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5.

Enzyme Linked Immunoassays

ELISA was developed in 1970 and became rapidly accepted. A wide variety of assay principles can be
used in ELISA techniques. Currently the most important ones are;-

1. Competitive methods
2. Sandwich methods
3. Antibody capture methods

Competitive methods

One component of the immune reaction is insolubilized and the other one labeled with an enzyme. The
analyte can then be quantified by its ability to prevent the formation of the complex between the
insolublized and the labelled reagent. Advantages of this approach are that only one incubation step is
necessary and that the "prozone effect" at high analyte concentrations cannot occur. Disadvantages are that
the concentration range in which the analyte can be quantified without sample dilution is rather narrow and
that the antigen or antibody (in cases where either may be present in a sample e.g. hepatitis B) produce the
same response, and can therefore cannot be distinguished in a one step assay.

Sandwich (Indirect) methods

1. The method in which the same component of the immune reaction (e.g. the antibody) is used in
the insolubilized and the enzyme labelled form. The other component, the analyte (i.e. the antigen
in the sample forms a bridge between the two reagents.)
2. The method in which one component (usually the antigen) is used in an insolubilized form to bind
the analyte from the sample (the antibody),which is subsequently determined by addition of
labelled second antibody against the same class of antibody as the analyte antibody or protein A.

In principle, quantification can be achieved over an extremely wide analyte concentration range in such
sandwich methods. The "prozone effect" can be avoided in the following ways;- (i) using sequential
incubation steps for sample and label, or (ii) by using monoclonal antibodies. Modification of the test in (2)
so that antibodies of a specific class such as IgM, can give spurious results if antibodies from other
immunoglobulin classes are also present in the sample. Also RF ( rheumatoid factor ) is known to be a
potentially interfering factor.

Sandwich inhibition methods

The sample containing the analyte (usually antibody) is pre-incubated with a fixed amount of its binding
partner (ie. the antigen ) after which the remaining amount of antigen is determined in a sandwich assay.
These methods usually are complicated and have a limited measuring range. The method allows for
simultaneous detection of antibody or antigen, if either of these 2 analytes is present in the sample.
Antibody capture methods

These methods used to detect antibodies of specific immunoglobulin subclasses, by first reacting the
sample with e.g. insolubilized anti-IgM,and subsequently with either enzyme labelled antigen followed by
enzyme linked antibody. Neither antibodies from other immunoglobulin subclasses nor rheumatoid factor
interfere significantly in such assays. They are widely used for the diagnosis of acute infections by IgM
detection. These assays may be used for detecting IgG and IgA. Considering trends towards simplification
of assays and quantification of analytes over a wide concentration range. It must be expected that
competitive and sandwich inhibition methods will decrease in importance, and the sandwich and antibody
capture methods will be the main assay principles in the future.
Assay Characteristics

The use of monoclonal antibodies has lead to many improvements in ELISA systems.

1. Higher sensitivity ;- either by selection of antibodies with a extremely high affinity, or by


reduction of the height and variability of the background reaction, which makes very low
concentrations of analyte more readily detectable.
2. Higher specificity ;- by avoiding the presence of any antibody in the assay system with specific
reactivity against non-analyte epitopes, and by selecting combinations of monoclonal antibodies
which may further increase specificity.
3. Higher practicality ;- e.g. by introducing simultaneous incubation of label, solid phase and
sample without risk of "prozone effect".

The enzyme label ;- Most of the assays employ horse-radish peroxidase, alkaline phosphatase, or B-D-
galactosidase. The most interesting recent developments has been in new methods to detect these enzymes
rather than the use of new enzymes. Fluorimeters were introduced in 1984 for the detection of alkaline
phosphatase and B-D-galactosidase. Methods are available to detect horse radish peroxidase by means of
chemilumininescence. Fluorimetric and luminometric methods offer higher sensitivity and a wider
measuring range than conventional spectrometry. TMB is gradually replacing mutagenic substrates such as
OPD, leading to increased sensitivity and safety.

A sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen
present binds to capture antibody; (3) enzyme linked capture antibody used as detecting antibody is added,
and binds to antigen; (4) substrate is added, and is converted by enzyme to detectable form.

A less-common variant of this technique,

t of this technique,

Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody
in a sample, it is a useful tool for determining serum antibody concentrations (such as with the HIV test[3] or
West Nile Virus). It has also found applications in the food industry in detecting potential food allergens
such as milk, peanuts, walnuts, almonds, and eggs.[4] ELISA can also be used in toxicology as a rapid
presumptive screen for certain classes of drugs.

The ELISA was the first screening test widely used for HIV because of its high sensitivity. In an ELISA, a
person's serum is diluted 400-fold and applied to a plate to which HIV antigens are attached. If antibodies
to HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove
all other components of the serum. A specially prepared "secondary antibody" an antibody that binds to
other antibodies is then applied to the plate, followed by another wash. This secondary antibody is
chemically linked in advance to an enzyme. Thus, the plate will contain enzyme in proportion to the
amount of secondary antibody bound to the plate. A substrate for the enzyme is applied, and catalysis by
the enzyme leads to a change in color or fluorescence. ELISA results are reported as a number; the most
controversial aspect of this test is determining the "cut-off" point between a positive and negative result.

A cut-off point may be determined by comparing it with a known standard. If an ELISA test is used for
drug screening at workplace, a cut-off concentration, 50 ng/mL, for example, is established, and a sample
will be prepared which contains the standard concentration of analyte. Unknowns that generate a signal that
is stronger than the known sample are "positive". Those that generate weaker signal are "negative."

ELISA can also be used to determine the level of antibodies in faecal content...specifically the direct
method

Radioimmunoassay (RIA) is a very sensitive technique used to measure concentrations of antigens


(for example, hormone levels in the blood) without the need to use a bioassay.

Although the RIA technique is extremely sensitive and extremely specific, it requires specialized
equipment and is costly. It also requires special precautions, since radioactive substances are used.
Therefore, today it has been largely supplanted by the ELISA method, where the antigen-antibody reaction
is measured using colorimetric signals instead of a radioactive signal.

The RAST test (radioallergosorbent test) is an example of radioimmunoassay. It is used to detect the
causative allergen for an allergy.

Method

To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by


labeling it with gamma-radioactive isotopes of iodine attached to tyrosine. This radiolabeled antigen is then
mixed with a known amount of antibody for that antigen, and as a result, the two chemically bind to one
another. Then, a sample of serum from a patient containing an unknown quantity of that same antigen is
added. This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled
antigen ("hot") for antibody binding sites.

As the concentration of "cold" antigen is increased, more of it binds to the antibody, displacing the
radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled antigen to free radiolabeled
antigen. The bound antigens are then separated from the unbound ones, and the radioactivity of the free
antigen remaining in the supernatant is measured. Using known standards, a binding curve can then be
generated which allows the amount of antigen in the patient's serum to be derived.

Hiv virus

History
It was developed by Rosalyn Yalow and Solomon Aaron Berson in the 1950s.[1] In 1977, Rosalyn Sussman
Yalow received the Nobel Prize in Medicine for the development of the RIA for insulin: the precise
measurement of minute amounts of such a hormone was considered a breakthrough in endocrinology.

With this technique, separating bound from unbound antigen is crucial. Initially, the method of separation
employed was the use of a second "anti-antibody" directed against the first for precipitation and
centrifugation. The use of charcoal suspension for precipitation was extended but replaced later by Drs.
Werner and Acebedo at Columbia University for RIA of T3 and T4.[2] An ultramicro RIA for human TSH
was published in BBRC (1975) by Drs. Acebedo, Hayek et al
In 1981, the Centres for Disease Control reported cases of Pneumocystis carinii pneumonia and Kaposi's
sarcoma in previously healthy young male homosexuals. Before then, pneumocystis carinii was mainly
known to occur in immunodepressed patients following organ transplantation or suffering from congenital
immunodeficiencies. Shortly afterwards, the same condition was seen in IV drug abusers, haemophilliacs
and babies of IV drug abusing mothers. It was recognized that these patients had profound
immunosuppression due to the depletion of T4 helper lymphocytes. The name acquired immunodeficiency
was coined for this syndrome. Epidemiological studies soon established that this disease was infectious,
transmitted by sexual intercourse, blood or blood products. There were initial difficulties in isolating the
virus due to the fact that the lymphocytes of patients died early. Eventually the virus was isolated by
Montagnier and Gallo in 1984. In 1986, HIV-2 was isolated from West Africa which did not cross-react
serologically with HIV-1 in screening tests.

Basic Properties

Belong to the lentivirus subfamily of the retroviridae


Enveloped RNA virus, 120nm in diameter, contains 2 copies of the RNA genome
Replication depends on reverse transcriptase
cytopathic viruses, in contrast to the oncornaviruses
HIV-2 less cytopathic for in cell culture than HIV-1, and shares 40% nucleotide homology with HIV-1
HIV-II is more related to SIV than to HIV-I
genome consists of 9200 nucleotides (HIV-1)
gag core proteins - p15, p17 and p24
pol - p16 (protease), p31 (integrase/endonuclease)
env - gp160 (gp120:outer membrane part, gp41:transmembrane part)
other regulatory genes are present in addition to the above ie. tat, rev, vif, nef, vpr and vpu

The replication cycle is as follows; The first step of infection is the binding of gp120 to the CD4 receptor of
the cell, which is followed by the fusion of the virus and cell membrane and is mediated by the gp41
molecule. The virus then penetrates into the cell and uncoating, reverse transcription, provirus synthesis
and integration takes place. This is followed by the synthesis and maturation of virus progeny.

Pathogenesis_of_AIDS

The profound immunosuppression seen in AIDS is due to the depletion of T4 helper lymphocytes. In the
immediate period following exposure, HIV is present at a high level in the blood (as detected by HIV
Antigen and HIV-RNA assays). It then settles down to a certain low level (set-point) during the incubation
period. During the incubation period, there is a massive turnover of CD4 cells, whereby CD4 cells killed by
HIV are replaced efficiently. Eventually, the immune system succumbs and AIDS develop when killed
CD4 cells can no longer be replaced (witnessed by high HIV-RNA, HIV-Antigen, and low CD4 counts).

HIV-1 Half-Lives. Activated cells that become infected with HIV produce virus immediately and die
within one to two days. Production of virus by short-lived, activated cells accounts for the vast majority of
virus present in the plasma. The time required to complete a single HIV life-cycle is approximately 1.5
days. Resting cells that become infected produce virus only after immune stimulation; these cells have a
half-life of at least 5-6 months. Some cells are infected with defective virus that cannot complete the virus
life-cycle. Such cells are very long lived, and have an estimated half-life of approximately three to six
months. For a more detailed article on HIV pathogenesis, go to the Medscape article "HIV Pathogenesis
and Viral Markers" Daniel R. Kuritzkes
The HIV replication cycle

Tobacco mosaic virus

Tobacco mosaic virus (TMV) is an RNA virus that infects plants, especially tobacco and
other members of the family Solanaceae. The infection causes characteristic patterns
(mottling and discoloration) on the leaves (hence the name). TMV was the first virus to
be discovered. Although it was known from the late 19th century that an infectious
disease was damaging tobacco crops, it was not until 1930 that the infectious agent was
determined to be a virus.
History
In 1883, Adolf Mayer first described the disease that could be transferred between plants, similar to
bacterial infections[1]. Dimitri Ivanovski gave the first concrete evidence for the existence of a non-bacterial
infectious agent, showing that infected sap remained infectious even after filtering through Chamberland
filter candles, in 1892. However, he remained convinced despite repeated failures to produce evidence, that
bacteria were the infectious agents. In 1898, Martinus Beijerinck showed that a filtered, bacteria-free
culture medium still contained the infectious agent[1]. Wendell Meredith Stanley crystallized the virus in
1935 and showed that it remains active even after crystallization[1]. For his work, he was awarded 1/4 of the
Nobel Prize in Chemistry in 1946[2], even though it was later shown some of his conclusions (in particular,
that the crystals were pure protein, and assembled by autocatalysis) were incorrect.[3] The first electron
microscopical images of TMV were made in 1939 by Gustav Kausche, Edgar Pfankuch and Helmut Ruska
- the brother of Nobel Prize winner Ernst Ruska.[4] In 1955, Heinz Fraenkel-Conrat and Robley Williams
showed that purified TMV RNA and its capsid (coat) protein assemble by themselves to functional viruses,
indicating that this is the most stable structure (the one with the lowest free energy), and likely the natural
assembly mechanism within the host cell.

The crystallographer Rosalind Franklin worked for Stanley for about a month at Berkeley, and later
designed and built a model of TMV for the 1958 World's Fair at Brussels. In 1958, she speculated that the
virus was hollow, not solid, and hypothesized that the RNA of TMV is single-stranded. This conjecture was
proven to be correct after her death and is now known to be the + strand.

Structure

Schematic model of TMV: 1. nucleic acid (RNA), 2. capsomer (protomer), 3. capsid

Tobacco mosaic virus has a rod-like appearance. Its capsid is made from 2130 molecules
of coat protein (see image to the left) and one molecule of genomic RNA 6400 bases
long. The coat protein self-assembles into the rod like helical structure (16.3 proteins per
helix turn) around the RNA which forms a hairpin loop structure (see the electron
micrograph above). The protein monomer consists of 158 amino acids which are
assembled into four main alpha-helices, which are joined by a prominent loop proximal
to the axis of the virion. Virions are ~300 nm in length and ~18 nm in diameter.[6]
Negatively stained electron microphotographs show a distinct inner channel of ~4 nm.
The RNA is located at a radius of ~6 nm and is protected from the action of cellular
enzymes by the coat protein. There are three RNA nucleotides per protein monomer.[7] X-
ray fiber diffraction structure of the intact virus based on an electron density map at 3.6
resolution.[5]
Physicochemical Properties

TMV is a thermostable virus. On a dried leaf, it can withstand up to 120 degrees


Fahrenheit (50 C) for 30 minutes.TMV has an index of refraction of about 1.57.[8]

Replication

Following entry into its host via mechanical inoculation, the TMV RNA genome is not immediately
translated. Instead the RNA is processed by a mechanism that is not yet understood. The resulting mRNAs
encode several proteins, including the coat protein and an RNA-dependent RNA polymerase (RdRp). Thus
TMV can replicate its own genome. After the coat protein and RNA genome of TMV have been
synthesized, they spontaneously assemble into complete TMV virions in a highly organized process. The
protomers come together to form disks composed of two layers of protomers arranged in a helical spiral.
The helical capsid grows by the addition of protomers to the end of the rod. As the rod lengthens, the RNA
passes through a channel in its center and forms a loop at the growing end. In this way the RNA can easily
fit as a spiral into the interior of the helical capsid.[9]

Infection
When TMV infects a tobacco plant, the virus enters mechanically (for example, through a ruptured plant
cell wall) and replicates. After its multiplication, it enters the neighboring cells through plasmodesmata. For
its smooth entry, TMV produces a 30 kDa movement protein called P30 which tends to enlarge the
plasmodesmata. TMV most likely moves from cell-to-cell as a complex of the RNA, P30, and replicase
proteins. The first symptom of this virus disease is a light green coloration between the veins of young
leaves. This is followed quickly by the development of a "mosaic" or mottled pattern of light and dark
green areas in the leaves. These symptoms develop quickly and are more pronounced on younger leaves.
Mosaic does not result in plant death, but if infection occurs early in the season, plants are stunted. Lower
leaves are subjected to "mosaic burn" especially during periods of hot and dry weather. In these cases, large
dead areas develop in the leaves. This constitutes one of the most destructive phases of tobacco mosaic
virus infection. Infected leaves may be crinkled, puckered, or elongated.

Transference to Humans

Consumption of tobacco products infected with the tobacco mosaic virus has been found to have no effect
on humans that is due to the virus.

Scientific and environmental impact

The tobacco mosaic virus has been known to cause a production loss for flue cured tobacco of up to two
percent in North Carolina.[10] It is known to infect members of nine plant families, and at least 125
individual species, including tobacco, tomato, pepper (all members of the useful Solanaceae), cucumbers,
and a number of ornamental flowers.[11] There are many different strains.

The large amount of literature about TMV and its choice for many pioneering investigations in structural
biology (including X-ray diffraction), virus assembly and disassembly, and so on, are fundamentally due to
the large quantities that can be obtained, plus the fact that it does not infect animals. After growing a few
infected tobacco plants in a greenhouse and a few simple laboratory procedures, a scientist can easily
produce several grams of virus. As a result of this, TMV can be treated almost as an organic chemical,
rather than an infective agent.[verific