Principles, Classification and Optimization of Immunoassay

1. IMMUNOASSAY
THEORTICAL BASIS &
OPTIMIZATION
SUBMITTED TO:-
DR. ABHILASHA AHLAWAT
ASSOCIATE PROFESSOR M. D. U.
SUBMITTED BY:-
SAHIL
Class- M. Pharmacy 1st sem
Roll no:-1809
Department of Pharmaceutical
Sciences
M.D.U. ROHTAK

2. INTRODUCTION
• ► An immunoassay is a test that uses antibody and antigen complexes as a means of generating a
measurable result. An antibody: antigen complex is also known as an immuno- complex.
• "Immuno" refers to an immune response that causes the body to generate antibodies, and "assay"
refers to a test. Thus, an immunoassay is a test that utilizes immuno-complexing when antibodies and
antigens are brought together.
• An immunoassay is a laboratory technique used to detect or measure specific substances, such as
hormones, proteins, drugs, or antibodies, in biological samples.
• It relies on the binding between an antibody and an antigen to identify or quantify the target
molecule. Immunoassays are widely used in medical diagnostics, research, and various industries due
to their sensitivity and specificity in detecting substances within biological samples.
• There are different types of immunoassays, including ELISA (Enzyme-Linked Immunosorbent Assay)
and western blotting.

3. PRINCIPLE OF IMMUNOASSAY
• Immunoassays work on the principle of using antibodies to detect and quantify specific substances.
The process involves the interaction between an antigen (the substance being measured) and an
antibody that selectively binds to it. This binding forms the basis for detecting and measuring the
target substance, often through various methods like enzyme-linked immunosorbent assay (ELISA),
radioimmunoassay (RIA), or Chemiluminescent Immunoassay (CLIA). The amount of signal produced in
these assays correlates with the concentration of the substance being tested.
• In addition to the binding of an antibody to its antigen, the other key feature of all immunoassays is a
means to produce a measurable signal in response to the binding. Most, though not all, immunoassays
involve chemically linking antibodies or antigens with some kind of detectable label. A large number
of labels exist in modern immunoassays, and they allow for detection through different means. Many
labels are detectable because they either emit radiation, produce a color change in a solution,
fluorescence under light, or can be induced to emit light.

4. COMPONENTS OF IMMUNOASSAY
Immunoassays typically require several key components:
• Antigens or analytes: These are the substances being measured or detected, which could be
hormones, proteins, drugs, or infectious agents.
• Antibodies: These are specific proteins that bind to the antigens or analytes. They can be produced in
the laboratory or obtained commercially. Antibody (Ab) also know as Immunoglobulin (Ig) is the large
Y shaped protein produced by the body's immune system when it detects harmful substances, called
antigens like bacteria and viruses.The production of antibodies is a major function of the immune
system and is carried out by a type of white blood cell called a B cell (B lymphocyte).

5. • Solid support or surfaces: These are platforms where the immunochemical reactions take place, such
as microplates, beads, or membranes.
• Washing solutions: These are used to remove unbound substances and reduce background noise in
the assay.
• Detection labels: These could be enzymes, fluorescent molecules, radioactive isotopes, or
chemiluminescent compounds. They help visualize or quantify the binding interactions between
antibodies and antigens.
• Calibrators and controls: These are samples of known concentrations used to create a standard curve
for quantification and to ensure the accuracy and reliability of the assay.
• Reader or detection system: This could be a spectrophotometer, luminometer, or other instrument to
measure and interpret the signals produced during the assay.

6. STRUCTURE OF ANTIBODY
• There are four polypeptide chains: two identical heavy chains and
two identical light chains connected by disulfide bonds. Light Chain
(L) consists polypeptides of about 22,000 Da and Heavy Chain (H)
consists larger polypeptides of around 50,000 Da or more.
• An antibody is made up of a variable region and a constant region,
and the region that changes to various structures depending on
differences in antigens is called the variable region, and the region
that has a constant structure is called the constant region.

7. TYPES OF ANTIBODY
• Antibody may be either
1.) Polyclonoal or
2.) Monoclonal
For immunoassay development, monoclonal antibodies are more advantageous than polyclonal ones
because they differ from polyclonal antibodies in that they are highly specific for a single epitope on a
monovalent antigen.

8. POLYCLONAL ANTIBODY PRODUCTION
• If the agent is a foreign to the animal, the animal will
develop antibodies to the agent and release these
antibodies into its blood.
• After a few months, blood is removed from the animal
and the antibodies produced are collected for use.
• Antibodies produced in this fashion are typically very
heterogeneous and known as polyclonal antibodies.
• Recognize a number of different sites on the
analyte(antigen), binding with a range of affinities.
• Arise from several different lines of antibody-
producing cells within the animal.

9. MONOCLON ANTIBODY PRODUCTION
• Monoclonal antibodies differ from
polyclonal antibodies in that they are
produced by a single cell line within the
body.
• All monoclonal antibodies from the same
cell line recognize the same site on an
analyte and bind with an identical
binding affinity.

10. GENERAL PROCEDURE & CLASSIFICATIONFOR
IMMUNOASSAY
• When these immuno-analytical reagents are mixed and incubated, the analyte is bound to the antibody forming an
immune complex.
• This complex is separated from the unbound reagent fraction by physical or chemical separation technique.
• Analysis is achieved by measuring the label activity (e.g., radiation, fluorescence or enzyme) in either bound or free
fraction.
CLASSIFICATION OF IMMUNOASSAY
1.) ENZYME IMMUNOASSAY (EIA) or ENZYME LINKED IMMUNOASSAY (ELISA)
2.) RADIOIMMUNEASSAY (RIA)
3.) FLUOROIMMUNEASSAY (FIA)
4.) CHEMILUMINESCENCE IMMUNOASSAY (CIA)
5.) LABEL-FREE IMMUNOASSAYS

11. ENZYME IMMUNOASSAY (EIA) OR ENZYME-LINKED
IMMUNOSORBENT ASSAY (ELISA)
• Enzyme immunoassays, or ELISAs, are simple, widely used tests that rely on highly specific antibody-antigen
interactions. An ELISA is designed to detect and quantify soluble molecules such as antibodies, proteins and
hormones. There are four main kinds of ELISA: sandwich, competitive, direct and indirect assays. These methods
differ in how the antibody or antigen is attached to the solid plate, and how the signal is detected.
• In a sandwich ELISA, for example, an antibody is immobilized on a plate. The sample containing the target
antigen is added, which binds to the antibody and so is immobilized on the plate. Next, a second type of
antibody is added, which also binds to the target antigen on the plate, forming a ‘sandwich’ with the target
antigen in the middle. The second antibody is linked to an enzyme, called a reporter enzyme, which allows the
binding reaction to be measured by creating a color signal. To create this signal, first any unbound antibody is
washed away, and a colorimetric substrate is added. The enzyme catalyzes a reaction of the substrate, creating
a color change. A stronger color signal indicates more target antigen is present. An example of this is a home
pregnancy test. Other types of ELISAs also have vital applications in clinical practice, such as magneto ELISA
that is used for the detection of CD4+ cells for the diagnosis and treatment of AIDS.

12. Fig. Flow chart for direct ELISA Fig. Flow chart for Indirect ELISA

13. RADIOIMMUNEASSAY (RIA)
• A radioimmunoassay (RIA) uses radiolabeled antigens to measure concentrations of substances in
body fluids such as blood and saliva. A radioisotope is attached to an antigen of interest and bound
with its complementary antibody. A sample with the target antigen is then added, which competes
with the radioactive antigen, kicks it out of the binding spot and replaces it.
• After washing away unbound antigens the radioactivity of the sample is measured. The smaller the
radioactive signal, the more target antigen is present. Compared to other immunoassay techniques,
extra precautions are needed due to the radioactive substances involved, but RIA’s high sensitivity and
specificity means that it remains in use today.

14. Fig. Flow chart for RADIOIMMUNEASSAY

15. FLUORESCENTIMMUNEASSAY (FIA)
• Fluorescent immunoassays (FIA) use a
fluorescent compound as the detection
reagent to detect and quantify a variety of
compounds. FIA is widely used in the in vitro
diagnostics (IVD) industry and has the
advantage of being fast and highly sensitive
compared to other methods. In FIA,
antibodies are labeled with fluorescent
probes. FIA fluorescent dyes illuminate in UV
light and are used to detect a specific
antigen-antibody binding. After incubation
with the antigen, the antibody-antigen
complexes are isolated, and the fluorescent
intensity is measured.

16. CHEMILUMINESCENT IMMUNOASSAY (CLIA)
• The principles of a chemiluminescent immunoassay (CLIA) are the same as an ELISA or
fluoroimmunoassay, but the reporter is different. Luminescence is the release of light due to an
electron being kicked up to a higher energy state and emitting a photon as it relaxes down. CLIA uses
a chemical reaction to kick the electron up to a higher energy, whereas FIA uses certain light
frequencies.
• Due to its high sensitivity and specificity, CLIA is used in a range of different fields, including
environmental monitoring, disease diagnosis, life sciences and food safety. Newer techniques, such as
magnetic-bead based CLIA have also widened its potential. For example, magneto-actuated
chemiluminescence assays were developed to detect the presence of Zika virus in patient samples.

17. LABEL-FREE IMMUNOASSAYS
• The emerging field of photonic biosensors is driving development of a new generation of label-free
assays that don’t rely on central laboratory facilities. Photonics is the science of detecting and
manipulating light. By using technologies developed by the electronics industry in the production of
microprocessors and adapting them to light, photonic biosensors combine photonic sensing with bio
recognition technology to create label-free testing on-chip. Instead of moving electrons around on
silicon chips, light is moved around on silicon chips via waveguides.
• This technology has allowed the development of miniature lab-on-a-chip label-free immunoassay
(LFIA) devices. These devices are functionalized with capture antibodies and have a resonance
condition of light. This resonance wavelength will be shifted by a reaction between the capture
antibody and the target antigen due to the change in refractive index. Measuring the shift in resonance
wavelength provides a readout of a binding event. Label-free assays therefore enable the detection of
antigen-antibody binding without the use of an additional label, resulting in increased assay sensitivity
and decreased working time.

18. OPTIMIZATION OF IMMUNOASSAY
The large number of types and subtypes of immunoassays can make choosing the right immunoassay for
your application challenging. Here are some things to consider when making your decision:
• Decide what sensitivity you need; This is determined by the concentration of antigens you’ll be
working with low concentrations need higher sensitivity to ensure detection. Also consider your
throughput requirements and cost.
• Decide on the antibody-antigen pairs that you will test; You should check their availability
commercially, or alternatively produce reagents in the laboratory.
• Decide what surface or environment you want to use; For example, you can bind antibodies or
antigens to a solid plate or a magnetic bead, depending on which target needs to be observed.

19. MINIMIZE BACKGROUND AND PROMOTE HIGHER
SPECIFIC SIGNAL
• There are a variety of methods to promote a strong signal while minimizing nonspecific binding and
background noise. First, use an efficient plate coating buffer to provide a stabilized coating of the
antibody or antigen on the microtiter plate. These plate coating buffers stabilize coated proteins by
maintaining their tertiary three-dimensional structure, allowing for greater binding reactivity with the
detection molecule and enhancing the specific signal. Next, use a blocking buffer to block assay wells.
This blocking of unoccupied space in the plate wells prevents nonspecific binding of sample and assay
components and reduces the overall background signal. Similarly, using the proper sample diluents
will dilute the samples to read within the functional range, minimize sample matrix effects, block
nonspecific conjugate binding, and inhibit complement and thrombin activity, all of which reduce
background noise and optimize signal. Conjugate stabilizer diluents will also inhibit nonspecific
binding, minimizing background signal.
• Finally, effective wash buffers are needed to rinse microtiter plates between each reagent addition step
in an immunoassay. The use of these methods provides an overall improvement in assay sensitivity by
reducing background noise.

20. IMPROVE PRECISION
It is necessary to reduce plate to plate
variability when preparing immunoassays.
Protein stabilizing buffers allow plates to be
prepared in batches to be used over time,
which increases consistency and provides
increased plate to plate precision over
extensive storage periods.
INCREASE ASSAY
REPRODUCIBILITY
To increase the reproducibility of any custom
ELISA or immunoassay, it is important to source
reagents from a consistent and reliable supplier.
Look for established companies that offer strong
customer support, an easy-to-use purchasing
system, and fast shipping options.

21. STABALIZE PROTEIN
CONJUGATES
Not only do conjugate stabilizer
diluents inhibit nonspecific binding to
reduce background noise, but they also
preserve enzymatic activity and protein
conformation of enzyme conjugates. In
addition, stabilizing conjugates enables
you to store conjugated proteins and
antibodies for future use, prepare
batches of diluted, ready-to-use
conjugate aliquots, and reconstitute
lyophilized conjugates while
preserving native protein configuration
and activity.
Using dependable stabilizing and blocking buffers to stabilize proteins
allow you to store your prepared microtiter plates. Depending on the
type of protein, properly prepared plates can be stored under proper
conditions for several months or even years if prepared using reliable
reagents.
ICT’s Antibody-Sandwich ELISA Development Kit provides all the
necessary reagents to assist you in developing and optimizing your
immunoassays. From coating buffer to substrate stop solution, this kit
contains enough reagent to construct ten 96-well ELISA plates. In
addition, a comprehensive ELISA development manual is supplied with
protocols for assessing initial assay feasibility and optimizing ELISA
performance parameters, which will be beneficial to both novice or
experienced users. This kit is convenient, economical, and backed by the
dedicated ICT technical support team.
INCREASE SHELF-LIFE

22. REFERENCE
• https://www.immunochemistry.com/blogs/blog/5-essential-steps-optimize-
immunoassay-performance
• https://www.sepmag.eu/blog/different-types-of-immunoassays
• Christopher P. Price an David J. Newman’s“Principle and Practice of Immunoassay”
published by Palgrave Macmillan London
• Wiki description explanation, brief detail Biochemistry: Immunology

23. THANK YOU