Download In immunology, an adjuvant is an agent that may stimulate the

In immunology, an adjuvant is an agent that may stimulate the immune
system and increase the response to a vaccine, without having any specific
antigenic effect in itself. The word “adjuvant” comes from the Latin word
adiuvare, meaning to help or aid. "An immunologic adjuvant is defined as any
substance that acts to accelerate, prolong, or enhance antigen-specific
immune responses when used in combination with specific vaccine antigens."
Adjuvants have been whimsically called the dirty little secret of vaccines in
the scientific community. This dates from the early days of commercial
vaccine manufacture, when significant variations in the effectiveness of
different batches of the same vaccine were observed, correctly assumed
to be due to contamination of the reaction vessels. However, it was soon
found that more scrupulous attention to cleanliness actually seemed to
reduce the effectiveness of the vaccines, and that the contaminants –
"dirt" – actually enhanced the immune response. There are many known
adjuvants in widespread use, including oils, aluminium salts, and virosomes,
although precisely how they work is still not entirely understood.
A virosome is a drug or vaccine delivery mechanism consisting of unilamellar
phospholipid bilayer vesicle incorporating virus derived proteins to allow the
virosomes to fuse with target cells. Virosomes are not able to replicate but
are pure fusion-active vesicles.
Antigens can be incorporated into virosomes, adsorbed to the virosome
surface, or integrated into the lipid membrane, either via hydrophobic
domains or lipid moieties cross-linked to the antigen.
Virosomes therefore represent an innovative, broadly applicable adjuvant
and carrier system with prospective applications in areas beyond
conventional vaccines. They are one of only three adjuvant systems widely
approved by regulatory authorities and the only one that has carrier
capabilities
Antigens can be incorporated into virosomes, adsorbed to the virosome
surface, or integrated into the lipid membrane, either via hydrophobic
domains or lipid moieties cross-linked to the antigen.
Virosomes therefore represent an innovative, broadly applicable adjuvant
and carrier system with prospective applications in areas beyond
conventional vaccines. They are one of only three adjuvant systems widely
approved by regulatory authorities and the only one that has carrier
capabilities
An assay is a procedure in molecular biology for testing or measuring the
activity of a drug or biochemical in an organism or organic sample. A
quantitative assay may also measure the amount of a substance in a sample.
Bioassays and immunoassays are among the many varieties of specialized
biochemical assays. Other assays measure processes such as enzyme
activity, antigen capture, stem cell activity, and competitive protein
binding.
An immunoassay is a biochemical test that measures the presence or
concentration of a substance in solutions that frequently contain a complex
mixture of substances. Analytes in biological liquids such as serum or urine
are frequently assayed using immunoassay methods. Such assays are based
on the unique ability of an antibody to bind with high specificity to one or a
very limited group of molecules. A molecule that binds to an antibody is
called an antigen. Immunoassays can be carried out for either member of
an antigen/antibody pair. For antigen analytes, an antibody that specifically
binds to that antigen can frequently be prepared for use as an analytical
reagent. When the analyte is a specific antibody its cognate antigen can be
used as the analytical reagent. In either case the specificity of the assay
depends on the degree to which the analytical reagent is able to bind to its
specific binding partner to the exclusion of all other substances that might
be present in the sample to be analyzed. In addition to the need for
specificity, a binding partner must be selected that has a sufficiently high
affinity for the analyte to permit an accurate measurement. The affinity
requirements depend on the particular assay format that is used.
In addition to binding specificity, the other key feature of all
immunoassays is a means to produce a measurable signal in response to a
specific binding. Historically this was accomplished by measuring a change
in some physical characteristic such as light scattering or changes in
refractive index. With modern instrumentation such methods are again
becoming increasingly popular. Nevertheless most immunoassays today
depend on the use of an analytical reagent that is associated with a
detectable label. A large variety of labels have been demonstrated
including radioactive elements used in radioimmunoassays; enzymes;
fluorescent, phosphorescent, and chemiluminescent dyes; latex and
magnetic particles; dye crystalites, gold, silver, and selenium colloidal
particles; metal chelates; coenzymes; electroactive groups;
oligonucleotides, stable radicals,and others. Such labels serve for detection
and quantitation of binding events either after separating free and bound
labeled reagents or by designing the system in such a way that a binding
event effects a change in the signal produced by the label. Immunoassays
requiring a separation step, often called separation immunoassays or
heterogeneous immunoassays, are popular because they are easy to design,
but they frequently require multiple steps including careful washing of a
surface onto which the labeled reagent has bound. Immunoassays in which
the signal is affected by binding can often be run without a separation
step. Such assays can frequently be carried out simply by mixing the
reagents and sample and making a physical measurement. Such assays are
called homogenous immunoassays or less frequently non-separation
immunoassays.
Regardless of the method used, interpretation of the signal produced in an
immunoassay requires reference to a calibrator that mimics the
characteristics of the sample medium. For qualitative assays the
calibrators may consist of a negative sample with no analyte and a positive
sample having the lowest concentration of the analyte that is considered
detectable. Quantitative assays require additional calibrators with known
analyte concentrations. Comparison of the assay response of a real sample
to the assay responses produced by the calibrators makes it possible to
interpret the signal strength in terms of the presence or concentration of
analyte in the sample.
An antigen is a substance/molecule that, when introduced into the body,
triggers the production of an antibody by the immune system, which will
then kill or neutralize the antigen that is recognized as a foreign and
potentially harmful invader. These invaders can be molecules such as pollen
or cells such as bacteria. The term originally came from antibody generator
and was a molecule that binds specifically to an antibody, but the term now
also refers to any molecule or molecular fragment that can be bound by a
major histocompatibility complex (MHC) and presented to a T-cell receptor.
"Self" antigens are usually tolerated by the immune system; whereas "Nonself" antigens are identified as intruders and attacked by the immune
system. Autoimmune disorders arise from the immune system reacting to
its own antigens.
A sporozoite (G. sporos, seed + zoon, animal) is the cell form that infects
new hosts. In Plasmodium, for instance, the sporozoites are cells that
develop in the mosquito's salivary glands, leave the mosquito during a blood
meal, and enter the liver where they multiply. Cells infected with
sporozoites eventually burst, releasing merozoites into the bloodstream
A merozoite (G. meros, part [of a series], +zoon, animal) are the result of
merogony that takes place within a host cell. In coccidiosis, merozoites
form the first phase of the internal life cycle of coccidian. In the case of
Plasmodium, merozoites infect red blood cells and then rapidly reproduce
asexually. The red blood cell host is destroyed by this process, which
releases many new merozoites that go on to find new blood-borne hosts.
ELISA - Enzyme-Linked Immunosorbent Assay; this is an assay that uses an
enzyme linked to an antibody. In this experiment, a colorless substrate is
turned into a colored product by the bound enzyme. The amount of activity
of this enzyme (as determined by detection of the amount of colored
product) is used as a measurement of the amount of bound antibody.
Generalized ELISA protocol for detecting a target antigen. Enzyme (E)
is conjugated to secondary antibody.
The interaction of antigen and antibody outside the body--in the
laboratory--can be used to determine if a patient has an infectious or
an autoimmune disease. The test measures whether a specific antibody
associated with an illness can be found in a patient's blood. A positive
result indicates that the antibody is there and implies that the person
has encountered a particular disease.
This exercise begins with removing red and white blood cells, which can
interfere with the test, from a patient's blood sample. The watery
fluid that remains is called serum. A portion of serum possibly
containing the antibody is allowed to react with the target antigen. A
correct match causes the antigen and antibody to bind together.
Detection becomes possible when a second antibody is added. This
antibody is prepared from the serum of an animal injected previously
with human antibody; the human antibody in this case serves as an
antigen and the animal thus produces an antibody against the human
antibody. Once isolated, the second antibody can be chemically linked
to a system that can produce a detectable signal.
In ELISAs, the antigen antibody complex is exposed to the second
antibody, which binds to the antibody portion of the complex (against
which it was formed), creating a sandwich-type structure. The signaling
system consists of an enzyme attached to the second antibody. When
the appropriate chemical is added, the enzyme converts it to a colored
substance that can be measured.
This test quantifies how much enzyme is present by the amount of
color produced. The more enzyme present, the more secondary
antibody must be attached. The amount of secondary antibody present
is determined by the amount of target, or first antibody, available.
Finally, because the first antibody binds to antigen, the more antigen
that is accessible, the more first antibody will be retained. The
measure of color, therefore, reflects the amount of antigen initially
present.
A Bind sample to support
B Add primary antibody; incubate and then wash
Incubating serum samples in antigen-coated wells helps ensure that the
antibody present in the sample will interact correctly with the antigen.
Because SLE is a disease of humans, the reaction usually occurs at the
temperature of the human body, which is 37 degrees C. Time is important:
the reaction must proceed long enough for adequate binding to occur or the
measurement will be artificially low.
Problem: If the timer is set to less than 15 minutes, the proper reaction
will not occur and no color will be evident at the end of the assay. The
observer will record the results incorrectly as false negatives.
Problem: If the temperature is set too low, the reaction will not be
completed in the allowed time. If the temperature is set too high, protein
(antigen and antibody) will be adversely affected via a process known as
denaturation, which diminishes its ability to interact. The results will again
be recorded as false negatives.
Washing helps remove any antibody that did not react with the SLE antigen
in the well. When the fluid is removed from the well, antibody that has
reacted with antigen remains attached to the well surface. Unreacted
(unbound) antibody may also remain in the well in the small amount of fluid
that is left behind. This unbound antibody must be removed, because the
anti-human antibody added in the next step will recognize and react with
any antibody remaining in the well, regardless of whether that antibody is
specific for the SLE antigen. A reaction with non-SLE antibody will produce
a false-positive result.
C Add secondary antibody-enzyme conjugate; wash
The second antibody, unlike the first, does not recognize the SLE antigen.
Instead, rabbit anti-human antibody reacts with human antibody. SLE
antibody is a human antibody that may be present in a well because it is
being held by antigen. The second antibody (from rabbit) will therefore
recognize this antibody and bind to it. If the well has not been washed
thoroughly, other human antibody may still be there and will also react with
the second antibody. Reaction of a non-SLE human antibody with the
second antibody will produce a false-positive result.
Washing helps remove any antibody that did not react with the SLE antigen
in the well. When the fluid is removed from the well, antibody that has
reacted with antigen remains attached to the well surface. Unreacted
(unbound) antibody may also remain in the well in the small amount of fluid
that is left behind. This unbound antibody must be removed, because the
anti-human antibody added in the next step will recognize and react with
any antibody remaining in the well, regardless of whether that antibody is
specific for the SLE antigen. A reaction with non-SLE antibody will produce
a false-positive result.
D Add substrate
One of the most commonly used enzymes that can be attached to antibody
is called horseradish peroxidase. The enzyme together with hydrogen
peroxide acts on a chemical called ABTS (2,2'-azinobis-3ethylbenzothiazoleine-6-sulfonic acid) to produce a yellow solution that can
be estimated by eye or quantitatively measured in a spectrometer at 414
nanometers.
An ELISA may be subject to many errors. One is that the biological and
chemical reagents used in ELISA can change with time. Another is that the
ELISA is not always conducted under appropriate conditions. To rule out
such problems, two controls are used. One control should always produce a
positive response if the reagents and conditions are correct. The second
control should never produce a positive response. If either control sample
fails to react as expected, then the results for the patients' samples
cannot be trusted and the assay must be repeated.
Any serum from a patient that contains the antibody for SLE will recognize
the antigen in the well and bind to it. Each serum sample contains many
different types of antibodies, but because they are so specific in how they
react, usually no antibody will recognize the SLE antigen except the SLE
antibody.
anti-DNA antibody test: a blood test that is useful for the diagnosis and
follow-up of systemic lupus erythematosus (SLE). The test uses doublestranded deoxyribonucleic acid (DNA) as antigen to detect anti-DNA
antibodies. High titers characterize SLE, and low to moderate levels may
indicate other rheumatic diseases as well as chronic hepatitis, infectious
mononucleosis, and biliary cirrhosis.
Substrate - any substance on which an enzyme can act. In this example,
HRP (the enzyme) will interact with a substrate called ABTS (2,2'-azinobis3-ethylbenzothiazoleine-6-sulfonic acid) to produce a yellow solution.
Sera/Serum - a clear watery fluid obtained after removing blood cells and
other components from blood by centrifugation that will contain antibodies
Titer - the concentration of a substance in a solution. For instance, the
amount of a specific antibody in the serum.
Antibody - a type of protein called an immunoglobulin found in the blood
that is produced by immune cells in response to the presence of a foreign
particle (antigen). Antibodies are specific to each different type of foreign
particle.
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Primary : first antibody used in an immunoassay to detect the foreign
particle; in this case, we are testing to see if the serum from the
patients contains primary antibodies to SLE.
Secondary antibody: the second antibody used in an immunoassay
that detects the primary antibody. Note, this antibody must be made
in a different species (rabbit, donkey, horse) than the primary
antibody, in order to recognize the primary antibody as "foreign". In
this case, we are using HRP-tagged rabbit anti-human antibodies as
our secondary antibody.