ANTIGEN ANTIBODY REACTION

THE INTERACTION OF THE ANTIBODY MOLECULE WITH SPECIFIC ANTIGEN

Antigen-Binding Site Form From Localized Region Of Hypervariable Sequence
V regions of antibody molecule differ from one to another as compare to the
sequence variability which is likely the same. Sequence variability is distributed
evenly throughout the V regions but it is concentrated in certain segments as seen in
variability plot (Fig 1). The amino acids sequences are compared to different antibody
V regions. In V H and V L domains, three particularly variable segments can be
identified. They are the hypervariable regions and denoted by HV1, HV2 and HV3. In
the light chains they are located at residues 28 to 35, 49 to 59 and 92 to 103
respectively. While at the heavy chains they run roughly from 30 to 36, 49 to 65, and
95 to 103 respectively. The most variable part of the domain is in the HV3 regions.
The termed framework regions are the regions between hypervariable regions, which
comprise the rest of the V domain and show less variability. There are four
framework regions in each V domain designated FR1, FR2, FR3 and FR4.
The hypervariable sequences correspond to three loops at the outer edge of the
barrel, which are juxtaposed in the folded domain whereas the framework
regions form the sheets provide structural framework of the domain. Hence,
diversity is not only concentrated in particular parts of the V domain sequence, but it
is also localized to a particular region on surface of the molecule. Three
hypervariable loops from each domain are brought together when V H and V L
immunoglobin domains paired in the antibody molecule and it also create a single
hypervariable site at the tip of each arm of the molecule. This is called the antibody
combining complex or antigen-binding site, which regulate the antigen specificity of
the antibody.
The term complementarily-determining regions or CDRs are a common term for the
six hypervariable loops because the surface they form is complementary to that of
the antigen they bind. There are three CDRs from each of light and heavy chains,
namely CDR1, CDR2 and CDR3. Because from both V H and V L domains the
CDRs contribute to the antigen binding site, it is the combination of the heavy and
light chain, and not either alone of the chain, that determine the final antigen
specificity. Hence, the immune system is able to generate antibodies of different
specificity through one way which is by generating different combinations of heavychain and light-chain V regions.

FIGURE 1

FIGURE 2

Antibodies Bind To Conformational Shapes On Surfaces Of Antigens

Antibodies biological functions are to bind to pathogens and their products and to
promote the removal from the body of host. Antibody generally able to recognize only
a small region on surface of a large molecule such as polysaccharides or protein.
Epitope or antigenic determinant is the term of the structure that is recognized by
an antibody. Some of the most important pathogens have polysaccharides coats and
an antibody that recognize epitopes formed by the sugar subunits of these molecules
are essential in providing immune protection against such pathogens.
In many cases, the antigens that provoke an immune response are proteins. An
example include viral coat proteins are recognize by protective antibodies against
viruses. In all such cases, located on the surface of the protein are the structures that
able the antibody to recognize it. Protein folding brought together such sites that are
likely to be composed by amino acids from different parts of the polypeptide chain.
Antigenic determinants of this kind are known as conformational or discontinuous
epitopes. This is because the structure recognized is composed of segments of the
protein that are discontinues in the amino acids sequence of the antigen but are
brought together in the three-dimensional structure.
On the other hand, continuous or linear epitope are an epitope composed of a
single segment of polypeptide chain. Although most antibodies raised against intact,
fully folded proteins recognize discontinues epitope, some will bind to peptide
fragments of proteins. Contrarily, antibodies raised against peptides of a protein or
against synthetic peptides corresponding to part of its sequence are occasionally
found to bind to the natural folded proteins. In some cases, this makes it possible to
use synthetic peptides in vaccines that aim at raising antibodies against a pathogen
protein.

PRECIPITATION
In an aqueous solution antibody and antigen interact to form a lattice that will
develops into a visible precipitate. These antibodies that aggregate a soluble
antigens are called precipitins. The formations of Ag-Ab complex occurs within
minutes, however formations of the visible precipitate occurs more slowly and
sometimes takes a day or two to reach completion.
The formation of Ag-Ab lattice depend on the valency of antigen and antibody:

Antibody needs to be bivalent. A precipitate will not form with monovalent Fab
fragments.
Antigen must be either bivalent or polyvalent. Meaning that it must have at
least two copies of the same epitope or have different epitopes that react with
different antibodies present in polyclonal antisera.

Experiments conducted with myoglobin illustrate on the requirement that protein

antigen need to be either bivalent or polyvalent for a precipitations reactions to occur.
Myoglobin fails to precipitates with a specific monoclonal antibodies because it
contains multiple, distinct epitope but only a single copy of each epitopes. While with
specific polyclonal antisera myoglobin precipitates well. Thus it can form a crosslinked lattice structure with polyclonal antisera but not with monoclonal antisera.
Through the time, faster methods are invented because they are far more sensitive
and require only very small quantities of antigen or antibody. Table 1 shows
comparison of sensitivity or minimum amount of antibody detectable by a number of
immunoassays.

TABLE 1

Precipitation Reactions in Fluid Yield a Precipitin Curve

By placing a constant amount of antibody in a series of tubes and adding increasing
amount of antigen to the tubes a quantitative precipitation reaction can be performed.
This method was used to calculate the amount of antigen or antibody in a sample of
interest. When the precipitate had formed, each tube is then centrifuged to pellet the
precipitate, the supernatant is then poured off and the amount of precipitate is
measured. A precipitin curve is plot by amount of precipitate against increasing
antigen concentrations. Figure 3-b shows, excess of either antibody or antigen
interferes with maximal precipitation, which occurs in equivalence zone, which shows
ratio of antibody to antigen is optimal. The complex increases in size and precipitates
out of the solution as large multimolecular lattice is formed at equivalence. As shown
in Figure 3, under conditions which antibody or antigen is excess, extensive lattices
do not form and precipitation is inhibited. Today, the quantitative reactions is seldom
used in experiments, however the principles of antigen excess, antibody excess and
equivalence apply to many Ag-Ab reactions.

FIGURE 3

Precipitation Reactions in Gels Yield Visible Precipitin Lines

When an antibody is incorporated into agar and an antigen diffuses into the antibodycontaining matrix or when an antigen and antibody diffuse toward one another in an
agar, a visible line of precipitation will form. As in a precipitation reactions in fluid,
visible precipitation occurs in the region of equivalence, whereas no visible
precipitate forms in regions of antibody or antigen excess. To determine relative
concentrations of an antibodies or an antigens, to compare antigens or to determine
the relative purity of an antigen preparation two types of immunodiffusion reactions
can be used. The radial immunodiffusion (Mancini method) and double
immunodiffusion (Ouchterlony method); are both carried out in a semisolid medium
such as agar.
In radial immunodiffusion, an antigen sample is placed in a well and allowed to
diffuse into agar containing a suitable dilution of an antiserum. The region of
equivalence is established and a ring of precipitation, a precipitin ring, forms around
the well when an antigen diffuses into the agar (Figure 4-upper panel). The area of
the precipitin ring is proportional to the concentration of an antigen. By comparing the
area of the precipitin ring with a standard curve (this is obtained by measuring the
precipitin areas of known concentrations of the antigen), the concentration of the
antigen sample can be determined. Both antigen and antibody diffuse radially from
wells toward each other in the Ouchterlony method, thereby establishing a
concentrations gradient. When equivalent is reached, a visible line of precipitation, a
precipitin line, forms (Figure 4-lower panel)

FIGURE 4

AGGLUTINATION
An interaction that occurs between antibody and a specific antigen will results in
visible clumping known as agglutination. Agglutinins are antibodies that produce such
reactions. The principle of agglutinations are similar to precipitation reactions which is
they depend on the crosslinking of polyvalent antigens. Prozone effect is the
inhibition of agglutination when there is an excess of antibody. Because prone effect
can be encountered in many types of immunoassays, to understand the basis of the
phenomenon is importance.
There are several mechanism that can cause prozone effect. Firstly, number of
binding site may exceed the number of epitopes at high antibody concentrations.
This resulted in most antibodies bind to antigen univalently instead of instead of
multivalently. When an antibody bind univalently they cannot cross-linked one
antigen to another. Prozone effects are readily diagnosed by performing the assay at
a variety of antibody/antigen concentrations. As one dilutes to an optimum antibody
concentrations, one sees higher lever of agglutinations being measured in the assay.
When one is using polyclonal antibodies, the prozone effect can only occur for
another reason.
Antiserum may contain high concentrations of antibodies that bind to antigen
however it don not induce agglutination; these antibodies called as incomplete
antibodies, are one of the IgG class. At high concentrations of IgG incomplete
antibodies will occupy the antigenic sites, thus blocking access by IgM, which is a
good agglutinin. This effect is not seen with agglutinating monoclonal antibodies. It is
because of lacking of agglutinating activity of an incomplete antibody that may be
due to restricted flexibility in the hinge region making it difficult for the antibody to
assume the required angle for optimal cross-linking of epitopes on two or more
particulate antigens. Alternatively, the density of epitope distribution in deep pockets
of particulate antigens may make it difficult for the antibodies specific for theses
epitopes to agglutinate certain particulate antigens. When feasible, the solution to
both of these problems is to try different antibodies that may react with other epitopes
of the antigen that do not present in these limitations.

COMPLEMENT FIXATION REACTIONS

A German scientist name Richard Pfeiffer had discover in 1894 that when cholera
were injected into peritoneum of a guinea pig immunized against the infection, the pig
would die rapidly. Bordet discovered this bacteriolysis, it did not occur when the
bacteria was injected into a non immunized guinea pig but did so when the same
animal had received the antiserum from an immunized animal. When the bacteria
and the antiserum were mixed in a test tube the bacteriolysis did not take place
unless fresh antiserum was used. However, when Bordet heated the antiserum to 55
degrees centigrade, it lost its power to kill the bacteria. He find out that he could
restore the bacteriolytic power of the antiserum if he add a little fresh serum from a
non immunized animal, Bordet then concluded that the bacteria-killing phenomenon
was due to the combined action of two distinct substances: an antibody in antiserum
and a non specific substance. The antibody specifically acted against a particular
kind of bacterium whereas the non specific substance is sensitive to heat and was
latter called as alexine by Bordet. (later named complement).
In a series of experiments conducted later, Bordet had also learned that injection of
red blood cells from one animal species (rabbit cells in the initial experiments) into
another species (guinea pigs) caused serum of the second species to quickly destroy
the red cells of the first. Although serum lost its power to kill the red cells when
heated to 55 degrees centigrade its potency was restored when alexine
(complement) was added. It become clear to Bordet that haemolytic serums acted
exactly as bacteriolytic serums; thus he had uncovered the basic mechanism by
which animal bodies defend or immunized themselves against the invasion of foreign
elements. Bordet and his colleague eventually find a way to implement their
discovery. They determined that alexine was bound to red blood cells or to bacteria
during immunizing process. When the red cells was added to a normal serum mixed
with specific form of bacteria in a test tube, the bacteria remained active while red
cells were destroyed through fixation of the alexine. However, while antiserumcontained antibody that specific to the bacteria destroy the alexine and solution was
separated into a layer of clear serum overlaying the intact red cells. Hence, it was
possible to visually determine the presence of bacteria in patient blood. This was
later known as complement fixation test.
Bordet and his associates applied these findings to various other infections, like
typhoid fever, carbuncle, and hog cholera.

FIGURE 5

RADIOIMMUNOASSAY (RIA)
One of the most sensitive techniques for detecting antigen or antibody is
radioimmunoassay (RIA). The technique was first developed in 1960 by two
endocrinologists, S. A. Berson and Rosalyn Yalow, to determine levels of insulinantiinsulin complexes in diabetics. Although their technique encountered some
skepticism, it soon proved its value for measuring hormones, serum proteins, drugs,
and vitamins at concentrations of 0.001 micrograms per milliliter or less.
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 be displaced 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.

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

The principle of ELISA is that 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.
Types of ELISA (FIGURE 6)
1. Indirect ELISA: Serum or some other sample containing primary antibody (Ab 1) is
added to an antigen-coated microtiter well and allowed to react with the antigen
attached to the well. After any free Ab 1 is washed away, the presence of antibody
bound to the antigen is detected by adding an enzyme-conjugated secondary antiisotype antibody (Ab2), which binds to the primary antibody. Any free Ab 2 then is
washed away, and a substrate for the enzyme is added. The amount of colored
reaction product that forms is measured by specialized spectrophotometric plate
readers, which can measure the absorbance of all of the wells of a 96-well plate in
seconds.
2. Sandwich ELISA: The antibody (rather than the antigen) is immobilized on a
microtiter well. A sample containing antigen is added and allowed to react with the
immobilized antibody. After the well is washed, a second enzyme-linked antibody
specific for a different epitope on the antigen is added and allowed to react with the
bound antigen. After any free second antibody is removed by washing, substrate is
added, and the colored reaction product is measured.

3. Competitive ELISA: antibody is first incubated in solution with a sample containing

antigen. The antigen-antibody mixture is then added to an antigen-coated microtiter
well. The more antigen present in the sample, the less free antibody will be available
to bind to the antigen-coated well. Addition of an enzyme-conjugated secondary
antibody (Ab2) specific for the isotype of the primary antibody can be used to
determine the amount of primary antibody bound to the well as in an indirect ELISA.
In the competitive assay, however, the higher the concentration of antigen in the
original sample, the lower the absorbance.

FIGURE 6

IMMUNOASSAYS
Immunoassays are bioanalytical methods in which the quantitation of the analyte
depends on the reaction of an antigen (analyte) and an antibody. Principally, these
methods are based on a competitive binding reaction between a fixed amount of
labelled form of an analyte and a variable amount of unlabelled sample analyte for a
limited amount of binding sites on a highly specific anti-analyte antibody. When these
immunoanalytical reagents are mixed an incubated, the analyte is bound to the
antibody forming an immune complex. This complex is separated from the unbound
reagent fraction by physical or chemical separation technique. Analysis is achieved
by measuring the label activity (e.g. radiation, fluorescence, or enzyme) in either of
the bound or free fraction.
Immunoassay methods have been widely used in many important areas of
pharmaceutical analysis such as diagnosis of diseases, therapeutic drug monitoring,
clinical pharmacokinetic and bioequivalence studies in drug discovery and
pharmaceutical industries. The analysis in these areas usually involves measurement
of very low concentrations of low molecular weight drugs, macromolecular
biomolecules of pharmaceutical interest, metabolites, and/or biomarkers which
indicate disease diagnosis or prognosis.
The importance and widespread of immunoassay methods in pharmaceutical
analysis are attributed to their inherent specificity, high throughput, and high
sensitivity for the analysis of wide range of analytes in biological samples. The
detection system in immunoassays depends on readily detectable labels (e.g.
radioisotopes or enzymes) coupled to one of the immunoanalytical reagents (i.e.
analyte or antibody).

FLUORESCENT ANTIBODIES
In 1944, Albert Coons showed that antibodies could be labeled with molecules that
have the property of fluorescence. Fluorescent molecules absorb light of one
wavelength (excitation) and emit light of another wavelength (emission). If antibody
molecules are tagged with a fluorescent dye, or fluorochrome, immune complexes
containing these fluorescently labeled antibodies (FA) can be detected by colored
light emission when excited by light of the appropriate wave- length.
Antibody molecules bound to antigens in cells or tissue sections can similarly be
visualized. The emitted light can be viewed with a fluorescence microscope, which is
equipped with a UV light source. In this technique, known as immunofluorescence,
fluorescent compounds such as fluorescein and rhodamine are in common use, but
other highly fluorescent substances are also routinely used, such as phycoerythrin,
an intensely colored and highly fluorescent pigment obtained from algae. These
molecules can be conjugated to the Fc region of an antibody molecule without
affecting the specificity of the antibody. Each of the fluochromes below absorbs light
at one wavelength and emits light at a longer wavelength:
Fluorescein an organic dye that is widely used label for immunofluorescence
procedures absorbs blue light (490 nm) and emits an intense yellow-green
fluorescence (517 nm).
Rhodamine another organic dye, absorbs in the yellow-green range (515 nm)
and emits a deep red fluorescence (546 nm). Because it emits fluorescence
at a longer wavelength than fluorescein, it can be used in two-color
immunofluorescence assays. An antibody specific to one determinant is
labeled with fluorescein, and an antibody recognizing a different antigen is
labeled with rhodamine.
Phycoerythrin is an efficient absorber of light (~30-fold greater than
fluorescein) and a brilliant emitter of red fluorescence, stimulating its wide use
as a label for immunofluorescence.
Immunofluorescence has been applied to identify a number of subpopulations of
lymphocytes, notably the

+
CD 4

and

+
CD 8

T-cell subpopulations. The

technique is also suit- able for identifying bacterial species, detecting Ag-Ab
complexes in autoimmune disease, detecting complement components in tissues,
and localizing hormones and other cellular products stained in situ. Indeed, a major
application of the fluorescent-antibody technique is the localization of antigens in
tissue sections or in subcellular compartments. Because it can be used to map the
actual location of target antigens, fluorescence microscopy is a powerful tool for
relating the molecular architecture of tissues and organs to their overall gross
anatomy.

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I.

(2006).

Immunoassay

Methods

and

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Applications

in

Pharmaceutical Analysis: Basic Methodology and Recent Advances. InternatIonal

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Complement

Fixation

Test.

(n.d.).

Retrieved

January

14,

2016,

from

http://www.nos.org/media/documents/dmlt/Microbiology/Lesson-61.pdf
Murphy, K., & Travers, P. (2012). Janeway's immunobiology (8th ed.). New York:
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Rabson, A., & Roitt, I. (2005). Really essential medical immunology (2nd ed.).
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