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ANTIBODIES AND IMMUNODIAGNOSIS

POLYCLONAL ANTIBODY PRODUCTION

Polyclonal antibodies (or antisera) are antibodies that are obtained from
different B cell resources. They are a combination of immunoglobulin
molecules secreted against a specific antigen, each identifying a different
epitope.
These antibodies are typically produced by inoculation of a suitable mammal,
such as a mouse, rabbit or goat. Larger mammals are often preferred as the
amount of serum that can be collected is greater. An antigen is injected into
the mammal. This induces the B-lymphocytes to produce IgG
immunoglobulins specific for the antigen. This polyclonal IgG is purified from
the mammals serum.
By contrast, monoclonal antibodies are derived from a single cell line.
Many methodologies exist for polyclonal antibody production in laboratory
animals. Institutional guidelines governing animal use and procedures
relating to these methodologies are generally oriented around humane
considerations and appropriate conduct for adjuvant (agents which modify

the effect of other agents while having few if any direct effects when given
by themselves) use.
This includes adjuvant selection, routes and sites of administration, injection
volumes per site and number of sites per animal. Institutional policies
generally include allowable volumes of blood per collection and safety
precautions including appropriate restraint and sedation or anesthesia of
animals for injury prevention to animals or personnel.
The primary goal of antibody production in laboratory animals is to obtain
high titer, high affinity antisera for use in experimentation or diagnostic
tests. Adjuvants are used to improve or enhance an immune response to
antigens. Most adjuvants provide for an injection site, antigen depot which
allows for a slow release of antigen into draining lymph nodes.

Many adjuvants also contain or act directly as:


surfactants which promote concentration of protein antigens molecules over
a large surface area, and immunostimulatory molecules or properties.
Adjuvants are generally used with soluble protein antigens to increase
antibody titers and induce a prolonged response with accompanying memory
Such antigens by themselves are generally poor immunogens. Most complex
protein antigens induce multiple B-cell clones during the immune response,
thus, the response is polyclonal. Immune responses to non-protein antigens
are generally poorly or enhanced by adjuvants and there is no system
memory.
Antibodies are currently also being produced from isolation of human Blymphocytes to produce specific recombinant polyclonal antibodies. The
biotechnology company, Symphogen, produces this type of antibody for
therapeutic applications. They are the first research company to develop
recombinant polyclonal antibody drugs to reach phase two trials. This
production prevents viral and prion transmission.

MONOCLONAL ANTIBODY PRODUCTION

Monoclonal antibodies are typically made by fusing myeloma cells with the
spleen cells from a mouse that has been immunized with the desired
antigen. However, recent advances have allowed the use of rabbit B-cells to
form a Rabbit Hybridoma. Polyethylene glycol is used to fuse adjacent
plasma membranes, but the success rate is low so a selective medium in
which only fused cells can grow is used. This is because myeloma cells have
lost the ability to synthesize hypoxanthine-guanine-phosphoribosyl
transferase (HGPRT), an enzyme necessary for the salvage synthesis of
nucleic acids. The absence of HGPRT is not a problem for these cells unless
the de novo purine synthesis pathway is also disrupted. By exposing cells to

aminopterin (a folic acid analogue, which inhibits dihydrofolate reductase,


DHFR), they are unable to use the de novo pathway and become fully
auxotrophic for nucleic acids requiring supplementation to survive.

The selective culture medium is called HAT medium because it contains


hypoxanthine, aminopterin, and thymidine. This medium is selective for
fused (hybridoma) cells. Unfused myeloma cells cannot grow because they
lack HGPRT, and thus cannot replicate their DNA. Unfused spleen cells cannot
grow indefinitely because of their limited life span. Only fused hybrid cells,
referred to as hybridomas, are able to grow indefinitely in the media because
the spleen cell partner supplies HGPRT and the myeloma partner has traits
that make it immortal (as it is a cancer cell).

This mixture of cells is then diluted and clones are grown from single parent
cells on microtitre wells. The antibodies secreted by the different clones are
then assayed for their ability to bind to the antigen (with a test such as ELISA
or Antigen Microarray Assay) or immuno-dot blot. The most productive and
stable clone is then selected for future use.
The hybridomas can be grown indefinitely in a suitable cell culture medium.
They can also be injected into mice (in the peritoneal cavity, surrounding the
gut). There, they produce tumors secreting an antibody-rich fluid called
ascites fluid.
The medium must be enriched during in-vitro selection to further favour
hybridoma growth. This can be achieved by the use of a layer of feeder
fibrocyte cells or supplement medium such as briclone. Culture-medium
conditioned by macrophages can also be used. Production in cell culture is
usually preferred as the ascites technique is painful to the animal. Where
alternate techniques exist, this method (ascites) is considered unethical.

RADIOIMMUNOASSAY
The technique was first developed in 1960 by two endocrinologists, S. A.
Berson and Rosalyn Yalow, to determine levels of insulinanti-insulin
complexes in diabetics.
PRINCIPLE

The principle of RIA involves competitive binding of radiolabeled antigen and


unlabeled antigen to a high-affinity antibody.
The labeled antigen is mixed with antibody at a concentration that saturates
the antigen-binding sites of the antibody.

Then test samples of unlabeled antigen of unknown concentration are added


in progressively larger amounts.
The antibody does not distinguish labeled from unlabeled antigen, so the
two kinds of antigen compete for available binding sites on the antibody.
As the concentration of unlabeled antigen increases, more labeled antigen
will bdisplaced from the binding sites.
The decrease in the amount of radiolabeled antigen bound to specific
antibody in the presence of the test sample is measured in order to
determine the amount of antigen present in the test sample.
The antigen is generally labeled with a gamma-emitting isotope such as
125I, but beta-emitting isotopes such as tritium (3H) are also routinely used
as labels.
The radiolabeled antigen is part of the assay mixture; the test sample may
be a complex mixture, such as serum or other body fluids, that contains the
unlabeled antigen.
The first step in Antigen-Antibody Interactions: Principles and Applications
setting up an RIA is to determine the amount of antibody needed to bind
50%70% of a fixed quantity of radioactive antigen (Ag) in the assay
mixture.
This ratio of antibody to Ag is chosen to ensure that the number of epitopes
presented by the labeled antigen always exceeds the total number of
antibody binding sites.
Consequently, unlabeled antigen added to the sample mixture will compete
with radiolabeled antigen for the limited supply of antibody.
Even a small amount of unlabeled antigen added to the assay mixture of
labeled antigen and antibody will cause a decrease in the amount of
radioactive antigen bound, and this decrease will be proportional to the
amount of unlabeled antigen added.

To determine the amount of labeled antigen bound, the Ag-Ab complex is


precipitated to separate it from free antigen (antigen not bound to Ab), and
the radioactivity in the precipitate is measured.

A standard curve can be generated using unlabeled antigen samples of


known concentration (in place of the test sample), and from this plot the
amount of antigen in the test mixture may be precisely determined.
Several methods have been developed for separating the bound antigen
from the free antigen in RIA.
One method involves precipitating the Ag-Ab complex with a secondary antiisotype antiserum.
For example, if the Ag-Ab complex contains rabbit IgG antibody, then goat
anti-rabbit IgG will bind to the rabbit IgG and precipitate the complex.
Another method makes use of the fact that protein A of Staphylococcus
aureus has high affinity for IgG. If the Ag-Ab complex contains an IgG
antibody, the complex can be precipitated by mixing with formalin-killed S.
aureus.
After removal of the complex by either of these methods, the amount of free
labeled antigen remaining in the supernatant can be measured in a radiation
counter; subtracting this value from the total amount of labeled antigen
added yields the amount of labeled
antigen bound.
Various solid-phase RIAs have been developed that make it easier to
separate the Ag-Ab complex from the unbound antigen.
The amount of radiolabeled antigen bound to the beads can be measured
after the beads have been centrifuged and washed.
Alternatively, the antibody can be immobilized on polystyrene or
polyvinylchloride wells and the amount of free labeled antigen in the
supernatant can be determined in a radiation counter.
In another approach, the antibody is immobilized on the walls of microtiter
wells and the amount of bound antigen determined.
Because the procedure requires only small amounts of sample and can be
conducted in small 96-well microtiter plates (slightly larger than a 3 5 card),
this procedure is well suited for determining the concentration of a particular
antigen in large numbers of samples

RIA screening of donor blood has sharply reduced the incidence of


hepatitis B infections in recipients of blood transfusions.

In Clinical Immunology Antibodies for Inhalant Allergens,Allergy


Diagnosis
In Oncology Carcinoembryonic Antigen and Early Cancer Detection
and Diagnosis

ENZYME-LINKED IMMUNOSORBENT ASSAY


Enzyme-linked immunosorbent assay, commonly known as ELISA (or EIA), is
similar in principle to RIA but depends on an enzyme rather than a
radioactive label.
An enzyme conjugated with an antibody reacts with a colorless substrate to
generate a colored reaction product. Such a substrate is called a
chromogenic substrate.
A number of enzymes have been employed for ELISA, including alkaline
phosphatase, horseradish peroxidase, and -galactosidase.
These assays approach the sensitivity of RIAs and have the advantage of
being safer and less costly.

INDIRECT ELISA
A buffered solution of the antigen to be tested for is added to each well of a
microtiter plate, where it is given time to adhere to the plastic through
charge interactions.
A solution of non-reacting protein, such as bovine serum albumin or casein,
is added to block any plastic surface in the well that remains uncoated by the
antigen.
Next the primary antibody is added, which binds specifically to the test
antigen that is coating the well. This primary antibody could also be in the
serum of a donor to be tested for reactivity towards the antigen.
Afterwards, a secondary antibody is added, which will bind the primary
antibody. This secondary antibody often has an enzyme attached to it, which
has a negligible effect on the binding properties of the antibody.
A substrate for this enzyme is then added. Often, this substrate changes
color upon reaction with the enzyme. The color change shows that secondary
antibody has bound to primary antibody, which strongly implies that the
donor has had an immune reaction to the test antigen. This can be helpful in
a clinical setting, and in R&D.
The higher the concentration of the primary antibody that was present in the
serum, the stronger the color change. Often a spectrometer is used to give
quantitative values for color strength

SANDWICHED ELISA
A less-common variant of this technique, called "sandwich" ELISA, is used to
detect sample antigen. The steps are as follows:

Prepare a surface to which a known quantity of capture antibody is bound.


Block any nonspecific binding sites on the surface.
Apply the antigen-containing sample to the plate.
Wash the plate, so that unbound antigen is removed.

A specific antibody is added, and binds to antigen (hence the 'sandwich':


the Ag is stuck between two antibodies);
Apply enzyme-linked secondary antibodies as detection antibodies that
also bind specifically to the antibody's Fc region (non-specific).
Wash the plate, so that the unbound antibody-enzyme conjugates are
removed.
Apply a chemical that is converted by the enzyme into a color or
fluorescent or electrochemical signal.
Measure the absorbency or fluorescence or electrochemical signal (e.g.,
current) of the plate wells to determine the presence and quantity of antigen.

The image to the right includes the use of a secondary antibody conjugated
to an enzyme, though, in the technical sense, this is not necessary if the
primary antibody is conjugated to an enzyme.
COMPETITIVE ELISA
A third use of ELISA is through competitive binding. The steps for this ELISA
are somewhat different than the first two examples:
Unlabeled antibody is incubated in the presence of its antigen (Sample).
These bound antibody/antigen complexes are then added to an antigencoated well.
The plate is washed, so that unbound antibody is removed. (The more
antigen in the sample, the less antibody will be able to bind to the antigen in
the well, hence "competition.")
The secondary antibody, specific to the primary antibody is added. This
second antibody is coupled to the enzyme.
A substrate is added, and remaining enzymes elicit a chromogenic or
fluorescent signal.
The reaction is stopped in order to prevent eventual saturation of the signal

WESTERN BLOTTING

Identification of a specific protein in a complex mixture of proteins can be


accomplished by a technique known as Western blotting
In Western blotting, a protein mixture is electrophoretically separated on an
SDS-polyacrylamide gel (SDS-PAGE), a slab gel infused with sodium dodecyl
sulfate (SDS), a dissociating agent (Figure 6-12).
The protein bands are transferred to a nylon membrane by electrophoresis
and the individual protein bands are identified by flooding the nitrocellulose
membrane with radiolabeled or enzymelinked polyclonal or monoclonal
antibody specific for the protein of interest.
The Ag-Ab complexes that form on the band containing the protein
recognized by the antibody can be visualized in a variety of ways.
If the protein of interest was bound by a radioactive antibody, its position on
the blot can

be determined by exposing the membrane to a sheet of x-ray film, a


procedure called autoradiography. However, the most generally used
detection procedures employ enzyme-linked antibodies against the protein.
After binding of the enzymeantibody conjugate, addition of a chromogenic
substrate that produces a highly colored and insoluble product causes the
appearance of a colored band at the site of the target antigen.
The site of the protein of interest can be determined with much higher
sensitivity if a chemiluminescent compound along with suitable enhancing
agents is used to produce light at the antigen site.
Western blotting can also identify a specific antibody in a mixture.
In this case, known antigens of well-defined molecular weight are separated
by SDS-PAGE and blotted onto nitrocellulose.
The separated bands of known antigens are then probed with the sample
suspected of containing antibody specific for one or more of these antigens.
Reaction of an antibody with a band is detected by using either radiolabeled
or enzyme-linked secondary antibody that is specific for the species of the
antibodies in the test sample.
The most widely used application of this procedure is in confirmatory testing
for HIV, where Western blotting is used to determine whether the patient has
antibodies that react with one or more viral proteins.

IMMUNOELECTROPHORESIS
In immunoelectrophoresis, the antigen mixture is first electrophoresed to
separate its components by charge.
Troughs are then cut into the agar gel parallel to the direction of the electric
field, and antiserum is added to the troughs.
Antibody and antigen then diffuse toward each other and produce lines of
precipitation where they meet in appropriate proportions

An antigen preparation
(orange) is first
electrophoresed, which
separates the
component antigens on
the basis of charge.
Antiserum (blue) is then
added to troughs on one
or both sides of the

Immunoelectrophoresis is used in clinical laboratories to detect the presence


or absence of proteins in the serum.
A sample of serum is electrophoresed, and the individual serum components
are identified with antisera specific for a given protein or immunoglobulin
class (Figure 6-6b).
This technique is useful in determining whether a patient produces
abnormally low amounts of one or more isotypes, characteristic of certain
immunodeficiency diseases.

It can also show whether a patient overproduces some serum protein, such
as albumin, immunoglobulin, or transferrin.
Immunoelectrophoresis is a strictly qualitative technique that only detects
relatively high antibody concentrations (greater than several hundred g/ml),
it utility is limited to the detection
Rocket electrophoresis, does permit measurement of antigen levels.
In rocket electrophoresis, a negatively charged antigen is electrophoresed in
a gel containing antibody.
The precipitate formed between antigen and antibody has the shape of a
rocket, the height of which is proportional to the concentration of antigen in
the well.
One limitation of rocket electrophoresis is the need for the antigen to be
negatively charged for electrophoretic movement within the agar matrix.
Some proteins, immunoglobulins for example, are not sufficiently charged to
be quantitatively analyzed by rocket electrophoresis; nor is it possible to
measure the amounts of several antigens in a mixture at the same time.

SDS-PAGE
Polyacrylamide gels are prepared by the free radical polymerization of
acrylamide and the cross linking agent N
N methylene bis acrylamide
Acrylamide + N N methylene bis acrylamide Polyacrylamide
[Chemical PolymerizationAmmonium persulfate (catalyst) + TEMED
(N,N N N tetramethylethylene diamine)]

Procedure
Sample preparation

Samples may be any material containing proteins, for example prokaryotic or


eukaryotic cells, tissues, viruses, environmental samples, or purified
proteins. In the case of solid tissues, these are often first broken down
mechanically using a blender (for larger sample volumes), using a
homogenizer (smaller volumes), by sonicator or by using cycling of high
pressure. Cells may also be broken open by one of the above mechanical
methods.

In the case of tissues or cells, a combination of biochemical and mechanical


techniques including various types of filtration and centrifugation may be
used to separate different cell compartments and organelles prior to
electrophoresis.

The sample to be analyzed is mixed with SDS, an anionic detergent which


denatures secondary and nondisulfidelinked tertiary structures, and applies
a negative charge to each protein in proportion to its mass. Heating the
samples to at least 60C further promotes protein denaturation, helping SDS
to bind

A tracking dye may be added to the protein solution. This typically has a
higher electrophoretic mobility than the proteins to allow the experimenter to
track the progress of the protein solution through the gel during the
electrophoretic run.

Preparing acrylamide gels


The gels typically consist of acrylamide, bisacrylamide, SDS, and a buffer
with an adjusted pH. The solution may be degassed under a vacuum to
prevent the formation of air bubbles during polymerization. [5] A source of
free radicals and a stabilizer such as ammonium persulfate and TEMED are
added to initiate polymerization.[6] The polymerization reaction results in a
gel because of the added bisacrylamide, generally about 1 part in 35 relative
to acrylamide, which can form cross-links between two polyacrylamide
molecules. The ratio of acrylamide to bisacrylamide can be varied for special
purposes. The acrylamide concentration of the gel can also be varied,
generally in the range from 5% to 25%. Lower percentage gels are better for
resolving very high molecular weight proteins, while much higher
percentages are needed to resolve smaller proteins. Determining how much
of the various solutions to mix together to make gels of particular acrylamide
concentration is possible.

Gels are usually polymerized between two glass plates in a gel caster, with a
comb inserted at the top to create the sample wells. After the gel is
polymerized the comb can be removed and the gel is ready for
electrophoresis.

Electrophoresis

Various buffer systems are used in SDS-PAGE depending on the nature of the
sample and the experimental objective. The buffers used at the anode and

cathode may be the same or different

An electric field is applied across the gel, causing the negatively-charged


proteins to migrate across the gel towards the positive (+) electrode (anode).
Depending on their size, each protein will move differently through the gel
matrix: short proteins will more easily fit through the pores in the gel, while
larger ones will have more difficulty (they encounter more resistance). After
a set amount of time (usually a few hours- though this depends on the
voltage applied across the gel; higher voltages run faster but tend to
produce somewhat poorer resolution), the proteins will have differentially
migrated based on their size; smaller proteins will have traveled farther
down the gel, while larger ones will have remained closer to the point of
origin. Proteins may therefore be separated roughly according to size (and
thus molecular weight); however certain glycoproteins behave anomalously
on SDS gels.

ENHANCED CHEMILUMINESCENCE

Enhanced chemiluminescence is a common technique for a variety of


detection assays in biology. A horseradish peroxidase enzyme (HRP) is
tethered to the molecule of interest (usually through labeling an
immunoglobulin that specifically recognizes the molecule). This enzyme
complex then catalyzes the conversion of the enhanced chemiluminescent
substrate into a sensitized reagent in the vicinity of the molecule of interest,
which on further oxidation by hydrogen peroxide, produces a triplet (excited)
carbonyl, which emits light when it decays to the singlet carbonyl. Enhanced
chemiluminescence allows detection of minute quantities of a biomolecule.
Proteins can be detected down to femtomole quantities, well below the
detection limit for most assay system