Biochemistry data

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IMMUNOASSAY
Group members
Mahnoor Ateqad,
Bushra Faryal,
Afeera Ruman,
Fiza Irshad,
Ayesha Nawal,
Mr khursheed

2. IMMUNE & ASSAY
• Immuno refers to an immune response that causes the
body to generate antibodies.
• Assay refers to a test.
• Thus, Immunoassay is a test that utilizes
immunocomplexing when antibodies and antigens are
brought together.
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3. INTRODUCTION TO
IMMUNOASSAY
• Immunoassay are analytical methods based on the
specific immuno-reaction between antibody(Ab) and
antigen(Ag) for the determination of amount of either
reactant in the solution. An antigen-antibody complex is
known as Immuno-complex.
• An Immunoassay is a test that uses antibody and
antigen complexes as a mean of generating a
measureable result to quantitate specific analytes.
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4. PRINCIPLE
• Immunoassay methods are based on a competitive
binding between a fixed amount of labelled form of an
analyte and a variable amount of unlabeled sample
analyte for a limited amount of binding sites on a highly
specific anti-analyte antibody.
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5. ANTI-BODIES
Antibodies, also known
as immunoglobulins, are Y-
shaped proteins that are
produced by the immune system
to help stop invaders from
harming the body.
When an invader enters the
body, the immune system
springs into action. These
invaders, which are
called antigens, can be viruses,
bacteria, or other chemicals.
When an antigen is found in the
body, the immune system will
create antibodies to mark the
antigen for the body to destroy.
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6. CLASSES OF ANTIBODIES
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7. Types of
Immuno-
assay
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• Competitive immunoassay
• Non-competitive
immunoassay
• Homogenous immunoassay
• Heterogenous immunoassay
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8. Competitive assay
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• In competitive formats, unlabelled
analyte (usually antigen) in the test
sample is measured by its ability to
compete with labeled antigen in the
immunoassay.
• The unlabeled antigen blocks the
ability of the labeled antigen to
bind because that binding site on
the antibody is already occupied.
• Thus, in a competitive
immunoassay, less label measured
in the assay means more of the
unlabeled (test sample) antigen is
present.
• The amount of antigen in the test
sample is inversely related to the
amount of label measured in the
competitive format.
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10. One step
competitive
format
In the one step competitive format both the
labeled antigen reagent (Ag*) and the unlabeled
specimen (or test sample analyte) compete for a
limited amount of antibody
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11. Two step competitive
format
• In the two step competitive format, the
antibody concentration of the reaction
solution is present in excess in
comparison to the concentration of
antigen.
•Antibody reagent is first incubated with
specimen containing antigens of interest;
then in the second step, labeled antigen is
added.
• Remember that in the competitive
format, less bound labeled antigen
indicates more antigen present in the test
sample. Assay formats provide several
fold improved assay sensitivity compared
to one step assay formats.
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13. Noncompetitive
(Sandwich)
Method
Noncompetitive assay formats generally
provide the highest level of assay sensitivity
and specificity and are applied to the
measurement of critical analytes such as
cardiac and hepatitis markers. This format is
referred to as a “sandwich” assay because
analyte is bound (sandwiched) between two
highly specific antibody reagents.
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14. Cont…
The reaction mixture typically
includes an excess of labeled
antibody, so that all analyte is
bound
The amount of antibody-
antigen complex is then
measured to determine the
amount of analyte present in
the sample
The labeled antibody, is
directly proportional to the
amount of antigen present in
the sample
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15. Heterogeneous and Homogenous
Immunoassay Methods
Methods that require separation of bound Ab-Ag* complex
are referred as heterogenous immunoassays.
Those that do not require separation are referred to as
homogeneous immunoassays.
Homogeneous methods have been generally applied to the
measurement of small analytes such as abused and
therapeutic drugs.
Since homogeneous methods do not require the separation
of the bound Ab-Ag* from the free Ag*, they are generally
much easier and faster to perform.
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18. Types of Immunoassays
(Heterogeneous)
• Radioimmunoassay's (RIAs)
• utilize a radioactive label (usually I125,
H3 or C14), which emits radiation that can
be measured with a beta or gamma
counter.
• Usually used for the measurements of
trace elements present in blood e.g.
hormones.
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19. Enzyme linked immunosorbant assay (ELISA):
Direct, sandwich and competitive
Reaction components are absorbed or bound to the surface of a
solid phase, commonly a well of a microtiter plate
Absorbance is measured using a micro-plate reader
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20. Types of Immunoassays (Homogeneous)
Enzyme Multiplied Immunoassay (EMIT)
The drug in the sample and the drug labeled with G6PD compete for
antibody binding sites.Binding inhibits enzyme activity, while free
enzyme remains active to interact with the substrate.Enzyme
activity/absorbance is directly proportiotional to drug concentration.
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21. Application of immunoassay
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For detection of naturally occurring
constituent
Clinical pharmacokinetics
Clinical bioequivalence in drugs
discovery

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Basic reagent required for
immunoassay in pharmaceutical
analysis
o Antibody (mono-clonal/poly-clonal)
o Signal- generating label
o Separation metricx
 Methodology used in
immunoassay
1. Competitive Non -competitive
Antigen
capturing
Antibody capturing

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IMMUNOASSAY METHODS APPLIED IN
PHARMACEUTICAL ANALYSIS
Enzyme
immunoassay
Radio immunoassay
Chemo Luminas
immunoassay
Fluoroimmunoassay
Liposome
Immunoassay

24. Advances in immunoassays
for pharmaceutical
analysis

25. Advances in preparation of the
immunoanalytical reagents
• Monoclonal antibody-based assays,
• Molecular modeling of hapten structure
• Immunization technique for rapid production of monoclonal
antibodies
• An efficient shorter immunization regime for generating highly
specific monoclonal antibody

26. Advances in preparation of the
immunoanalytical reagents
• Production of high number of positive hybridomas was
obtained
• The use of only single injection in this technique saves not
only the time and effort given to the purification of the
immunogens but also the amount of the immunogen

27. Advances to Involve New ccategories of
compounds
• This technique is theoretically applicable to any analyte
• The ability of generating monoclonal antibodies that
recognize metal ions
• By this approach, specific monoclonal antibodies were
generated for U, Pb, Cd, Co and Hg
• These antibodies were used in the development of
competitive immunoassays for the accurate measurement of
metal ions in water and serum

28. Advances to Involve New ccategories of
compounds
• Recently, methods are being developed for lead (II) and
cadmium (II)
• The antibodies were raised against protein conjugates of the
metal
• This method are very sensitive

30. Advances to Involve New ccategories of
compounds
• These assays are
1. Highly sensitive
2. Remarkably quick
3. Easily performed
4. Reasonably portable to the analysis site
5. Require minimum sample pretreatment
6. Inexpensive.

31. CEDIA
• Cloned enzyme donor immunoassay is a novel method
to produce homogenous enzyme immunoassays for
drugs.
• Enzyme donor units combine with enzyme acceptor
units to form a complete fully active tetrameric enzyme
molecule which reacts with colorless molecule to
produced colored product.
• This method is precise and sensitive and is proved for
application in routine drug screening.
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32. Principle of CEDIA
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33. CEIA
• Capillary electrophoresis immunoassay combines
principle of capillary electrophoresis separation and
heterogenous immunoassay.
• This process has efficiency, small sample requirements,
and relatively high speed.
• In this technique, the antibody is attached covalently to
the modified interior surface of a microcapillary, which is
used as a solid phase immunoreactor. The detection is
based on the reaction process of the immunoassay. The
analyte and labelled analyte (tracers) are mixed and the
mixture is then injected into the system.
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34. FIIA
• Flow injection immunoassay is introduced to enhance
the efficiency of immunochemical reaction.
• FIIA is based on displacement were developed for
analysis of various componds.
• The column packed with immobilized solid phase
antibodies is incorporated into the system. The column
is saturated with a solution containing labelled analyte.
Injection of the sample into the column results in
displacement of the labelled analyte.
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36. IMMUNOSENSORS
• These sensors usually employ either
reusable/regenerable or disposable assay format.
• Immunosensors are developed for wide range of drug
and hormone analysis.
• Drawback: It is not possible to perform analysis in
complex biological samples (e.g serum) in a one step
procedure, due to interference resulting from non-specific
binding to sensor surface.
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