobilization of viral particles in a grid and theirposterior covering by specific antibodies.

3. Antigen capture

Conversely, the grid is previously covered by specific antibodies.

Recommended LiteratureBall, E.M. 1974. Serological test of identification of plant viruses.

Published by American Phytopathological Society Inc. USA.

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Devlin, T. 1985. Textbook of biochemistry with clinical correlation. JohnWiley & Sons Inc. New York, USA.

Harlow, E. and D. Lane. 1988. Antibodies: a laboratory manual. ColdSpring Harbor Laboratory. New York, USA.

Harper, H.A. 1980. Manual de Química Fisiológica. 7ma. Edición. ElManual Moderno, Mexico.

Landsteiner, K. 1969. The specifics of serological reactions. DoverPublications Inc. New York, USA.

Lehninger, A. 1978. Biochemistry. Second Edition. Worth Publisher Inc.New York, USA.

Nowotny A. 1969. Basic exercises in immuno-chemistry. Laboratorymanual. Springer-Verlag. Berlin, Germany.