Immunochemical techniques

Amrita Bhowmik
BSc & MSc, Biochemistry and Molecular Biology, JU
PhD Fellow, Biochemistry and Molecular Biology, DU
Lecturer
Dept of Applied Laboratory sciences
Bangladesh University of Health Sciences
 Basic concepts and definitions
Lymphocytes occurs 2 major types-
• B cells (develop in the bone marrow, and
produce antibody)
• T cells (derived from bone marrow, then
develop in the thymus, generate cytokines and
kill virus or other pathogen)
They are responsible for specific recognition of
Antigens.
Antibody:
• Antibodies are produced by plasma cells that
result from differentiation of B lymphocytes
following stimulation with antigen.
• Antibodies are Immunoglobulins (Ig).
• They are capable of binding specifically to a wide
array of natural and synthetic antigens (proteins,
CHOs, nucleic acids, lipids and other molecules)
• There are 5 Types of Ig-IgA, IgG, IgM, IgD and IgE.
 IgG
• IgG is the most prevalent immunochemical reagent in use.
• It is a Glycoprotein.
• Molecular weight (MW) 160 000.
• It is composed of two duplex chains with each set being
made up of a heavy (ɣ) and a light (α) chain joined by
disulfide bonds.
• Inter-chain disulfide bonds hold the duplex chains together
and create a symmetrical molecules.
• The variable amino acids sequence at the amino terminal
end if each chain determines the antigenic specificity of the
particular antibody.
• Each unique amino acid sequence is a product of a single
plasma line with a single specificity.
Schematic diagram of IgG antibody molecules
 Antigen
• Definition: A substance that when introduced into the body stimulates the
production of an antibody. Antigens include toxins, bacteria, foreign blood
cells, and the cells of transplanted organs.
• In immunology, an antigen, or antibody generator is any substance which
provokes an adaptive immune response. An antigen is often foreign or
toxic to the body (for example, a bacterium) which, once in the body,
attracts and is bound to a respective and specific antibody. That is to say, an
antigen is molecule that also induces an immune response in the body.
Following are some general properties requisite for immunogenicity:
• Ag has area of structural stability within the molecule
• Structure is random
• Structure is foreign
• Has minimal MW of 4000-5000
• Is able to be metabolized
• A particular immunogenic configuration is accessible to the antibody-
forming mechanism
Epitope:
• Each unique region of the
molecular antigen that binds
a complementary antibody is
termed an Epitope.
• It is also called Antigenic
Determinant.

Paratope
The paratope is the part of an antibody which recognizes
an antigen, the antigen-binding site of an antibody. It is a
small region (of 15–22 amino acids) of the antibody's Fv
region and contains parts of the antibody's heavy and light
chains.[1]
*1. Ref
Monoclonal Ab:
• Monoclonal antibodies are products of a single clone of plasma
cells derived from B-lymphocytes.
• The unique ability of a monoclonal Ab to react with a single
epitope on a multivalent antigen.
• The majority of monoclonal Ab do not cross-link and precipitate
macromolecular Ag. For this reason monoclonal antibodies have
not found broad applicability in the clinical laboratory.
• They typically display excellent specificity, but poor ability to
precipitate antigen.
Polyclonal Ab:
A complex antigen is capable of eliciting a
multiplicity of antibodies with different
specificities that are derived from different cell
lines. Antibodies derived in this manner are
termed Polyclonal and exhibit diverse
specificities in their reactivity with immunogen.
 Immunogen
• Antigens are recognized by specific lymphocytes or by antibodies,
not every antigen can evoke an immune response. Those antigens
that are capable of inducing an immune response are called
immunogens.
• Immunogen is an immunogenic material, that is either a protein or a
substance, coupled to a carrier.
• It is usually a protein, that when introduced into a foreign host is
capable of inducing the formation of an antibody in the host.
• We can define an immunogen as a complete antigen which is
composed of the macromolecular carrier and epitopes
(determinants) that can induce immune response.
 Haptan
• The Haptens are low-molecular-weight compounds that may be
bound by antibodies, but cannot elicit an immune response.
Consequently the haptens themselves are nonimmunogenic and
they cannot evoke an immune response until they bind with a
larger carrier immunogenic molecule. The hapten-carrier complex,
unlike free hapten, can act as an immunogen and can induce an
immune response .
• It is a chemically defined determinant, that when conjugated to an
immunogenic carrier, stimulates the synthesis of antibody specific
to the haptan.
The strength or energy of interaction between the
Ag & Ab is described by two terms:
Affinity:
A thermodynamic quantity, defining the energy of interaction of
a single Ab-combining site and its corresponding epitope on
the antigen.
Avidity:
The overall strength of binding of Ab and Ag, and includes the
sum of the binding affinities of all the individual combining
sites on the antibody.
Example:
• IgG has two affinity binding sites and IgM has 10 per Ab
molecules.
• Affinity is a property of the substance bound (Ag).
• Avidity is a property of the binder (Ab)
IgM
• Immunochemical reactions form the basis of a
diverse range of sensitive and specific clinical
assays.
• The principles of the methods most commonly
used in the laboratory are – (next slide)

Book Reference:
Tietz
Fundamentals of Clinical Chemistry
4th or higher Edition
 Principles
1. In a Typical immunochemical analysis, an
antibody is used as a reagent to detect the
chemical substance (Antigen) of interest.
2. The specificity and the high affinity of
antibodies for specific antigens coupled with
the unique ability of antibodies to cross-link
antigens allows identification and quantitation of
specific substances by a variety of methods.
 Ag-Ab binding
Reaction mechanism
Several forces act cooperatively to produce Ag-Ab
binding. Three major contributing forces are-
1. Electrostatic Vandar Waals – London dipole-
dipole interaction (The interaction of two atoms, molecules, or nuclei by
means of their electric or magnetic dipole moments).

2. Hydrophobic interaction, and

3. Ionic coulombic bonding (primarily between
COO- and NH4+ groups)
 The binding of Ag to Ab is not static but is an
equilibrium reaction that proceeds in 3 phases:
Phage 1: the initial reaction of a multivalent antigen
(Agn) and a bivalent Ab occurs very rapidly.
Phage 2: the subsequent growth of the complexes is
slower than phage 1 and the depicted by the
following equation-

Where k1 (the rate constant for forward reaction)>>>k2 (the rate constant for
reverse reaction),
n is the number of epitopes per molecules
a and b are the number of Ag and Ab molecules per complex.

Phage 3: The reaction involves the precipitation of the

complex after a critical size is reached.
The speed of these reactions depends on –
• Electrolyte concentration
• pH
• Temperature
• Types of Ag and Ab
• Binding affinity of the Ab
 The Precipitin Reaction
A. If the number of Ab combining sites, [Ab], is significantly
greater than the Ag binding sites [Ag], then Ag binding
sites are quickly saturated by Ab before cross-linking can
occur and the formation small Ag-Ab complexes (soluble)
of the composition AgAb results.
B. For the case in which antibody is in moderate excess
(Ab>Ag), the probability of cross-linking of Ag by Ab is
more likely and hence, large complex (insoluble)
formation is favored.
C. In the case where [Ag] is in great excess, large complexes
are less probable, and the theoretical minimum size of
complexes (soluble) would be Ag2Ab.
 Classical Quanitative precipitin curve

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 (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 an 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.
 Effects of Ionic species and Ionic strength
 Cationic salts produce an inhibition of the binding of
Ab with cationic haptan.
Cs+> Rb+> NH4+> K+> Na+>Li+
This order corresponds to the decreasing ionic radius
and increasing radius of hydrogen.

 Similar results were found for anionic haptan and

anionic salts. The order of inhibition of binding is-
CNS->NO3-> I-> Br-> Cl-> F-
again in the order of decreasing ionic radius and
increasing radius of hydration.
 Polymer Effect
The addition of a linear polymer to a mixture of Ag and Ab
causes a significant increase in the rate of immune
complex growth and enhances the precipitation of
immune complex, specially with low avidity Ab.

↑MW of polymer=
↑degree of linearity of polymer (minimum branching)=
↑aqueous solubility of protien
 Qualitative Methods
Precipitation methods in gel

1. Passive Gel Diffusion

2. Immuno-electrophoresis
3. Western Blotting
 Passive Gel Diffusion
Principle:
• This methods performed in a semisolid medium (such
as agar or agarose), that stabilizes the diffusion
process with regard to mixing caused by vibration or
convection and allows visualization of precipitin
bands for qualitative and quantitative evaluation of
the reaction.
• Ag-Ab ratio, salt concentration, and polymer
enhancement have the same influence on the Ag-Ab
reaction in gels as they have on reactions in solution.
It the matrix does not interact with molecular
species under investigation, passive diffusion
of reactants in a semisolid matrix can be
described by Fick’s equation:
dQ/dt=-DA dC/dx
Where dQ= the amount of diffusing substance that passes
through the area A during time t
dC/dx= the concentration gradient
D= the diffusion coefficient
D is a directly proportional function of Temp and
is an inversely proportional function to hydrated molecular
volume of the diffusing species.
dQ/dt is clearly a function of dC/dx, the concentration
gradient; the amount of diffusing species transferred from
the origin to a distant point (over the migration distance) is
dependent on the length of time diffusion is allowed to
 Two basic approaches to passive diffusion
Simple diffusion/ Single immunodiffusion:

A concentration gradient is established for only a single reactant and usually

depends on diffusion of an Antigen into agar impregnated with Ab.
• The antigen is applied into wells that are cut in the agarose gel containing
dispersed corresponding monospecific antibody.
• 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),
• 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.
This technique has been, however, currently largely replaced with
immunoturbidimetry or nephelometry
Double immunodiffusion/ Ouchterlony technique:

In double immunodiffusion reaction, the antigen and the antibody diffuse

towards each other.
• a reaction of identity –It is based on wells punched into the agarose
gel 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.
• a reaction of non-identity –If antiserum (antiserum, blood serum that contains
specific antibodies against an infective organism or poisonous substance) to several possible
antigens is placed into the central well and the outer wells are filled by
different antigens, the precipitin bands form lines that intersect.
• a reaction of partial identity – if two antigenic mixtures are applied
into two adjacent wells, it is characterized by a formation of a spur.
The spur means that the second antigen lacks an epitope present in the
first antigen that is recognized by one of the antibodies in the
antiserum.
 2. Immunoelectrophoresis (IEP)
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).
Eg-, evaluation of human myeloma proteins.
Crossed
immunoelectrophoresis
(CRIE)
Two dimensional
Electrophoresis is used in
the 2nd dimension to derive
the Ag into a gel
that contains Ab specific
for the Ag of interest
More sensitive
Higher resolution
Counter immunoelectrophoresis
(CIE)
Two parallel lines of wells are
punched in the agar
One row is filled with Ag solution
and the opposing row is filled with
Ab solution,
Voltage is applied across the gel so
that the Ag and Ab move toward
the anode, and Ab moves in the
opposite direction as a result of
electrophoresis.
A precipitin line is formed where
they meet.
Provided within 1 -2 hrs
Detection of bacterial Ag in blood, Used for the evaluation of
urine and Cerebrospinalfluid (CSF) myeloma protein
Immunofixation
(IF)
First dimensional
electrophoresis is performed
in gel to separate the proteins
in the mixture
Antiserum (Ab) spread
directly on the gel causes the
proteins of interest to
precipitate which trapped
within gel matrix and
nonprecipitate proteins are
removed by washing gel.
The gel may be stained for
protein identification
 3. Western Blotting

• Direct immunoprecipitation of the protein in the

gel.
• Transfer of the Protein from the medium in
which the electrophoresis was performed onto a
solid phase (nitrocellulose/nylon based
membrane), acts to fix the proteins after
electrophoretic separation.
• The proteins are fixed to the membrane, they
can be detected using Ab probes labeled with
either radioactive isotopes or enzyme.
• Sensitivity is 10-100 times higher than other
methods.
 Qualitative or Semiquantitative Assay:
Agglutination
• Agglutination (glue/ attach) is an immunochemical technique in which a
specific antibody reacts with the corpuscular antigen (small particles of living organism) .
• 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.
• Agglutination reactions are performed on slides, in test tubes or microtiter
plates. They are more sensitive in comparison with immunoprecipitation
methods.
 Agglutination assays may be classified as direct
or indirect tests.

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 or detection of specific antibodies
directed against naturally occurring antigens on the surface of some microbes.
Indirect agglutination
• Indirect agglutination assay utilizes particles (latex, colloid
gold) with the antigens (RBC are used as carrier) that have
been passively attached to their surface.(fig 9)
• Instead of the antigens, particles can also be coated by
specific antibodies. This technique is called reverse
agglutination and can be utilized for the detection of soluble
antigens (for example C-reactive protein or human chorionic
gonadotrophin /hCG) (Fig. 10).
 Quantitative Methods
Precipitation methods

1. Radial Immunodiffusion (RID) and Electroimmunoassay

(Rocket technique)(in gel)
2. Turbidi metric and Nephelo metric Assays (in solution)
3. Labeled immunochemical Assays, eg-
• Radioimmunoassay (RIA)
• Enzyme Immunoassay (EIA)
 1. Radial Immunodiffusion and Electroimmunoassay
• Both are Gel based methods.
RID:
1. It is a passive diffusion method in which a concentration gradient
is established for a single reactant, usually the Ag.
2. The Ab is uniformly dispersed in the gel matrix.
3. Ag is allowed to diffuse from a well into gel until Ab excess
exists and immunoprecipitation occurs.
4. The Ag-Ab interaction is manifested by a well-defined ring of
precipitation around the Ag well.
5. The ring diameter continues to increase until equilibrium is
reached.
6. Calibrators are run at the same time as the sample, and a
calibration curve of ring area or diameter vs concentration is
plotted.
 Electroimmunoassay
• As in RID, a single concentration gradient is established for the Ag,
but in this case an applied voltage is used to drive the Ag from the
application well into homogeneous suspension of antibody in the gel.

• In distinction to RID, this produces a unidirectional migration of Ag and

results in increased sensitivity.
• The height of the resulting rocket-shaped precipitin line is proportional
to the Ag concentration.
• Quantitation is effected by using calibrators on the same plate along
with the unknowns, and the estimating the concentrations of unkhowns
from the heights of the “rockets” obtained.
•The calibration curve is linear only over a narrow concentration range,
so samples may have to be diluted or concentrated as needed.
 2. Turbidi metric and Nephelo metric Assays
Precipitation methods in solution
Two techniques are used to quantitate this precipitate: immunoturbidimetry and
immunonephelometry. (for determination of proteins in serum, plasma, urine
and cerebrospinal fluid )
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) (SEE: Precipitation Reaction Type A) 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. Turbidimetry methods follow the
Beer-lamberts Law. 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). Exp- determination of several blood plasma proteins or in diseases
such as multiple myeloma
• 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 (see the precipitation curve). 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.
• The immunoprecipitation techniques in
solution are much faster than radial
immunodiffusion, but also more expensive.
 3. Labeled Immunochemical assays
• The method rely on examining the immune complex
formation as an index of Ag-Ab reaction.
• The initial binding of the Ab and Ag has been
demonstrated to be very useful analytically and has
been used with labeled Ag and Ab to develop many
sensitive and specific immunochemical assays.
• The reaction describing this initial binding and the
kinetic constant for the overall reactions are shown
respectively-
• As would be predicted from the law of mass
action, the concentration of Ab, Ag and AgAb
will be dependent on the magnitude of k1 and
k(-1) .
• The original assays employed radioactive
labels, but concerns over the safe handling
and disposal of radioactive reagents and
waste have led to the development of
alternative nonisotopic labels.
• The label can be a=
• Radioisotope (radioimmunoassay, RIA): isotopes of
iodine (125I, 131I) and tritium (3H) as labels.
Combination of labels (57Co and 125I) are used for
simultaneous assays; eg- Vit B12 and folate
• Nonisotopic immunoassay –
An enzyme (enzyme immunoassay, EIA),
Fluorescent or chemiluminiscent substance.

Detection limits of these techniques can be as low as

10−15 – 10−20 mol/L.
 Methodological Principles of
Labeled Immunochemical assays
A. The reaction scheme in these immunochemical assays can
follow either competitive, or non-competitive approach.
Competitive enzyme immunoassay (limited reagent assay)
• 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 quantity of
specific antibodies bound to the solid phase.
• The avidity of the Ab for both the labeled and unlabeled Ag must be the
same.
• Under these conditions, the probability of the Ab binding the labeled Ag
is inversely proportional to the concentration of unlabeled Ag; hence,
bound label is inversely proportional to unlabeled Ag concentration.

Note: Precipitin reaction C

The reaction involved in two steps-
Step 1: Unlabeled Ag is first mixed with excess Ab, and binding is
allowed to reach equilibrium.
Step 2: Labeled Ag is then sequentially added and allowed to
equilibrate
After separation, the bound label is determined and used to
calculate the unlabeled Ag concentration.
• 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 for determination of
antibody:
• Excess reagent, two-site, or sandwich assays.
Step 1: This assay for an Ag , a capture Ab is first passively adsorbed or
covalently bound to the surface of a solid phage.
The Ag from the sample is allowed to react with the solid-phage
Ab, other proteins are wash away.
Step 2: A labeled Ab (conjugate) is added that reacts with the bound
Ag through a second and distinct epitope.
After washing again, the bound label is determined, and its
concentration or activity is directly proportional to the
concentration of Ag.

Note: Precipitin reaction A

• 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 assay procedure are similar to that described for the
measurement of antigens.
 B. Heterogenous and Homogenous
Immunochemical Assays
• A heterogeneous enzyme immunoassay method is also called enzyme-
linked immunosorbent assay (ELISA).
• This physical separation techniques are used to separate the free label
(Ag*) from the bound labeled Ag (AbAg*).
• There are several types of techniques named-
1. Liquid phage adsorption: The free Ag is adsorbed onto particles of
activated charcoal or dextran-coated charcoal that are direactly to the
reaction mixture.
2. Precipitation: Addition of a protein-precipitating chemical, (NH4)2SO4 or
double Ab precipitation.
3. Solid-Phage Antibiotics: most popular, widely used
4. Miscellaneous: Electrophoresis, Gel Filtration, Ion exchange
• Precipitation and Solid phage Adsorption are mostly used.
• Tubes, wells of microtiter plates, and magnetic particles may be used as
the solid phases.
 ELISA
• An aliquot of sample (serum/whole blood) or calibrator containing
the Ag (virus/parasites) to be quantitated is added to and allowed
to bind with a solid phase Ab (micro titer well).
• After washing, Enzyme labeled Ab (different from the bound Ab) is
added and forms a “sandwich complex” of solid phage AbAgAb-
enzyme.
• Excess (unbound) Ab is then washed away, and enzyme substrate is
added; the enzyme catalytically converts the substrate to visible
products(s), the amount of which is proportional to the quantity of
Ag in the sample.
• Ab in a sample can also be quantitated using an ELISA procedure in
which Ag, instead of Ab, is bound to a solid phage and the 2nd
reagent is an enzyme-labeled Ab specific for the analyte Ab. (Fig 14
and 15)
biotinylation is the process of covalently attaching biotin to a protein, nucleic acid or other molecule. Biotin binds to streptavidin protein for extremely high affinity,
fast on-rate, and high specificity.

Membrane-bound EIA (lateral flow) tests are commonly used for rapid detection of
hormones (hCG, LH), viruses (hepatitis B and C), bacteria (Streptococcus,
Chlamydia, Helicobacter pylori), bacterial toxins, parasites (malaria),
therapeutic and illicit drugs, as well as biomarkers such as troponin
(cardiovascular disease) or prostate specific antigen(PSA, prostate cancer).
 A homogeneous enzyme immunoassay :
A. Enzyme multiplied immunoassay technique (EMIT)
• It 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.(competitive immunoassay)
• 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 method can be used for whole blood, serum, or urine. Enzyme multiplied
immunoassays can be fully automated with a fast throughput of clinical samples
especially in laboratories specializing in monitoring therapeutic drugs like
cyclosporin in transplant recipients.
 B. Cloned enzyme donor immunoassay (CEDIA)
• This assay was the first RIA designed and developed using genetic
engineering techniques.
• Two active fragments (Enzyme donor ED and Enzyme Acceptor
EA) spontaneously reassemble to form active enzyme even if the
enzyme donor is attached to an Ag.
• Binding of Ab to the enzyme donor, Ag conjugates inhibits
reassembly. And no active enzyme is formed.
• Thus competition between Ag and the enzyme donor-Ag conjugate
for a fixed amount of Ab in the presence of the enzyme acceptor
modulates the measured enzyme activity.
• High Concentration of Ag produce the least (less) inhibition of
enzyme activity; low concentrations produce the greater inhibition.
One Example of Labeled Immunochemical Assays
eg- EIA

• EIA utilizes the catalytic properties of enzymes

(Alkaline phophatase, horseradish per oxidase etc) to
detect the quantitate immunological reactions.
• EIA also utilize fluorescent or chemiluminescent-
labeled substrate or products are often preferred to
photometry-based assays owing to the inherent
sensitivity of fluorescence and chemiluminescence
measurement.
 A

A. Chemiluminescent assay for horseradish peroxidase label using luminol

B. Photometric assay for an alkaline phosphatase label using a cascade
detection reaction. INT= pera iodonitrotetrazolium
 Examples of fast immunochemical diagnostic methods
Detection of narcotics (psychoactive compound with any sleep-inducing properties) 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.
• Positive Result: 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.
• Negative result: 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.
 Detection of human chorionic gonadotropin (hCG)
(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 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..
1. the antigen-antibody binding is highly specific, allowing for specific detection
of analytes with a particular epitope. However, some antibodies bind to non-
target compounds that have structural similarities with the target analyte—in
other words, they have cross-reactivity against these related compounds. Cross-
reactivity makes assays less specific but at the same time more robust, and
suitable for detection of different classes of compounds, like pyrethroid
metabolites in human urine samples.
2. Nonspecific binding of primary or secondary antibodies onto solid support can
lead to false positive readings. Usually, nonspecific binding is eliminated
using blocking agents like bovine serum albumin (BSA) or non-fat dry milk,
which bind to potential non-specific binding sites without interfering with the
assay itself.
3. One of the problems with the analysis of biofluids (blood, urine, saliva) is the
interference caused by the structurally similar compounds in the sample matrix.
Sometimes this matrix interference is so strong that the sample has to be
cleaned up prior to analysis. Common clean-up techniques that eliminate
structurally similar, interefering substances include liquid-liquid or solid phase
extraction (SPE).
1. The future for clinical immunochemistry looks very promising. We
have come a long way from the most simple antibody-based tests
like the PPD skin test for tuberculosis to fully automated ELISAs with
fluorescent markers for quantification of small molecules in patient
serum to a pico or femto mole level.
2. Future development will undoubtedly include more micro-scale
instrumentation with ultrasensitive detection, faster turnaround
time, and increased throughput in clinical laboratories.
3. Rapid development in the area of quantum dots and other
fluorescent nanoparticles will produce more applications in which
multiple analytes can be detected simultaneously.
4. Advances in the fields of multiplex immunoassays and sensitive
cellular imaging will eventually benefit routine clinical laboratory
analysis as well.
There are many advantages to using immunochemical methods in clinical
laboratories.
1. Specificity,
2. sensitivity,
3. speed,
4. portability,
5. ease of use,
6. low cost, and
7. the range of analytes that can be measured are the main reasons for the
increased use of these techniques in laboratories and
8. point-of-care facilities.
There are many advantages to using immunochemical methods in clinical
laboratories.
1. Specificity,
2. sensitivity,
3. speed,
4. portability,
5. ease of use,
6. low cost, and
7. the range of analytes that can be measured are the main reasons for the
increased use of these techniques in laboratories and
8. point-of-care facilities.
The End