lecture notes on Immunochemical techniques like, Precipitation, Agglutination, ELISA, RIA, Complement fixation, Immunofluresceneetc

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Lecture notes in Immunology
Topic: Immunochemical techniques
By,
Mrs. K. P. Komal
Assistant professor in Biochemistry
Government Science College, Chitradurga
Karnataka. 577501

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antigen
IMMUNOCHEMICAL TECHNIQUES
Antigens
Antigens are macromolecules of natural or synthetic origin; chemically they consist of
various polymers – proteins, polypeptides, polysaccharides or nucleoproteins. Antigens
display two essential properties: first, they are able to evoke a specific immune response,
either cellular or humoral type; and, second, they specifically interact with products of
this immune response, i.e. antibodies or immunocompetent cells. A complete antigen –
immunogen – consists of a macromolecule that bears antigenic determinants (epitopes)
on its surface (Fig. 1). The antigenic determinant (epitope) is a certain group of atoms on the
antigen surface that actually interacts with the binding site on the antibody or lymphocyte
receptor for the antigen. Number of epitopes on the antigen surface determines its valency.
Low-molecular-weight compound that cannot as such elicit production of antibodies, but is
able to react specifically with the products of immune response, is called hapten
(incomplete antigen).
epitopes
Fig. 1. Antigen and epitopes
Antibodies
Antibodies are produced by plasma cells that result from differentiation of B lymphocytes
following stimulation with antigen.
Antibodies are heterogeneous group of animal glycoproteins with electrophoretic mobility β
- γ, and are also called immunoglobulins (Ig). Every immunoglobulin molecule contains at
least two light (L) and two heavy (H) chains connected with disulphidic bridges (Fig. 2). One
antibody molecule contains only one type of light as well as heavy chain. There are two
types of light chains - k and λ - that determine type of immunoglobulin molecule; while
heavy chains exist in 5 isotypes - γ, μ, α, δ, £; and determine class of immunoglobulins - IgG,
IgM, IgA, IgD and IgE. The C terminal parts of both chains form constant regions on an
antibody molecule, while the N-terminal parts are designated as variable regions and
constitute the portion of antibody molecule that binds antigen – binding site or paratope.
Except for IgM that has 10 binding sites and IgA with 4 binding sites, immunoglobulin

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molecules possess 2 antigen binding sites.
Fig. 2. Structure of antibody of IgG class
Immunochemical techniques
Immunochemical techniques are based on a reaction of antigen with antibody, or more
exactly, on a reaction of an antigenic determinants with the binding site of the antibody. The
antibodies used are produced by various ways.
Monoclonal antibodies are products of a single clone of plasma cells derived from B-
lymphocytes, prepared in the laboratory by hybridoma technology, based on cellular fusion
of tumour (myeloma) cells with splenic lymphocytes of immunised mice. Monoclonal
antibodies are directed against single epitope; and are all identical copies of immunoglobulin
molecule with the same primary structure and specificity of antigen binding site. They
typically display excellent specificity, but poor ability to precipitate antigen.
Polyclonal antibodies (conventional antibodies) are prepared by immunisation of animals
(rabbits, goats, sheep) with the antigen. Blood serum of the immunised animal that contains
antibodies against the antigen used, is called an antiserum. If one antigen (e.g. one protein)
is used for immunisation, monospecific antibodies (antiserum) result. However, as every
epitope stimulates different clone of B cells, and complex antigens bear several epitopes, the

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antiserum contains mixture of monoclonal antibodies, differing in their affinity and
specificity towards particular epitopes on the antigen used for immunisation.
Immunisation of an animal with mixture of antigens results in production of polyspecific
antibodies, containing immunoglobulins against many antigens (e.g. antiserum against
human serum proteins used in immunoelectrophoresis).
Quantitative precipitin curve
Measurement of antigen-antibody complex formation has proved extremely useful for
analysing many constituents of body fluids. A wide variety of immunochemical methods has
been developed based on the fundamental principle of quantitative precipitin curve
described by Heidelberger and Kendall in 1935.
A soluble antigen possessing multiple antigenic determinants reacts with the corresponding
antibody and the resulting antigen-antibody complex precipitates out of solution. The
precipitin curve describes the relationship between the antigen concentration and the
amount of precipitate for a constant quantity of antibody.
Three zones can be distinguished on the precipitin curve (see Fig. 3):
The antibody excess zone: The amount of precipitate increases proportionally as the
concentration of antigen increases. If the antibody is present in an excess, all the antigen
binding sites are covered with the antibody and only small soluble antigen-antibody
complexes are formed. No free antigen is detected in the supernatant, but free (unbound)
antibodies can be found. Such conditions are useful for immunoturbidimetry,
immunonephelometry and non-competitive immunoassays.
The equivalence zone: Molecules of antigen and antibody are cross-linked forming large,
insoluble complexes. The complexes further aggregate and precipitate. Neither free antigen
nor free antibody can be detected in the supernatant. Equivalence is reached in
immunodiffusion techniques.
The antigen excess zone: The amount of precipitate decreases due to the high antigen
concentration. Large aggregated immunocomplexes decay. As all the antibody sites are
saturated by antigen, small soluble complexes prevail. No free antibody but an increasing
amount of free antigen may be found in the liquid phase. Excess of free antigen is required
for competitive immunoassays.

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This immunoprecipitin curve forms the basis of many immunochemical assays that can be
performed in a solution as well as in a gel.
Precipitation methods in gel
As a support matrix, agar or agarose gels are used most often. In single immunodiffusion
only one component (i.e. antigen or antibody) diffuses from the place of sample application,
while the other reaction partner is dispersed evenly in the gel. If both components of the
immunochemical reaction diffuse in the gel against each other from places of their
application, the technique is called double immunodiffusion. In the area of antigen-
antibody reaction a precipitation zone appears as line, crescent or circle. The
immunodiffusion methods in gel are represented e.g. by the Mancini’s single radial
immunodiffusion and the Ouchterlony’s double immunodiffusion. Other techniques
combine an immunochemical reaction with electrophoretic separation, such as
Fig. 3. Precipitin curve

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immunoelectrophoresis, Laurell’s (rocket) immunoelectrophoresis, and immunofixation.
Single radial immunodiffusion
Single radial immunodiffusion represents a simple method without requirements for
expensive instruments. The antigen is applied into wells that are cut in the agarose gel
containing dispersed corresponding monospecific antibody. The agarose plate is incubated
at room temperature for 48-72 hours depending on the specific protein in question. The
antigen from the sample diffuses out from the wells into the agarose where the antibody
concentration is constant. In a distance from the well where antigen concentration is
equivalent to the antibody concentration (i.e., zone of equivalence is reached in terms of the
precipitin curve discussed above), the complex antigen-antibody precipitates and appears as
a strong white ring around the well. The square of the ring diameter is directly proportional
to the antigen concentration.
The protocol involves several steps:
• Boiled agarose is cooled to 50°C and monospecific serum is added to it. The gel is
poured onto a glass plate or a Petri dish.
• After the agarose has solidified, round-shaped wells (starts) are cut out. Samples and
standards are applied to the wells.
• The plate is incubated in a wet chamber. The antigen diffuses radially to the gel and
reacts with antibody.
• Then, the plate is stained in order to visualise the precipitate rings, and dried.
• Diameters of the rings are measured and squared values are calculated. Calibration
curve is plotted using standards. Finally, concentration of antigen in samples is read
from the plot.
The sensitivity of this technique is about 10-200 mg/L, and therefore it is suitable for
estimation of most serum proteins (e.g. immunoglobulin IgG, IgM, IgA, prealbumin,
transferrin, α2-macroglobulin, ceruloplasmin). This technique has been, however, currently
largely replaced with immunoturbidimetry or nephelometry

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Fig. 4: Single radial imunodiffusion (S1 – S7 – standards, V1-V2 – samples)
Double immunodiffusion
In double immunodiffusion reaction, the antigen and the antibody diffuse towards each
other. The Ouchterlony’s modification is the one most often used. It is based on wells
punched into the agarose gel in a rosette pattern that are filled with antigen or antibody
solutions, respectively. Both antigen and antibody molecules are allowed to diffuse radially
into the gel surrounding the wells; and where the antigen and specific reactive antibody
meet, a precipitin line forms. If antiserum to several possible antigens is placed into the
central well and the outer wells are filled by different antigens, precipitin lines of various
shapes can arise. For instance, if two antigenic mixtures are applied into two adjacent wells,
the following patterns of precipitin lines can be observed, in dependence on the relationship
between the two antigenic mixtures (fig. 5):
a reaction of identity – if two identical antigens are applied into two adjacent wells, the
precipitin bands form a continuous arc.
a reaction of non-identity – the precipitin bands form lines that intersect.
a reaction of partial identity – it is characterised by a formation of a spur. The common
hooked precipitation line arises from the reaction of the common antigenic determinants on
both antigens with the antibody. The spur means that the second antigen lacks an epitope
present in the first antigen that is recognised by one of the antibodies in the antiserum.
Fig. 5: Double immunodiffusion according to Ouchterlony (Aga, Agb, Agc – antigens with
epitopes a, b, c; Aba, Abb – antibodies against epitopes a and b)

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Double immunodiffusion is a qualitative technique suitable for the identification of antigens
and the estimation of their mutual immunochemical relationships if the specific antisera are
available. On the other hand, this method may be used for characterisation of antibodies
using known purified antigens.
Immunoelectrophoresis
Immunoelectrophoresis is a qualitative method that combines protein electrophoresis with
immunodiffusion.
It is performed in two steps. The first one involves the separation of antigens according to
their charges/size in an electrical field. In the second step, a suitable antiserum (polyspecific
or monospecific) is applied to grooves running parallel to the electrophoresis migration
zone. The separated antigens and antibodies are allowed to diffuse into the gel towards one
another. The precipitation line is formed in the area when the antigen with the reacting
antibody meets (Fig. 6).
Fig.6: Immunoelectrophoresis (polyspecific antiserum used)

9. Immunofixation
Immunofixation is a method used for the detection and isotyping of monoclonal
immunoglobulins in serum, urine and cerebrospinal fluid. Similarly to
immunoelectrophoresis, immunofixation is carried out in two stages. In the first one, serum
proteins are separated by electrophoresis. In the second step, the monoclonal
immunoglobulins are identified by means of immunoprecipitation with specific antibodies.
The typical procedure of immunofixation is following:
• The aliquots of an identical patient serum are applied into six indicated origins in an agarose
gel. All the specimens are separated by electrophoresis. At the end of electrophoresis, the gel
is covered with the template, in which the areas of the electrophoresis migration zones are
cut out. A fixative solution is applied on the first track to denaturate and immobilise the
serum protein and to create an electrophoresis reference pattern. The monospecific
antibodies, directed against constant regions of IgG, IgM, and IgA heavy chains, and light
chains k and λ, are added to each of the following tracks. The specific antibodies diffuse into
the gel. The antigen- antibody complexes are formed in the zone where appropriate antibody
and antigen meet. The immunocomplexes are retained in the porous structure of gel and
form a marked band, while all the non- precipited components are subsequently removed by
washing. Finally, the gel is stained for protein in order to visualise the formed
immunoprecipitates.
• Use of appropriately diluted sample is the critical step. It follows from the immunoprecipitin
curve that excess of either antigen or antibody leads to decay of the immunocomplex. Thus, if
both over-diluted or over-concentrated sample is examined, a false negative result may be
obtained.
Clinical application of immunofixation
In clinical practice, immunofixation is employed especially for detection of monoclonal
immunoglobulins. This technique has replaced immunoelectrophoresis that was formerly
used for the same purpose. Immunofixation is easier to perform, more sensitive, and its
evaluation is less complicated.
Finding any abnormal band on the conventional serum protein electrophoresis – so called M-
component – should be confirmed and further characterised by immunofixation.
Immunoglobulin molecules are produced by the plasma cells. Every plasma cell clone
synthesises only one type of immunoglobulin in terms of antibody specificity; the produced
antibody molecules are therefore uniform in their structure and function. Monoclonal

10. immunoglobulins (paraproteins) may be in form of polymers, monomers or fragments of
immunoglobulin molecules. The finding of monoclonal immunoglobulins indicates an
extreme uncontrolled proliferation of a B-cell clone producing Ig molecules. The clinical
conditions associated with monoclonal immnunoglubulins - monoclonal gammapathies –
include a wide spectrum from a benign form to the malignant diseases. Multiple myeloma
(tumour from plasma cells) is the commonest cause of malignant paraproteinemia. Mostly,
the myeloma secretes IgG and IgA molecules, which contain only one class of light chain.
Occassionally myelomas produce also monomers or dimers of light chains only – the Bence-
Jones protein. It is found predominantly in urine, since the light chains have a low molecular
weight and therefore, unlike complete Ig molecules, easily pass the glomerular filtration
membrane. Waldenstrom´s macroglobulinemia is another malignancy from plasmatic cells
that produces IgM.
Evaluation
Polyclonal immunoglobulins form diffusely stained precipitations detectable in the
reaction with one or several heavy or light chain antibodies.In contrast, the presence of
potentially pathologic monoclonal immunoglobulin is characterised by a narrow
homogeneous sharp band within the diffuse immunoprecipitate detected with one of the
anti-heavy chain antiserum as well as with one of the anti-light chain antiserum. Usually,
one monoclonal protein corresponds to one type of heavy chain and one type of light chain
(Fig. 7). The Bence-Jones protein (light chains only) forms a narrow band in a precipitate
produced by one of the light chain antisera.
Serum Urine
Fig. 7: Immunofixation – example of IgG monoclonal immunoglobulin (serum: IgG k
paraproteins, urine: free light kchains)

11. Precipitation methods in solution
The precipitate, which shapes in an agarose gel, can also form in a solution. Two techniques
are used to quantitate this precipitate: immunoturbidimetry and immunonephelometry.
Turbidimetry and nephelometry:
When a diluted antigen solution is combined with a solution of the corresponding antibody,
the formation of small aggregates (immunoprecipitates) results in turbidity (cloudy
appearance) of the solution. Two approaches can be used to quantify this turbidity:
Turbidimetry is based on the measurement of intensity of light transmission; i.e., light that
passes through the cuvette is measured. Such measurement can be performed on a
conventional spectrophotometer. In contrast, nephelometry measures directly the intensity
of scattered light. Nephelometry requires a special apparatus – the nephelometer, which
uses laser as the light source (Fig. 8).
A very critical point in these assays is to work in the range of the antibody excess in the
reaction mixture, because two different antigen concentrations can in principle give the
same value of absorbance – one in the antibody excess and another in the antigen excess
zone. This can easily lead to erroneous results. The conventional way of checking whether
the measured absorbance lies within the antibody excess or antigen excess zone of precipitin
curve is to repeat the measurement with a higher dilution of the sample. With a new
sophisticated equipment we can measure the rate of turbidity formation, which is directly
proportional to the concentration of antigen.
Practical performance of both techniques is simple, and therefore amenable for automation.
The suitably diluted monospecific antiserum is mixed with diluted sample; after incubation
the light scatter or absorbance of light passing the sample is measured.
Sensitivity of these techniques can be increased by binding of antibody or antigen onto
latex particles, which considerably increase light scatter due to immunocomplex formation.

12. Immunonephelometry
The immunoprecipitation techniques in solution are much faster than radial
immunodiffusion, but also more expensive.
Fig. 8: Immunoturbidimetry and immunonephelometry
Agglutination
Agglutination (from latin ‘agglutino’ – to glue, to attach) is an immunochemical technique in
which a specific antibody reacts with the corpuscular antigen.
Agglutination reaction is based on the formation of bridges between bivalent (IgG) or
multivalent (IgM) antibodies and antigenic particles with multiple epitopes. Bivalency or
multivalency of the used antibody and multiple antigenic determinants on the surface of
particles are necessary for the creation of cross-linking and the formation of a high-
molecular-weight lattice that is observable macroscopically. IgM antibodies with ten
antigenic-combining sites permit a more effective bridging than IgG. Some antibodies react
with the corpuscular antigen, but may not produce agglutination. In this case, the
agglutination may be achieved if an anti- immunoglobulin is added into the reaction mixture
(the Coombs test).
Hemagglutination is a variant of agglutination technique in which red blood cells are used
as the antigen- bearing particles.
Agglutination reactions are performed on slides, in test tubes or microtiter plates. They are
more sensitive in comparison with immunoprecipitation methods. The agglutination
methods produce qualitative or semiquantitative results.
Agglutination assays may be classified as direct or indirect tests.

13. Direct agglutination
In a direct agglutination test, the antigen is an integral part of the cell surface (red blood
cells, bacteria). A suspension of particles is directly agglutinated by specific antibodies
present in the examined sample. This assay is frequently used in the hematology for the
determination of blood group or in the immunological diagnostics for detection of specific
antibodies directed against naturally occurring antigens on the surface of some microbes
(for example against Salmonella typhi – the Widal test). For the examination of antibodies,
the test is usually performed with serial dilutions of the sample. The highest dilution of
serum that still causes agglutination is denoted as a titer of the antibody.
Indirect agglutination
Indirect agglutination assay utilises particles with the antigens that have been passively
attached to their surface. Originally, red blood cells were used as carriers for antigens; lately,
inert particles such as latex, colloid gold and other substances have been shown to be more
versatile for the agglutination technique. Many proteins, bacterial and viral antigens are
easily adsorbed onto the particle, while other substances require modification by tannic acid
or chromium chloride.
The particles coated with the specific antigens are used for the detection of antibodies
against surface antigens in some pathogens and some autoantibodies(e.g. rheumatoid
factor)(Fig. 9). Instead of the antigens, particles can also be coated by specific antibodies.
This technique is called reverse agglutination and can be utilised for the detection of
soluble antigens (for example C-reactive protein or human chorionic gonadotrophin)
(Fig. 10).

14. Fig. 9: Indirect agglutination for the determination of antibodies
1st reaction – antigen is adsorbed onto the surface of inertparticles
2nd reaction – particles coated with antigens are agglutinated by specific antibodies present
in the sample
Fig. 10: Indirect agglutination for the determination of antigens
1st reaction – antibodies are adsorbed onto the surface of inert particles
2nd reaction – particles coated with antibodies are agglutinated by the corresponding
antigens present in the sample

15. Agglutination inhibition test
Agglutination inhibition test is another form of agglutination reaction that permits the
determination of soluble antigens. It is based on the competition between the antigens in
solution and the same antigens on the particle surface for limited amount of antibodies. The
specific antibodies are incubated with the test solution containing the soluble antigens.
Following the addition of the particles coated with the antigens, the agglutination does not
occur because most of the antibodies have been already saturated with a soluble form of the
same antigen from the sample. Therefore, antibody binding sites are unavailable for bridging
the coated particles. A lack of agglutination indicates a positive result (Fig. 11). On the other
hand, if the soluble antigen is not present in the tested sample, after the addition of the
corpuscular antigen the agglutination develops.
Fig. 11: Agglutination inhibition
1st reaction – a sample containing the soluble antigens is added to the specific
antibodies, the antigen- antibody complexes arise
2nd reaction – after the addition of particles coated with antigens, the agglutination
does not occur

16. Enzyme immunoassay
State-of-the-art immunoanalytic techniques achieve high sensitivity by labelling of one
reacting component – antibody or antigen – with a substance which detection is more
sensitive than detection of immunoprecipitate. The label can be a radioisotope
(radioimmunoassay, RIA), an enzyme (enzyme immunoassay, EIA), a fluorescent or
chemiluminiscent substance. Detection limits of these techniques can be as low as 10-15 –
10-20 mol/L.
Enzyme immunoassays utilise enzymes, usually peroxidase or alkaline phosphatase, to detect
and quantify immunochemical reactions. Both antibodies or antigens can be labelled with an
enzyme in order to aid detection.
A heterogeneous enzyme immunoassay method is also called enzyme-linked
immunosorbent assay (ELISA). In this type of assay, one of the immunochemical reaction
components (antigen or antibody) is first non-specifically adsorbed to the surface of a solid
phase. Tubes, wells of microtiter plates, and magnetic particles may be used as the solid
phases. The solid phase facilitates separation of bound-and free-labelled reactants.
A homogeneous enzyme immunoassay is a sort of enzyme multiplied immunoassay
technique (EMIT) that does not require a separation of bound and free labelled antibodies or
antigens. It is simple to perform and has been used for estimation of drugs, hormones and
metabolites. Sample containing the estimated antigen is mixed with a known quantity of the
same antigen labelled with enzyme (conjugate); and limited amount of specific antibody is
added. The unlabelled antigen from the sample competes with the conjugate for the antibody.
Binding of antibody on the conjugate results in loss of enzyme activity due to blocking the
enzyme active site or change of its conformation. The more unlabelled antigen is present in
the solution, the less conjugate will bind to the antibody, and more enzyme activity will be
preserved in the solution. Therefore, the enzyme activity is proportional to the antigen
concentration in the sample.
The reaction scheme in these immunochemical assays can follow either competitive, or non-
competitive approach.
Competitive enzyme immunoassay
This assay is always performed under condition of antigen excess. The enzyme-labelled
antigen (conjugate) is mixed with serum sample containing the unknown amount of antigen.
The serum antigen and enzyme-labelled antigen compete for binding sites of a limited

17. quantity of specific antibodies bound to the solid phase. Labelled and non-labelled antigen
bind to the antibody in the same proportion as is their proportion in the reaction mixture. In
other words, the more non-labelled antigen is contained in the mixture the less labelled
antigen is bound (Fig. 12). Under these conditions the probability of the antibody binding the
labelled antigen is inversely proportional to the concentration of unlabelled antigen. The
higher the amount of unlabelled antigen in the sample, the more labelled antigen remains free
(unbound). After an incubation, all the unbound both enzyme-labelled and unlabelled
antigens are removed by washing along with all other serum constituents. In the subsequent
indicator reaction for detection of enzyme activity a chromogenic substrate for the enzyme
label is added. The intensity of colour is inversely proportional to the concentration of the
antigen in serum sample. The results are obtained from a calibration curve constructed with
the standards of known concentration of antigen (Fig. 13).
This approach can be used to determine the concentration of small molecules having only
one epitope, e.g. steroid and thyroid hormones, or drugs in biological fluids.
Non-competitive enzyme immunoassay (sandwich methods)
This kind of enzyme immunoassay can be adopted for measurement of either antigens
or antibodies. It is a heterogeneous immunoassay using a solid phase coated with
antibody or antigen, which must always be in excess over the analyte being measured.
Non-competitive enzyme immunoassay for determination of antigen:
This non-competitive enzyme immunoassay is suitable for the measurement of large
antigens with several antibody-binding sites. Two different molecules of antibodies directed
against various epitopes are necessary for performing the assay. The first antibody is in
excess adsorbed to a solid phase. The serum sample or calibrators containing the desired
antigen are added to the well with immobilised antibody. The first imunochemical reaction
occurs. Since the antibody is in excess, all antigen molecules should bind. After an incubation,
all the non-reacting material in the sample is washed away. Then a second enzyme-labelled
antibody (different from the first antibody) is added in excess. In the second
immunochemical reaction, another antigen epitope binds to the second labelled antibody.
“Sandwich complex“ consisting of solid-phase antibody – antigen – enzyme- labelled
antibody is formed. After washing of all the unreacted enzyme-labelled antibody, the
substrate is added. The intensity of the finally measured coloured product of the enzyme

18. reaction is directly proportional to the amount of antigen (Fig. 14).
Fig. 12: Principle of competitive immunoassays – various proportions of antigen and
antibody
a)
-(
-(
-( +
-(
•
•
•
•
-(•
-(•
-(•
-(•
b)
-(
-(
-(
-(
•
•
•
•
O
O
+
-(•
-(O
-(•
-(•
+ • O
c)
-(
-(
-( +
-(
• O
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• O
• O
-(•
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+ • O
d)
-(
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• O O
• O O
• O O
• O O
-(•
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+
•
•
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O
O
O
-( - antibody
• - labelled antigen
O - non-labelled antigen
-(• immunocomplex with labelled antigen
-(O immunocomplex with non-labelled antigen

19. Non-competitive enzyme immunoassay for determination of antibody:
It has been used extensively for detection of serum antibodies to viruses and parasites in
serum, autoantibodies, and IgE antibodies specific for a particular allergen. In this case the
antigen must be first immobilised on the solid phase. After adding test samples or calibrators
the antibodies in the sample react with the immobilised antigen and forms
immunocomplexes. The next steps of the assay procedure are similar to that described for
the measurement of antigens (Fig. 15).
Fig. 13: Competitive enzyme immunoassay

20. Fig. 14: Non-competitive enzyme immunoassay for determination
of antigen

21. Fig 15: Non-competitive enzyme immunoassay for determination of
antibody

22. Immunochemical methods in fast diagnostics
Fast and tentative “point-of-care” or “bed-side” tests based on “dry chemistry” create
another important application of immunochemical methods. Reagents are anchored in a
porous substrate that is attached on a plastic pad or inserted in a frame with windows for
sample application and result reading. Various setups of tests can be used. Estimation of
human chorionic gonadotropin (hCG) for diagnostics of pregnancy, troponine T for acute
heart infarction, or tests for narcotics may serve as examples.
Examples of fast immunochemical diagnostic methods
Detection of narcotics in urine or saliva
Tests are based on competition of a drug in the sample with the same drug immobilised in
the detection area of the test for a limited amount of specific antibody. The sample is mixed
with antibodies in the reaction zone. The antibodies are labelled with stained micro-
particles. Then, both the sample and antibodies move by capillary action through the
reaction zone to the detection area which contains the immobilised drug. In case the sample
contains molecules of the drug it fully saturates the binding sites of colour-labelled
antibodies. The antibody molecules cannot then react with the immobilised drug and colour
of the detection area stays unchanged. On the other hand if no drug is contained in the
sample the binding sites of antibodies remain free and the stained molecules are trapped by
the immobilised drug. A coloured band displays in the detection area (Fig. 16).Tests for
individual drugs or for detection of several drugs at once are available. The most frequently
abused substances can be estimated by this technique – amphetamine, cocaine, opioids,
phencyclidine, and tricyclic antidepressants.

23. Sample (no drug)
Sample Drug
Labelled antibody against tested drug Immobilised
drug
Sample
area
Reaction
area
Detection
area
Negative resultPositive result
Fig. 16: Tentative test for narcotics in urine
Detection of human chorionic gonadotropin (pregnancy test)
Porous membrane that serves as a support for the test is divided into four areas. Three
different antibodies are employed. The first sample area contains specific anti-hCG antibody
labelled with microparticles of colloid gold or blue latex (Ab1). When urine sample is applied
molecules of this antibody flow to the second, detection area. Another specific antibody
against hCG (Ab2) is anchored in this area. If chorionic gonadotropin is contained in the
sample a combined immunocomplex with both labelled and anchored antibody is formed
(Ab2-hCG-Ab1) and a coloured band displays in the detection area. Excess of the labelled
antibody is caught in the third (control) area with immobilised antibodies against labelled

24. Antigen (hCG)
Labelled antibody against hCG (the
first antibody)
Immobilised antibody against hCG
(the second antibody)
Anti-immunoglobulin antibody
Sample
Sample (no
antigen)
Sample
area
Reaction
area
Detection
area
Control
area
Negative resultPositive result
anti-hCG. A band in the control area is formed, indicating that the test works properly (Fig.
17). Thus, two bands are interpreted as a positive result.
In case the urine sample contains no hCG the labelled antibody is bound in the third area
only. One band in the control zone only is therefore read as a negative result. If no band is
displayed in the control area the test is invalid.
Some tests are so designed that the control zone has a shape of a minus sign and the
detection zone crosses it perpendicularly. A plus sign therefore appears in case of positive
result..
Fig. 17: Tentative test for hCG in urine
Radio immuno assay:
When radioisotopes instead of enzymes are used as labels to be conjugated with antigens or
antibodies, the technique of detection of the antigen-antibody complex is called

25. radioimmunoassay (RIA). Radioimmunoassay (RIA) is an in vitro assay that measures the
presence of an antigen with very high sensitivity. RIA was first described in 1960 for the
measurement of endogenous plasma insulin by Solomon Berson and Rosalyn Yalow of the
Veterans Administration Hospital in New York.
The classical RIA methods are based on the principle of competitive binding. In this method, an
unlabeled antigen competes with a radiolabeled antigen for binding to an antibody with the
appropriate specificity. Thus, when mixtures of radiolabeled and unlabeled antigen are
incubated with the corresponding antibody, the amount of free (not bound to antibody)
radiolabeled antigen is directly proportional to the quantity of unlabeled antigen in the
mixture.
Principle of Radioimmunoassay:
It involves a combination of three principles.
1. An immune reaction i.e. antigen, antibody binding.
2. A competitive binding or competitive displacement reaction. (It gives specificity)
3. Measurement of radio emission. (It gives sensitivity)
Immune reaction:
When a foreign biological substance enters into the body bloodstream through a non-oral
route, the body recognizes the specific chemistry on the surface of foreign substance as antigen
and produces specific antibodies against the antigen so as nullify the effects and keep the body
safe. The antibodies are produced by the body’s immune system so, it is an immune reaction.
Here the antibodies or antigens bind move due to chemical influence. This is different from
principle of electrophoresis where proteins are separated due to charge.

26. Competittive binding and competitive displacement reaction:
This is a phenomenon wherein when there are two antigens that can bind to the same
antibody, the antigen with more concentration binds extensively with the limited antibody
displacing others. So here in the experiment, a radiolabelled antigen is allowed to bind to high-
affinity antibody. Then when the patient serum is added unlabeled antigens in it start binding
to the antibody displacing the labeled antigen.
Measurement of radio emission:
Once the incubation is over, then washings are done to remove any unbound antigens. Then
radio emission of the antigen-antibody complex is taken, the gamma rays from radiolabeled
antigen are measured.
The target antigen is labeled radioactively and bound to its specific antibodies (a limited and
known amount of the specific antibody has to be added). A sample, for e.g. blood-serum, is
added in order to initiate a competitive reaction of the labeled antigens from the preparation,
and the unlabeled antigens from the serum-sample, with the specific antibodies. The
competition for the antibodies will release a certain amount of labeled antigen. This amount is
proportional to the ratio of labeled to an unlabeled antigen. A binding curve can then be
generated which allows the amount of antigen in the patient’s serum to be derived. That means
as the concentration of unlabeled antigen is increased, more of it binds to the antibody,
displacing the labeled variant. The bound antigens are then separated from the unbound ones,
and the radioactivity of the free antigens remaining in the supernatant is measured.
Antigen-antibody complexes are precipitated either by crosslinking with a second antibody or
by means of the addition of reagents that promote the precipitation of antigen-antibody
complexes. Counting radioactivity in the precipitates allows the determination of the amount
of radiolabeled antigen precipitated with the antibody. A standard curve is constructed by
plotting the percentage of antibody-bound radiolabeled antigen against known concentrations
of a standardized unlabeled antigen, and the concentrations of antigen in patient samples are
extrapolated from that curve.
The extremely high sensitivity of RIA is its major advantage.
Uses of Radioimmunoassay:
1. The test can be used to determine very small quantities (e.g. nanogram) of antigens and
antibodies in the serum.
2. The test is used for quantitation of hormones, drugs, HBsAg, and other viral antigens.
3. Analyze nanomolar and picomolar concentrations of hormones in biological fluids.

27. Limitations of RIA includes:
1. The cost of equipment and reagents
2. Short shelf-life of radiolabeled compounds
3. The problems associated with the disposal of radioactive waste.
Immunofluorescence:
Immunofluorescence is an assay which is used primarily on biological samples and is
classically defined as a procedure to detect antigens in cellular contexts using antibodies. The
specificity of antibodies to their antigen is the base for immunofluorescence.
The property of certain dyes absorbing light rays at one particular wavelength (ultraviolet
light) and emitting them at a different wavelength (visible light) is known as fluorescence. In
immunofluorescence test, fluorescent dye which illuminates in UV light are used to
detect/show the specific combination of an antigen and antibody. The dye usually used
is fluorescein isothiocynate, which gives yellow-green fluorescence. Immunofluorescence
tests are also termed as fluorescent antibody test (FAT).
Fluorescent dyes, such as fluorescein isothiocyanate and lissamine rhodamine, can be tagged
with antibody molecules. They emit blue-green and orange-red fluorescence, respectively
under ultraviolet (UV) rays in the fluorescence microscope. This forms the basis of the
immunological test. Immunofluorescence tests have wide applications in research and
diagnostics. These tests are broadly of two types:
1. Direct immunofluorescence test

28. 2. Indirect immunofluorescence test
Direct immunofluorescence test:
Direct immunofluorescence test is used to detect unknown antigen in a cell or tissue by
employing a known labeled antibody that interacts directly with unknown antigen. If antigen is
present, it reacts with labeled antibody and the antibody coated antigen is observed under UV
light of the fluorescence. It involves use of labeled antiviral antibody.
Method:
The specimen is placed on slide; fluorescent labeled antibody is then added to it and allowed
for some time for Antigen-Antibody reaction. The preparation is then washed which will allow
the removal of other components except the complex of antigen and fluorescent labeled
antibody. On microscopy (Fluorescence Microscopy), Antigen- Antibody complex are observed
fluorescing due to the dye attached to antibody.
The need for preparation of separate labeled antibody for each pathogen is the major
disadvantage of the direct immunofluorescence test.
Indirect Immunofluorescence test
Indirect fluorescence is a double antibody technique. The unlabeled antibodies which have
bound to the antigens are visualized by a fluorescent antiglobulin reagent directed at the
unlabeled antibodies. The indirect immunofluorescence test is used for detection of specific
antibodies in the serum and other body fluids for sero-diagnosis of many infectious diseases.
Method
Indirect immunofluorescence is a two-stage process.
First stage: A known antigen is fixed on a slide. Then the patient’s serum to be tested is
applied to the slide, followed by careful washing. If the patient’s serum contains antibody
against the antigen, it will combine with antigen on the slide.
Second stage: The combination of antibody with antigen can be detected by addition of a
fluorescent dye-labeled antibody to human IgG, which is examined by a fluorescence
microscope.
The first step in the indirect immunofluorescence test is the incubation of a fixed antigen (e.g.,
in a cell or tissue) with unlabeled antibody, which becomes associated with the antigen. After
careful washing, a fluorescent antibody (e.g. fluorescent labeled anti-IgG) is added to the
smear. This second antibody will become associated to the first, and the antigen–antibody
complex can be visualized on the fluorescence microscope.

29. The indirect method has the advantage of using a single labeled antiglobulin (antibody to IgG)
as a “universal reagent” to detect many different specific antigen–antibody reactions. The test
is often more sensitive than the direct immunofluorescence test.
Indirect immunofluorescence test is used widely to:
1. Detect specific antibodies for serodiagnosis of syphilis, leptospirosis, amoebiasis,
toxoplasmosis, and many other infectious diseases;
2. Identify the class of a given antibody by using fluorescent antibodies specific for
different immunoglobulin isotypes;
3. Identify and enumerate lymphocyte subpopulations by employing monoclonal
antibodies and cytofluorographs; and
4. Detect autoantibodies, such as antinuclear antibodies in autoimmune diseases.
Immunofluorescence may also be used to analyze the distribution of proteins, glycans, and
small biological and non-biological molecules. Immunofluorescence has been widely used in
biological research and medical research.
The major limitations of immunofluorescence is that the technique requires
1. expensive fluorescence microscope and reagents,
2. trained personnel
3. have a factor of subjectivity that may result in erroneous results
The biological samples include tissue and cells. Immunofluorescence aid to evaluate whether
or not cells in a particular sample express the antigen in study. In cases where an immune-
positive signal is found, immunofluorescence also helps to determine which subcellular
compartments are expressing the antigen. Immunofluorescence can be used on cultured cell
lines, tissue sections, or individual cells.