Interactions between antigen and antibody

Interaction between antigen and antibody is a bimolecular association and it does not lead to an irreversible
chemical alteration in either the antibody or the antigen. The association between an antigen and antibody
involves various non-covalent interactions between the antigenic determinant (epitope) of the antigen and the
variable-region (V
H
/V
L
) domain of the antibody molecule. The specific association of antigens and antibodies
is dependent on hydrogen bonds, hydrophobic interactions, electrostatic forces, and van der Walls interactions,
which are all weak and non-covalent in nature. So a large number of such weak interactions are required to
form a strong antigen-antibody (Ag-Ab) interaction. These interactions can only take place if the antigen and
antibody molecules are close enough for some of the individual atoms to fit into complementary recesses. The
complementary regions of an antibody are its two antigen binding sites. Like antibodies, antigens can be
multivalent, either through multiple copies of the same epitope, or through the presence of multiple epitopes
that are recognized by multiple antibodies. Interactions involving multivalency can produce more stabilized
complexes.
Properties of antigen-antibody interaction
Affinity
A strong antigen-antibody interaction depends on a very close fit between antigen and antibody. The combined
strength of the non-covalent interactions between a single antigen-binding site on an antibody and a single
epitope is the affinity of the antibody for that epitope. Affinity is the sum of the attractive and repulsive forces
operating between the antigenic determinant and the combining site of the antibody. There exists a weak
association between low-affinity antibodies and antigen, which dissociates easily whereas high-affinity
antibodies bind antigen more tightly and remain bound longer.
The binding of an antibody (Ab) to its antigen (Ag) is reversible, so the binding reaction can be expressed as:
Ag + Ab Ag-Ab ..(1)
Where k
1
is the forward rate constant and k
-1
is the reverse rate constant. The ratio k
1
/k
-1
is the association
constant K
a
, a measure of affinity. K
a
is the strength of the interaction and is expressed as:
K
a
= [Ag-Ab]/ [Ab] [Ag]
In this equation, [Ag-Ab] is the molar concentration of the antibody-antigen complex, and [Ab] and [Ag] are
the molar concentrations of the antibody and antigen, respectively. Affinity constants can vary widely between
different antibodies and antigens, and are affected by pH, temperature, and solvent.
The dissociation of the antigen-antibody complex can be expressed as:
Ag-Ab Ab + Ag(2)
The equilibrium constant for the above reaction is expressed as K
d
, and which is the reciprocal of K
a
, and is
given by
K
d
= [Ab] [Ag]/ [Ab-Ag]
The affinity constants described above apply to single site interactions. However, all naturally occurring
antibodies are multivalent and their functional affinity is dependent not only on their intrinsic affinity for
antigen but also on the number of binding sites (2 for IgD,G and E and 10 for IgM). The association constant
K
a
, for binding of a univalent ligand to a multivalent antibody may be expressed as:
K
a
= [Ab-Ag]/ [Ab] [Ag] = r/c(n-r)
where, at equilibrium, c is the concentration of free ligand, r represents the ratio of the concentration of bound
ligand to total antibody concentration and n is the maximum number of binding sites per antibody molecule
(the antibody valence). This expression can be rearranged to give the Scatchard equation:
r/c = K
a
n - K
a
r
A set of values of r and c can be obtained from a series of experiments in which the concentration of antibody
is kept constant with a varying concentration of ligand and from these a plot (Scatchard plot) can be
constructed in which r/c is plotted against r. From a Scatchard plot. Both the equilibrium constant (K
a
) and the
number of binding sites per antibody molecule (n) or its valency can be obtained. If all antibodies have the
same affinity, then a Scatchard plot gives a straight line with a slope of K
a
(figure-1) and if the antibody
mixture has a range of affinities (polyclonal antibodies), a scatchard plot gives a curved line whose slope is
constantly changing.


Figure 1: Scatchard plot of the antibodies having same affinity

Avidity
When complex antigens containing multiple repeating antigenic determinants are mixed with antibodies
containing multiple binding sites, the interaction of an antibody with an antigen at one site will increase the
probability of reaction between those two molecules at a second site. The strength of such multiple interactions
between a multivalent antibody and antigen is called the avidity. Avidity is more than the sum of the individual
affinities. Affinity describes the strength of interaction between antibody and antigen at single antigenic sites.
Avidity is perhaps a more informative measure of the overall stability or strength of the antibody-antigen
complex, which is controlled by three major factors: antibody-epitope affinity; the valence of both the antigen
and antibody; and the structural arrangement of the interacting parts.
Specificity
Specificity of an antigen-antibody reaction refers to the ability of an individual antibody combining site to
react with only one antigenic determinant or the ability of a population of antibody molecules to react with
only one antigen. Antigen-antibody reactions are highly specific in nature, that means one antibody can react
with its own antigen. A strong antigen-antibody interaction depends on a very close fit between the antigen
and antibody which requires high degree of specificity.
Cross reactivity
The most striking feature of the antigenantibody interaction is its high specificity and affinity. But in some
cases, antibody elicited by one antigen can cross react with an unrelated antigen, which is known as cross-
reactivity. Cross reactions arise because the cross reacting antigen shares an epitope in common with the
immunizing antigen or because it has an epitope which is structurally similar to one on the immunizing
antigen.
Cross-reactivity is often observed among polysaccharide antigens that contain similar oligosaccharide
residues. The ABO blood group antigens are the glycoproteins expressed on red blood cells. Subtle differences
in the terminal residues of the sugars attached to these surface proteins distinguish the A and B blood group
antigens. An individual lacking one or both of these antigens will have serum antibodies to the missing
antigens. Thus, anti-A antibodies are found in the serum of group O and B individuals and anti-B antibodies
are found in the serum of group O and A individuals. Group AB individuals are believed not to have anti-A
nor anti-B antibodies because they express both antigens on their red cells. The antibodies are induced by
exposure to cross-reacting microbial antigens present on common intestinal bacteria. The blood-group
antibodies, although elicited by microbial antigens, will cross react with similar oligosaccharides on foreign
red blood cells, providing the basis for blood typing tests and accounting for the necessity of compatible blood
types during blood transfusions.
A number of viral and bacterial antigens elicit antibody that cross reacts with the host-cell components,
which results in a tissue damaging reaction. Cross-reactivity is also exhibited by some vaccines.

Types of antigen-antibody reaction
Precipitation reactions
Precipitation reaction is the reaction, in which a soluble antibody reacts with a soluble antigen to give an
insoluble product or the precipitate. Soluble antibodies that aggregate soluble antigens are called precipitins.
Soluble antigen that induces the formation of a specific precipitin is called a precipitinogen.
When the antigens, which must have at least two epitopes per molecule are cross-linked by the bivalent
antibodies, a lattice formation occurs which ultimately develops into a visible precipitate. For the lattice to be
formed, the bivalent antibody will bind to epitopes on two different antigens. A second antibody molecule
combines with the second epitope on one of the antigen molecules and a third epitope on another antigen
molecule, so that the complex is formed. The complex continues to grow and when it is sufficiently large, it
becomes insoluble and can be visible as a precipitate. Thus, formation of the visible precipitate takes
sufficiently longer time than formation of the soluble antigen-antibody complexes.
Precipitation reaction can occur using polyclonal antibodies or mixture of monoclonal antibodies.
Polyclonal antibodies can form large aggregates, that precipitate out of solution. If the antigen is monovalent
or a single monoclonal antibody is used, the antibody can link only two molecules of antigen and no
precipitate is formed.
There is a marked effect on the precipitation reaction by changing the concentration of the antigen. A
quantitative precipitation reaction can be performed by placing a constant amount of antibody in a series of
tubes and adding increasing amounts of antigens to the tubes. Plotting the amount of precipitate against
increasing antigen concentrations yields a precipitation curve. A precipitation curve for a system of antigen-
antibody in the figure (figure-2) shows three zones, among which the first one is the zone of antibody excess or
prozone, in which the antigen concentration is very low and that of the antibody is relatively high, as a result of
which precipitation is inhibited, formation of small complexes occur and residual antibodies will remain in the
supernatant.


Figure 2: A precipitation curve showing three zones

The second zone is the equivalence zone, also the zone of maximal precipitation in which antigen and
antibody form large insoluble complexes and there is neither antigen nor antibody present in the supernatant.
The third zone is the zone of antigen excess or postzone, in which the antigen concentration is very high, and
therefore with increasing the amounts of antigen, the lattice size becomes too small to precipitate as a result of
which precipitation is inhibited and binding of antigen-antibody is absent in the supernatant.
Types of precipitation reactions
Precipitation reactions occur both in solution and in gel phase, where antigen-antibody forms a precipitate.
Similar to the precipitation reaction in fluid, visible precipitation occurs in the region of equivalence and no
visible precipitate forms in regions of antibody or antigen excess in gel phase.
Fluid phase precipitation
Fluid phase precipitation is a double diffusion method, where in a capillary tube an antigen solution is layered
over an antibody solution. Both antigen and antibody will diffuse towards each other and at the interface,
when antibody recognizes antigen, precipitate forms. The amount of the precipitate is proportional to the
concentration of both the antigen and antibody. This method is used to identify unknown antigen or unknown
antibody.
Gel phase precipitation
In gel phase precipitation, instead of solution, gel is used as a semisolid medium. The gel contains pores that
allow the movement of molecules. In immunoprecipitation reactions, the gel is a derivative of agar and is
called agarose. Agarose gel allows soluble antigen and /or antibody to diffuse through the pores until the
antigen and antibody reach the optimal concentration for lattice formation. Smaller molecules move through
the gel faster than larger molecules.
When antibody is incorporated into the agar, and antigen diffuses into the antibody-containing matrix, or
when antigen and antibody diffuse toward one another in agar, a visible line of precipitation will form unlike a
precipitation curve in fluids. Two types of immunodiffusion reactions can be used to determine relative
concentrations of antibodies or antigens, i.e. radial immuno-diffusion and double immuno-diffusion.

Radial immunodiffusion (The Mancini method)
In radial immunodiffusion (figure-3), an antigen sample is placed in a well and allowed to diffuse into agar
containing a suitable dilution of an antiserum. The antigen diffuses in all directions from the well, and
accordingly the region of equivalence is established and a ring of precipitation (precipitin ring) forms around
the well. The area of the precipitin ring is proportional to the concentration of antigen. The diameter of the area
of precipitation (including the well diameter) is measured to determine the concentration of antigen.


Figure 3: Radial immunodiffusion

Double immunodiffusion (The Ouchterlony method)
In double immunodiffusion (figure-4), if antigen to be detected, a known reagent antibody is placed in the
center well and the unknown samples are placed in the surrounding well. If antibody is to be detected,
unknown antigen is placed in the center.


Figure 4: Double immunodiffusion

After each of the samples and reagents have been added to the appropriate wells, diffusion occurs and both
antigen and antibody diffuse radially from wells toward each other, thereby establishing a concentration
gradient. A line of precipitation forms at the zone of equivalence.

Immunoelectrophoresis
Immunoelectrophoresis is a gel electrophoretic technique which uses both electrophoresis and double
diffusion. The samples that contain the proteins (the antigen mixture) to be analyzed are added to the wells on
the gel plate. The mixture of samples could contain serum from healthy individuals as well as from infected
persons. In this process, the antigen mixture is first electrophoresed to separate its components by charge. A
trough is created parallel to the length of the electric field, into which a single purified species of antibody or
known mixture of antibodies is added. The antibody molecules diffuse outward from the trough solution into
the gel. When an antigen is encountered by an antibody, formation of a visual precipitate occurs. Precipitin
arcs form at the zone of equivalence between the antigen and specific antiserum (figure-5). The pattern of
precipitation can reveal antigenic differences between the normal serum and the serum from an infected
person. If a nonspecific antiserum is placed in the trough, then only one arc will be formed if the particular
serum component is present.


Figure 5: Immunoelectrophoresis

Immunoelectrophoresis is used in clinical laboratories to detect the presence or absence of proteins in the
serum. This process separates the various proteins in a sample in an electric field and then probes the separated
proteins using the desired antiserum. In the clinical laboratory setting, immunoelectophoresis is used to
examine alterations in the content of serum, especially changes concerned with immunoglobulins. Change in
the immunoglobulin profile can be the result of immunodeficiencies, chronic bacterial or viral infections, and
infections of a fetus.
Another electrophoretic precipitation technique, used primarily in research and coagulation laboratories, is
the rocket, or Laurell technique. Rocket electrophoresis is used to quantitate antigens other than
immunoglobulins. In this process, a negatively charged antigen is electrophoresed in a gel containing antibody.
As the antigen migrates through the gel, it combines with antibody, precipitation occurs. The precipitate
formed between the antigen and antibody has the shape of a rocket, the height of which is proportional to the
concentration of the antigen in the well.

Agglutination reactions
The interaction between antibody and a particulate antigen results in visible clumping, called agglutination.
The general term agglutinin is used to describe antibodies that agglutinate particulate antigens. Agglutination is
a serological reaction and is very similar to the precipitation reaction. Both reactions are highly specific
because they depend on the specific antibody and antigen pair. As an excess of antibody inhibits precipitation
reactions, such excess can also inhibit agglutination reactions, this inhibition is known as prozone effect. The
main difference between these two reactions is the size of antigens. For precipitation, antigens are soluble
molecules, and for agglutination, antigens are large, easily sedimented particles. Agglutination reactions can be
used to type blood cells for transfusion, to identify bacterial cultures, and to detect the presence and relative
amount of specific antibody in a patients serum.
Prozone effect
At high antibody concentrations, the number of antibody binding sites may greatly exceed the number of
epitopes. As a results, most antibodies bind antigen only univalently instead of multivalently. Antibodies that
bind univalently cannot crosslink one antigen to another. Prozone effects are observed by performing the assay
at a variety of antigen or antibody concentration. Higher levels of agglutination can be seen at optimum
dilution of antibody concentration. When using polyclonal antibodies incomplete antibodies (class IgG) also
causes prozone effect. The antibodies present in high concentration in the antiserum, which bind to the antigen
but do not induce agglutination, are known as incomplete antibodies.
Types of agglutination reactions

Quantitative agglutination (Bacterial agglutination)
Agglutination has been commonly used to determine whether a patient had or has a bacterial infection. This
type of agglutination reaction is also known as quantitative agglutination test as here the measurement of level
of antibodies to particulate antigens is done. The presence of serum antibodies in a person specific for surface
antigens on the bacterial cells can be detected by bacterial agglutination reactions. If a patient is suspected of
having typhoid fever, the patients serum is mixed with a culture of Salmonella typhi. If an agglutination
reaction occurs, shown as clumping of the bacteria, the patient either had or has an S. typhi infection. Since
certain antibodies can persist in a patients blood for years after the patent has recovered from the infection, a
positive reaction does not mean that the patient currently has the infection. To determine whether a patient is
currently suffering from typhoid fever, the amount or titer of the antibody will be determined at the onset of
illness and two weeks later. The serum from the suspected patient is serially diluted in an array of tubes to
which the bacteria is added. Visible agglutination can be seen in the last tube which will reflect the serum
antibody titer of the patient. The agglutination titer is defined as the reciprocal of the greatest serum dilution
that elicits a positive agglutination reaction. Naturally, the higher the titer, the greater is the antibody response
of the individual to the disease. The patient currently suffering from suspected typhoid fever shows a
significant rise in the agglutination titer to Salmonella typhi. Agglutination reactions also help to type bacteria.


Qualitative agglutination (Hemagglutination)
Agglutination tests can be used in a qualitative manner to assay for the presence of an antigen or an antibody.
The antibody is mixed with the particulate antigen and a positive test is indicated by the agglutination of the
particulate antigen. Hemagglutination is a specific form of agglutination that involves typing of red blood
cells. The ABO blood group antigens are intrinsic red blood cell antigens, with the A and B signs referring
to proteins on the surface of red blood cells. Individuals expressing the A antigen are designated as blood
type A. Similarly those express B antigens, are designated as blood type B. Individuals expressing both
A and B antigens are designated as blood type AB, and those not expressing either A or B antigen are
designated as blood type O. Blood type can be determined by using antibodies that bind to the A or B blood
group in a sample of blood. In typing for the ABO antigens, RBCs are mixed on a slide with antisera to the A
or B blood-group antigens. If the antigen is present on the cells, they agglutinate forming a visible clump on
the slide. For example, if antibodies that bind the A blood group are added and agglutination occurs, the blood
is either type A or type AB. To determine between type A or type AB, antibodies that bind the B group are
added and if agglutination does not occur, the blood is type A. In blood grouping, the patient's serum is tested
against RBCs of known blood groups and also the patient's RBCs are tested against known serum types. In this
way the patient's blood group is confirmed from both RBCs and serum.

Passive agglutination
Passive agglutination is like agglutination reaction but performed with soluble antigens. It is defined as the
agglutination of particles that have been coated with soluble antigen, by antiserum specific for the adsorbed
antigen. Passive hemagglutination is a kind of passive agglutination in which erythrocytes, usually modified by
mild treatment with tannic acid or chromium chloride, are used to adsorb soluble antigen onto their surface,
and which then agglutinate in the presence of antiserum specific for the adsorbed antigen. Serum containing
antibodies is serially diluted into microtiter plate wells and the antigen-coated red blood cells are then added to
each well. Agglutination is assayed by the size of the characteristic spread pattern of agglutinated red blood
cells on the bottom of the well. Passive agglutination can be performed with tanned erythrocytes or synthetic
particles, such as latex beads. The use of synthetic beads offers the advantages of consistency, uniformity and
stability. The initial step in the test is the linking together of the latex particle by the antibody molecules that
specifically attach to the antigenic determinants on the surface of the particles. There is a formation of large
lattices through these cross links and these large lattices sediment readily due to the large size of clumps and
are visible to the unaided eye within minutes. The degree of agglutination can be determined by plotting the
agglutinant concentration which gives a bell shaped curve. The antigen-antibody complexes can be magnified
using the latex particles.

Agglutination inhibition
Agglutination inhibition is the modification of the agglutination reaction. If the antibody is incubated with
antigen prior to mixing with latex, agglutination is inhibited; this is because free antibodies are not available
for agglutination. In agglutination inhibition, the absence of agglutination is diagnostic of antigen, provides a
high sensitive assay for small quantities of antigen. One example of which is the home pregnancy test kits
included latex particles coated with human chorionic gonadotropin (HCG) and antibody to HCG. When urine
of a pregnant woman containing HCG, added to it, agglutination of the latex particles inhibited when the anti-
HCG was added. The absence of agglutination is the indication of pregnancy. If the urine contains no HCG,
then visible clumping occurs and agglutination can be observed which indicates no pregnancy (figure-6).


Figure 6: Agglutination inhibition

Agglutination assays can be employed for detecting the presence of any illegal drugs in a persons blood
sample or urine sample. Agglutination inhibition assays are also widely used in clinical laboratories to
determine whether an individual has been exposed to certain types of viruses that cause agglutination of red
blood cells. This technique is commonly used to determine the immune status of women with respect to the
rubella virus.

Complement fixation
The complement fixation test is an immunological medical test that can be used to detect the presence of either
specific antibody or specific antigen in a patient's serum. Complement is the activity of blood serum that
completes the action of antibody. The complement system is a system of serum proteins that react with
antigen-antibody complexes.
The basic steps of complement fixation test (figure) includes:
Isolation of serum from the patient.
The complement proteins in the patient's serum must be destroyed and replaced by a known amount of
standardized complement proteins. This is done to negate any effect on the test as patients serum
naturally has different levels of complement proteins.
The serum is heated in such a way that all of the complement proteins but none of the antibodies
within it are destroyed.
A known amount of standard complement proteins are added to the serum.
The antigen of interest is added to the serum.
If the patients serum contains antibodies against the antigen of interest, then antigen-antibody
complex will be formed that will fix the complement (figure-7). The complement proteins will react
with these complexes and be depleted.
Sheep red blood cells (sRBCs), which have been pre-bound to anti-sRBC antibodies are added to the
serum. When complement fixation occurs, there will be no complement left in the serum to react with
the sRBC-antibody complexes. However, if the patients serum contains no antibodies against the
antigen of interest, the complement will not be depleted and it will react with the sRBC-antibody
complexes, lysing the sRBCs and spilling their contents into the solution, thereby turning the solution
pink. The solution when turns to pink, it confirms the test negative.
It is equally possible to detect antigen in a patients serum. In this case, the patient's serum is
supplemented with specific antibody to induce formation of complexes; addition of complement and
indicator sRBC is performed as the above mentioned antibody detection procedure.


Figure 7: Complement fixation


Enzyme-linked Immunosorbent Assay (ELISA)
The ELISA is a fundamental tool of clinical immunology, which has been used as a diagnostic tool in
medicine and plant pathology, as well as a quality-control check in various industries. ELISA, or Enzyme-
linked immunosorbent assay that uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a
substance, usually an antigen, or anibody in a liquid sample or wet sample. It depends on an enzyme-substrate
reaction that generates a colored reaction product. An enzyme conjugated with an antibody reacts with a
colorless substrate to generate a colored reaction product. A number of enzymes i.e., alkaline phosphatase,
horseradish peroxidase, and -galactosidase, have been employed for ELISA.
A number of variations of ELISA have been developed, allowing qualitative detection or quantitative
measurement of either antigen or antibody. Four types of ELISA methods are there, but generally three
methods are employed for detection of either antigen or antibody. Direct ELISA is the simplest type of ELISA.
Antibody can be determined with an indirect ELISA whereas antigen can be determined with a sandwitch
ELISA or competitive ELISA. Each assay can be used qualitatively or quantitatively by comparison with
standard curves prepared with known concentrations of antibody or antigen.
Direct ELISA
The direct ELISA is a method for detecting and measuring antigen concentration in a sample. It is the simplest
type of ELISA among the four types. In direct ELISA, the presence of a particular antigen in a sample, is
detected by using a capture monoclonal antibody.
The procedure for direct ELISA includes:
The wells of a microtiter plate are coated with a sample containing the target antigen. The antigen is
fixed to the surface to render it immobile.
The plate wells are then coated with a blocking buffer.
In a separate reaction, an enzyme is linked to an antibody.
The enzyme-antibody conjugate is added to the wells to adsorb to the antigen.
The plate is washed to remove any excess enzyme-antibody conjugate.
Then a substrate is applied for the enzyme, and which is converted by the enzyme to elicit a
chromogenic or fluorescent signal. So the substrate detects the presence of the enzyme and the
antigen.
The amount of colored product is measured by a specialized spectrophotometric plate readers.
The advantage of direct ELISA is that it is relatively quick because of the use of only one antibody but direct
ELISA requires the labeling of every primary antibody, which can be time-consuming and more expensive
than in indirect methods. Certain antibodies may be unsuitable for direct labeling. Direct methods do not allow
for signal amplification in contrast to methods that use a secondary antibody. Direct ELISA can be used to test
specific antibody-to-antigen reactions, and helps to eliminate cross-reactivity between other antibodies.
Indirect ELISA
Indirect ELISA helps detecting antibody. It is the method of choice to detect the presence of serum antibodies
against human immunodeficiency virus (HIV), the causative agent of AIDS.
The procedure of indirect ELISA includes:
A buffered solution of the antigen to be tested for is added to each well of a microtiter plate. Coating
is achieved through passive adsorption of the antigen to the assay microplate. This process occurs
though hydrophobic interactions between the micro titer plate and non-polar protein residues. After
incubation any excess antigen is removed by washing steps by flooding and emptying the wells with
neutral phosphate buffered saline ( PBS ) or deionized water. A solution of nonreacting protein, such
as bovine serum albumin or casein, is added to block any plastic surface in the well that remains
uncoated by the antigen.
Serum or some other sample containing primary antibody is added to the antigen-coated microtiter
well. The antibody specific to the test antigen, binds the coated antigen on incubation.
Excess antibody or any unbound antibodies are removed by washing and is followed by addition of
blocking solution.
The presence of antibody bound to the antigen is detected by adding an enzyme-conjugated secondary
anti-isotype antibody, which binds to the primary antibody (figure-8).
Any free secondary antibody is washed away, and a substrate for the enzyme is then added. Often, this
substrate changes color upon reaction with the enzyme, which shows the secondary antibody has
bound to primary antibody. The higher the concentration of the primary antibody present in the serum,
the stronger the color change. The amount of colored reaction product that forms is measured by
specialized spectrophotometric plate readers.
Serum antibodies to HIV can be detected by indirected ELISA within six weeks of infection. In this
assay, recombinant envelope and core proteins of HIV are adsorbed to solid-phase antigens to
microtiter wells. Individuals infected with HIV will produce serum antibodies to epitopes on these
viral proteins.


Figure 8: Indirect ELISA

Indirect ELISA has sensitivity, since more than one labeled antibody is bound per primary antibody. It has
flexibility too, since different primary detection antibodies can be used with a single labeled secondary
antibody. Apart from that, it is cost effective, since fewer labeled antibodies are required.
A major disadvantage of the indirect ELISA is the method of antigen immobilization is not specific; when
serum is used as the source of test antigen, all proteins in the sample may stick to the microtiter plate well, so
small concentrations of analyte in serum must compete with other serum proteins when binding to the well
surface. The sandwich or direct ELISA provides a solution to this problem, by using a "capture" antibody
specific for the test antigen to pull it out of the serum's molecular mixture.
Sandwitch ELISA
Sandwitch ELISA helps detecting presence of antigen in a sample and to use it as a diagnostic tool for
medicine. The Sandwich ELISA measures the amount of antigen between two layers of antibodies. The two
layers of antibody consist of capture and detection antibody. Here, either monoclonal antibodies or polyclonal
antibodies can be used as capture and detection antibodies. The antigen to be measured must contain at least
two antigenic sites capable of binding to antibody, since at least two antibodies act in the sandwich.
The procedure for sandwitch ELISA includes:
In sandwitch ELISA, the antibody (capture antibody) rather than the antigen is coated on the surface
of a microtiter well.
Any nonspecific binding sites on the surface are blocked with the help of blocking solution.
The sample containing antigen is added and allowed to react with the immobilized antibody on the
microtiter well.
The microtiter plate is washed to remove any unbound or excess antigen.
After washing, a second enzyme-linked antibody (detection antibody) specific for a different epitope
on the antigen is added and is allowed to react with the bound antigen.
The plate is washed to remove the unbound antibody-enzyme conjugates.
After any free second antibody is removed by washing, substrate is added and the colored reaction
product is measured (figure-9). A large selection of substrates is available for performing the ELISA
with an HRP or AP conjugate. TMB (3, 3, 5, 5-tetramethyl benzidine) is the most commonly used
substrate for the enzyme horseradish peroxidase (HRP).
The absorbency of the plate wells is measured to determine the presence and quantity of antigen.
Specially designed spectrophotometers are available which reads through the microtiter wells either
singly or in rows. Most ELISA readers can be set to measure the absorbance of the colors produced by
the action of antibody- conjugated enzymes on their respective substrates.


Figure 9: Sandwitch ELISA

The advantage of Sandwich ELISA is that the sample does not have to be purified before analysis, and the
assay can be very sensitive than indirect ELISA or competitive ELISA. Sandwitch ELISA has high specificity,
since the two antibodies help in capturing and detecting the antigen. The process both has flexibility and
sensitivity.

Competitive ELISA
Competitive ELISA is another variation for measuring amounts of antigen. The procedures of competitive
ELISA is different from that of sandwitch ELISA and indirect ELISA.
The procedure for competitive ELISA includes:
Unlabeled primary antibody is first incubated in solution with a sample containing antigen.
The antigen-antibody mixture is then added to an antigen coated microtiter well (figure-10).
The more antigen present in the sample, the less free antibody will be available to the antigen-coated
well. Therefore competition arises.
The plate is washed to remove any unbound antibody.
The enzyme-conjugated secondary antibody, specific for the isotype of the primary antibody is added
to determine the amount of primary antibody bound to the well.
A substrate is added, and color change is measured.
In competitive ELISA, the concentration of antigen in the original sample is inversely proportional to
the color produced.


Figure 10: Competitive ELISA

Some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked antibody. The
labeled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). The more
antigen in the sample, the less labeled antigen is retained in the well and the weaker the signal. So, here the
microtiter well is coated with an antibody.
In competitive ELISA, the advantage is that the antigen does not require purification prior to
measurement. Competitive ELISA has also high specificity compared to indirect ELISA.

The ELISPOT assay
The enzyme-linked immunospot (Elispot) assay is a modification of the ELISA assay, allows the quantitative
determination of the number of cells in a population that are specific for a given antigen or an antigen for
which one has a specific antibody. Based on the sandwich enzyme-linked immunosorbent assay (ELISA), the
ELISPOT assay derives its specificity and sensitivity by employing high affinity capture and detection
antibodies and enzyme-amplification.
The procedure for the ELISPOT assay (figure-11) includes:
The wells of the microtiter plate are coated with the antigen (capture antigen) recognized by the
antibody of interest or with the antibody (capture antibody) specific for the antigen whose producion
is being assayed.
This assay is commonly used to detect cytokine secreted from different cells.
A suspension of cell population thought to contain some members secreting cytokine are added to the
wells coated with relevant antibodies (capture antibodies). It is allowed to be incubated.
After the incubation period, the wells are washed and enzyme-labeled anti-cytokine antibodies
(detection antibodies) are added.
Then again the wells are washed to remove any unbound antibody. After washing the wells, a
chromogenic substrate that forms an insoluble colored product is added.
The colored product precipitates and forms a spot only on the areas of the wells, where cytokine-
secreting cells had been deposited.
The number of cytokine-secreting cells present in the added cell suspension were identified by
counting the number of colored spots.


Figure 11: The ELISPOT assay

There are several advantages of the ELISPOT assay. Elispot assay has high sensitivity. Frozen or thawed
biological samples can be applicable for the assay. This assay requires minimun biological samples. It is also
compatible with other assays.

Radioimmuoassay (RIA)
Radioimmunoassay is a very sensitive in vitro assay technique used for separation of a protein from a mixture
using the specificity of antibody-antigen binding and quantitation using radioactivity. The technique was first
developed in 1960 by two endocrinologists, S.A. Berson and Rosalyn Yalow, to determine levels of insulin-
anti-insulin complexes in diabetics. Although the RIA technique is extremely sensitive and extremely specific,
requiring specialized equipment, it remains the least expensive method to perform such tests. It requires special
precautions and licensing, since radioactive substances are used.
The principle of RIA involves competitive binding of radiolabeled antigen and unlabeled antigen to a
high-affinity antibody. The method of RIA starts with making a known quantity of antigen radioactive by
labeling it with gamma-radioactive isotopes of iodine attached to tyrosine. The labeled antigen is then 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 test sample may
be a complex mixture, such as serum or body fluids, that contains the unlabeled antigen. This causes the
unlabeled antigen to compete with the radio-labeled antigen for antibody binding sites (figure-12).


Figure 12: The radioimmunoassay (RIA)

As the concentration of unlabeled antigen increases, more labeled antigen will be displaced from the
binding sites. Then the bound antigens are separated by the unbound ones by precipitating the antigen-antibody
complex with a secondary anti-isotype antiserum. For example, if the antigen-antibody complex contains
rabbit IgG antibody, then goat anti-rabbit IgG will bind to the rabbit IgG and precipitate the complex. After
separation, the radioactivity of the free antigen remaining in the supernatant is measured using a gamma
counter. Using known standards, a binding curve can then be generated which allows the amount of antigen in
the test sample to be derived. A standard curve is obtained by adding increasing concentrations of unlabeled
antigen to a fixed quantity of radio-labeled antigen and specific antibody (figure-13).


Figure 13: A standard curve of RIA

From the plot of the percentage of labeled antigen bound versus the concentration of unlabeled antigen,
the concentration of antigen in unknown serum samples can be determined by using the linear part of the
curve.

Immunoflorescence
Immunofluorescence is an antigen-antibody reaction where the antibodies are tagged (labelled) with a
fluorescent dye and the antigen-antibody complex is visualized using ultra-violet (fluorescent) microscope.
Fluorescent molecules absorb light of one wavelength and emit light of another wavelength. If antibody
molecules are tagged with a fluorescent dye, or flurochrome, immune complexes containing these
fluorescently labeled antibodies can be detected by colored light emission when excited by light of appropriate
wavelength. In immunoflorescence, fluorescent compounds such as fluorescein and rhodamine are commonly
used. Phycoerythrin, an intensely colored and highly fluorescent pigment obtained from the algae, is also
routinely used. It can be categorised into direct and indirect immunoflorescence, given briefly below.
Direct immunoflorescence
This technique is used to detect antigen in clinical specimens using specific fluorochrome labeled antibody. In
direct immunoflorescence, the specific antibody is directly conjugated with a fluorescent dye. The procedure
begins with fixation of cells with membrane antigens (mAg) to a slide (figure-14a). Then the cells are stained
with anti-mAg antibodies that are labeled with flurochromes. After a period of incubation, the slide is washed
to remove any unbound excess labeled antibody. After washing, the slide is viewed under fluorescent
microscope. When viewed under fluorescent microscope, the field is dark and areas with bound antibody
fluoresce green.
This technique can be used to detect viral, parasitic, tumor antigens from patient specimens or monolayer of
cells.

Indirect immunoflorescence
Indirect immunofluorescence is employed to detect antibodies in a test sample, for example in a patient's
serum. Here, primary antibodies which are unlabeled, allowed to react with cells having membrane antigens.
After a period of incubation, the slide is washed to remove any unbound antibodies. After washing, the cells
are stained with flurochrome-labeled secondary antibodies (fluorescein-labeled goat anti-mouse antibodies).
This antibody binds to Fc portion of first antibody and persists despite washing. The presence of secondary
antibodies is detected by observing under fluorescent microscope (figure-14b).


Figure 14: Direct method and indirect method of immunoflorescence

Indirect immunoflorescence staining has two advantages over direct staining. First, the supply of primary
antibodies is often a limiting factor and loss of primary antibody occurs during conjugation reaction. Indirect
methods avoid the loss of Primary antibody which is not conjugated with flourochrome. Secondly, indirect
methods increase the sensitivity of staining because multiple molecules of the flurochrome reagent bind to
each primary antibody molecule, increasing the amount of light emitted at the location of each primary
antibody molecule.
Immunoflorescence has been applied to identify the CD4
+
and CD8
+
T-cell populations.
Immunoflorescence is suitable for detecting antigen-antibody complexes in autoimmune disease, detecting
complement components in tissues and the major application of it is localizing antigens in tissue sections or in
sub-cellular compartments.
Fluorescent antibody techniques are important qualitative tools but they do not give quantitative data.
Flow cytometer, automate the analysis and separation of cells stained with fluorescent antibody. The flow
cytometer uses a laser beam and light detector to count single intact cells in suspension. Every time a cell
passes the laser beam, light is deflected from the detector, and this interruption of the laser signal is recorded.
Those cells having a fluorescently labeled antibody bound to their cell surface antigens are excited by the laser
and emit light that is detected by a second detector system located at a right angle to the laser beam.
The flow cytometer has multiple applications to clinical and research problems. Flow cytometry can also
analyze cell population that have been labeled with two or three different fluorescent antibodies.


Immunoprecipitation
Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody
that specifically binds to that particular protein. It also provides a sensitive assay for the presence of a
particular antigen in a given cell or tissue type. Immunoprecipitation requires that the antibody be coupled to a
solid substrate at some point in the procedure. There are two general methods for immunoprecipitation, i.e., the
direct capture method and the indirect capture method.
Direct method
An antibody (monoclonal or polyclonal) against a specific protein is pre-immobilized onto an insoluble
support, such as agarose or magnetic beads, and then incubated with a cell lysate containing the target protein.
During the incubation period, the lysate is gently agitated so that the proteins that are targeted by the antibodies
are captured onto the beads via the antibodies, in other words, they become immunoprecipitated. The
immobilized antigen-antibody complexes are collected from the lysate, and then eluted from the support and
analyzed.

Indirect method
In indirect method, the antibody is not pre-immobilized onto an insoluble support rather, the antibody that is
specific for a particular protein antigen, added directly to a cell lysate containing the target protein. Free,
nonbound antibodies are allowed to form immune complexes in the lysate. After some time, secondary
antibodies, specific for the primary antibodies, are attached to synthetic beads and added to the antigen-
antibody complexes in the lysate. At this point, the antibodies, which are now bound to their targets, will stick
to the beads. The antigen-antibody complexes are then eluted from the support and analyzed (figure-14).


Figure 15: Immunoprecipitation

Both the direct method and the indirect method gives the same end-result with the protein or protein
complexes bound to the antibodies which themselves are immobilized onto the beads. The direct method is a
preferred choice, but the indirect approach is sometimes preferred when the concentration of the protein target
is low or when the specific affinity of the antibody for the protein is weak.
Types of immnoprecipitation
There are generally four types of immunoprecipitation (IP) techniques, i.e., individual protein
immunoprecipitation, protein complex immunoprecipitation (Co-IP), chromatin immunoprecipitation (Ch-IP)
and RNA immunoprecipitation (RNA-IP).

Individual protein immunoprecipitation
It Involves using an antibody that is specific for a known protein to isolate that particular protein out of a
solution containing many different proteins. These solutions will often be in the form of a crude lysate of a
plant or animal tissue.

Protein complex immunoprecipitation (Co-IP)
Immunoprecipitation of intact protein complexes (i.e. antigen along with any proteins or ligands that are bound
to it) is known as co-immunoprecipitation (Co-IP). Co-IP is a powerful technique that is used regularly by
molecular biologists to analyze proteinprotein interactions.
Co-IP works by selecting an antibody that targets a known protein that is believed to be a member of a
larger complex of proteins. By targeting this known member with an antibody it may become possible to pull
the entire protein complex out of solution and thereby identify unknown members of the complex. This works
when the proteins involved in the complex bind to each other tightly, making it possible to pull multiple
members of the complex out of solution by latching onto one member with an antibody. Immunoprecipitated
proteins and their binding partners are commonly detected by SDS-PAGE and Western blot analysis.

Chromatin immunoprecipitation (Ch-IP)
Chromatin immunoprecipitation (ChIP) is a method used to determine the location of DNA binding sites on
the genome for a particular protein of interest. This technique gives a picture of the proteinDNA interactions
that occur inside the nucleus of living cells or tissues.
DNA-binding proteins (including transcription factors and histones) in living cells can be cross-linked to
the DNA that they are binding. By using an antibody that is specific to a putative DNA binding protein, one
can immunoprecipitate the proteinDNA complex out of cellular lysates. The purified proteinDNA
complexes are then heated, allowing the separation of DNA from the proteins. The DNA is then identified by
PCR, sequenced and applied to microarrays or analyzed according to the requirement.

RNA immunoprecipitation
The principle of RNA immunoprecipitation is similar to Ch-IP, except that here RNA-binding proteins are
immunoprecipitated instead of DNA-binding proteins. RNA immunoprecipitation is also an in vivo method in
that live cells are lysed and the immunoprecipitation is performed with an antibody that targets the protein of
interest. By isolating the protein, the RNA will also be isolated as it is bound to the protein. By performing an
RNA extraction, the purified RNA-protein complexes can be separated and immunoprecipitated RNAs can
then be identified by RT-PCR and cDNA sequencing.

References
1. Kuby, Janis etal.(2003). Immunology, 5th Edn. W.H. Freeman & Company Publishers.

2. Lichtman, Andrew H., Abbas, Abul K., Basic Immunology, 3rd Edn. Saunders Elsevier Inc.

3. Roitt, Ivan M. etal. Essential Immunology, 12th Edn. Willey-Blackwell Publishers.