Unlock the mysteries of immunoassays with this comprehensive PowerPoint presentation. Delve into the fundamental principles that underpin immunoassay techniques, exploring the theoretical foundations and key concepts. From antigen-antibody interactions to signal amplification strategies, this presentation provides valuable insights into the world of immunoassay science. Key Topics: Basics of Immunoassay: Antigen-Antibody Interactions Types of Immunoassays: ELISA, Western Blot, and More Signal Detection and Amplification Techniques Factors Affecting Assay Sensitivity and Specificity Optimization Strategies for Enhanced Performance Emerging Trends in Immunoassay Technology Who Should View: Designed for scientists, researchers, and students in the fields of immunology, biochemistry, and medical diagnostics. Whether you're new to immunoassays or seeking advanced insights, this presentation caters to a broad audience. Presenter: Mr. Gadage Ashish Rambhau (M Pharm Pharmacology) Pravara Rural Education Society pravaranagar,Loni .

1. General principle of immunoassay : Theoretical
basis and optimization of immunoassay
Presented by : Gadage Ashish Rambhau Guided by : Dr. Santosh Dighe
F. Y. M. Pharm(Pharmacology) Head of Department(Pharmacology)

2. Content
• Introduction
• Defination
• Principle
• Component required for immunoassay
Antibody
Antigen
Signal-generating lable (labelled)antigen
Separation matrices
• General principle of immunoassay
• Classification of immunoassay
• Optimization of immunoassay
• Application

3. Introdution :-
• An immunoassay is a test that uses antibody complexes as a means of
generating a measurable result.
• An antibody : antigen complex is also known as an immuno-
complex.
• Immuno" referes to an immune response that causes the body to
generate antibodies and "assay" referes to a test.
• Thus ; an immunoassay is test that utilizes immuno- complexing
when antibodies and antigens are brought together.

4. Defination :-
• An immunoassay is a biochemical test that measures the presence or concentration of
a macro molecule or a micro molecule in a solution through the use of an antibody or
an antigen.
• The molecule to be detected by the Immunoassay is often referred as an "analyte”.
• Analytes in biological liquids such as serum or urine are Frequently measured using
immunoassays for medical and research purposes.

5. Principle :-
• Immunoassays rely on the ability of an antibody to recognize and bind a specific
macromolecule. In immunology the particular macromolecule bound by an
antibody is referred to as an antigen and the area on an antigen to which the
antibody binds are called an epitope.
• In some cases, an immunoassay may use an antigen to detect for the presence of
antibodies, which recognize that antigen, in a Solution. In other word in some
immunoassays, , the analyte may be an antibody rather than an antigen.
• 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.

6. Components Required for immunoaasy
• Antibodies
• Antigen
• signal-generating labels (labelled) antigen
• separation matrices

7. Antibody :-
• Antibody (Ab) also known as immuno globulin (Ig) is the large Y Shaped Protein
produced by the body's immune system when it detects harmful substance 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).
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 the
varible region, and region that has a constant structure is called the
constant region.

8. Fig. Antibody

9. Paratope
• A Paratope also called an antigen-binding site, is a past of an antibody which
recognizes and binds to an antigen.
Epitope:
• The past of the antigen to which the paratope binds is called an epitope.

10. Types of antibody :-
• Polycolnal antibody
• Monoclonal antibody
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.
Antibody production - Polyclonal antibodies :
• 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.

11. • 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.

12. Antibody Production- monoclonal antibodies (m Ab) :
• Monoclonal antibodies differ Form 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.

13. Antigen :-
• Antigen is a substance capable of Causing on immune response leading to the
production of antibodies and are also the target to Which antibodies will bind.
• The area on an antigen to which the antibody binds is called an epitope.
Signal Generating Labels :-
• A label is a molecule that will react as part of assay, so a change in signal can be
measured in the blood : Reagent solution.
• All immunoassay requires the Use of labeled material in order to measure the
amount of antigen or antibody Present.
• Example of Label include a radioactive compound that produce radiation,
an enzyme that cause a changes of color in solution or a substance that produce
light.
• The label can be applied during the manufacture of reagent to either the antibody
or antigen

14. Separation matrices :-
• They are used for separation of the immune complexes that Formed as a result of
immuno analytical (ag-ab) reactions Include Polyethylene glycol, charcoal, second
antibody, microbeads or the most useful 96-well microwell plate.
• each well of the plate serves as a separate reaction tube.
• one compound of the reaction (ab or ag) is coated onto the Surface of the bottom
of the Plate wells and the immune Complex is Formed on the surface of the wells.

15. General procedure for 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 (eg radiation, Fluoresence of
enzyme) in either bound of Free reaction.

16. Classification of immunoassay :-
1. Competative immunoassay
a) Homogeneous
b) Heterogenous
2. Non-competitive immunoassay

17. 1. Competitive Immunoassay :
• These are reagent (Ag) excess Immunoassay.
• In this assay a fixed amount antibody and fixed amount labelled antigen and
unlabeled antigen variable amount was taken.
• Here labelled and unlabeled antigen both competes with each other for binding to
a fixed amount antibody.
a) Homogeneous competitive Immunoassay
• In this, unlabeled antigen in a sample competes with labelled antigen to bind an
antibody.
• Unbound ag do not need to be separated prior to measurement.
• The amount of unbound labelled antigen is measured.
• The amount of labelled unbound antigen is proportional to the amount of analyte
in the sample.
• E.g. Enzyme multiplied immunoassay

18. b) Heterogeneous competitive Immunoassay :
• In this assay , Unlabeled antigen in a sample competes with labelled antigen to
bind an antibody.
• The unbound antigen is separated or washed away, and the remaining labelled,
bound analyte is measured
• E.g. ELISA

19. 2. Non-competitive Immunoassay :
• These are antibody excess Immunossay.
• In this, fixed amount of antigen and a variable amount of unlabeled antibody and
fixed amount of labelled antibody was taken.
• The unkown analyte in the sample bind with labelled antibodies.
• The unbound labelled antibodies are washed measured away and the bound
labelled antibodies are measured.
• The intensity of the signal is directly Proportional to the amount unknown analyte.
• The amount of labelled antibody on the site is then measured It will be to the
directly proportional Concentration of the analyte because labelled antibody will
not bind if analyte is not Present in the unknown sample.

20. Optimization of immunoassay :
Minimize background noise and promote higher specific signal
• There are variety of methods to promote a strong signal while minimizing
nonspecific binding the background noise.
• use an efficient plate coating buffer to provide a stabilized coating of the antibody
of antigen on the microtiters plate.
• use a blocking buffer to block assay wells. This blocking of unoccupied space in
the plate well prevents nonspecific binding of sample and assay components and
reduces the overall background signal.
• Using the proper sample diluents samples will dilute the samples to read within
the Functional range, block nonspecific Conjugate binding.

21.  Improve precision
• It is necessary to reduce plate to plate variability when prepared
Immunoassay.
• 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.

22.  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.
 Stabilize protein conjugates
• Stabilizing conjugates enables us to Store conjugated proteins and antibodies For
Future use, prepare batches, ready-to use conjugate aliquots and reconstitute.
• lyophilized conjugates while preserving native protein Configuration and activity.

23.  Increase shelf-life
• Using dependable stabilizing and blocking buffers to stabilize protein, allow us to
store prepared microtiter plates.
• Depending on the type of protein properly prepared plates can be Stored under
proper conditions For several months of even years if prepared using reliable
reagents.

24. Application of immunoassay :
• These measures the presence or Concentration of macromolecule of a small molecule in a
solution through the use of an antibody or an antigen. For example in analyte fluid urine
and serum.
• These are used in analysis of metabolites which indicate disease diagnosis.
• These are used in sports anti-doping laboratories to test athletes.
• used in measurement of very low concentration of low molecular weight drugs.
• Drugs testing.
• used in bioequivalence studies drug discovery and pharmaceutical industries.
• Hormone testing (insulin in diabetic patients).
• Bacterial of viral testing.
• environmental testing (herbicides, Pesticides).

25. Reference :-
• Hans-Gerhard Vogel; Drug discovery and evaluation; pharmacology
assay; volume 2, 3𝑟𝑑 edition; pg. no. 1157
• Robert A. Turner, Screening Methods on Pharmacology; Volume 2