IMMUNOASSAY

PRESENTED BY: UNDER THE GUIDANCE OF :

PRANAM K S Dr .VARADRAJ BHAT G
ROLL NO : 200620009 ASSOCIATE PROFESSOR
M pharm 1st sem

Department of pharmaceutical chemistry

 CONTENTS
• Introduction

• Basic principle of Immunoassay

• Production of antibodies

• Separation of bound and unbound drugs

• Applications of immunoassay

• Conclusion

• Reference
 Introduction
• Immunoassays are bioanalytical methods in which the
quantitation of the analyte depends on the reaction of an antigen
(analyte) and an antibody.

• Principally, these methods are based on a competitive binding

reaction between a fixed amount of labelled form of an analyte
and a variable amount of unlabelled sample analyte for a limited
amount of binding sites on a highly specific anti-analyte
antibody.
Immunoassays have been widely used in many important areas of
pharmaceutical analysis such as:

• Diagnosis of diseases

• Therapeutic drug monitoring

• Clinical pharmacokinetic and bioequivalence studies in drug

discovery and pharmaceutical industries.
 Importance of Immunoassay

• The immunoassay methods are highly specific, and highly sensitive for the
analysis of wide range of analytes in biological samples.

• The detection system in immunoassays depends on readily detectable labels

(e.g. radioisotopes or enzymes) coupled to one of the immunoanalytical
reagents (i.e. analyte or antibody).

• The use of these labels in immunoassays results in assay methods with

extremely high sensitivity and low limits of detection.
 Classification
Immunoassay

Immunoassay Immunoassay
without label with label

Homogeneous Heterogeneous
immunoassay immunoassay

Non Non
Competitive competitive
competitive competitive
 BASIC PRINCIPLE OF IMMUNOASSAY
• The basic technique consists of placing a fixed quantity of the labelled drug, and the
test sample that contains the drug to be assayed.

• The specific binding sites on the antibody bind both the labelled drug molecules and
the unlabelled drug molecules present in the test sample.

• The proportion of labelled drug molecules bound is inversely proportional to the

number of unlabelled drug molecules.

• The technique is capable of high sensitivity, with µg/L quantities easily detectable
and ng/L possible with specialised assays.
• The group using no label - It includes methods such as immunodiffusion and nephelometry.
• The group using labels - it includes radioisotopes, enzymes, fluorescent and luminescent substances,
etc., includes well-known methods such as radioimmunoassay and enzyme immunoassay.

• In Homogeneous immunoassays-
Do not require antibody-bound antigen to be separated before measurement of signal.
No separation and washing steps are necessary.
• Example: EMIT ( enzyme multiplied immunoassay), Chemiluminescence immunoassay, fluorescent
immunoassay.

• In Heterogeneous immunoassays –
Require bound/free separation steps but are in general more sensitive than homogeneous ones.
• Example: CEDIA ( cloned enzyme donor immunoassay), Fluorescence polarisation immunoassay and
ELISA.
Basic steps involved in Immunoassay
• When applied to drug testing, the immunoassay technique uses an antibody specific
for the drug or drug class being assayed, and a labelled form of the same drug or a
labelled form of the antibody to generate a measurable signal.

• The drug immunoassay involves setting up a competition for binding to the antibody
between antigen (drug) in the sample and a fixed amount of antigen added as a part
of the test system

• Labelling of this added antigen or antibody with a suitable marker and measuring the
signal generated by a suitable analytical measurement enables results to be compared
with a calibration curve made from a set of standards with known amounts of added
drug.
 Production of Antibodies
• Antibody is a protein that is produced by body in response to an
invading (foreign) substance.

• The antibodies can be either polyclonal or 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.
• Antibodies are proteins-immunoglobulins (Ig) which are produced by
B-lymphocytes in response to an immunogen.

• There are many different immunoglobulins (IgG, IgA, IgM, IgE and
IgD), but mostly IgG is used in immunoassay design.

• The IgG molecule can be represented simply by the letter ‘Y’ with two
antigen binding fragments, ‘Fab’ at the top and the ‘Fc’ at the base.
 A typical monoclonal antibody production process

• Immunization of mice & isolation of splenocytes - Mice are immunized with an antigen and later

their blood is screened for antibody production. The antibody-producing splenocytes are then

isolated for in vitro hybridoma production.

• Preparation of myeloma cells - Myeloma cells are immortalized cells that, once fused with spleen

cells, can result in hybridoma capable of unlimited growth. Myeloma cells are prepared for fusion.

• Fusion - Myeloma cells and isolated splenocytes are fused together to form hybridomas in the

presence of polyehthylene glycol(PEG), which causes cell membranes to fuse.

• Clone screening and picking - clones are screened and selected on the basis of

antigen specificity and immunoglobulin class.

• Functional characterization - Confirm, validate and characterize (e.g. ELISA) each

potentially high-producing colony.

• Scale up and wean - Scale up clones producing desired antibodies and wean off

selection agent(s).

• Expansion - Expand clones producing desired antibodies (e.g. bioreactors or large

flasks).
• Drug molecules are too small to provoke an immune response,
so drug immunogens are created by conjugation of a drug or
drug derivative to a larger carrier protein to give an
immunogen through a process called “haptenisation”.

• Suitable carrier proteins include Bovine serum albumin (BSA)

 Separation of bound and unbound drugs
• The first separation procedures in immunoassay were performed using the physicochemical
differences between the immunoglobulin and the antigen.

• Immunoassays are used for quantification of drugs based on antigen–antibody reaction. The drug
molecules are labelled and act as antigens to compete with other components of mixture for
binding on specific sites of antibodies and form an immune complex, which is separated by
physical or chemical methods.

• A partitioning step, or a way of separating reacted from unreacted analyte.

• Most immunoassays use a solid phase (polystyrene test tubes, microtiter plates , glass or
polystyrene beads , magnetic beads , and cellulose membranes) for separation.
• The bound and unbound fractions are usually separated by physical means ,
including decanting , centrifugation , or filtration. This is followed by a
washing step to remove any remaining unbound analyte.
• In the absence of drug, the maximum binding of the enzyme label occurs.
• By thorough washing unbound drug can be separated so that the binding of
drug with albumin / antibody can be detected easily.
• Increased amounts of drug in the sample results in decreased amounts of
antibody-bound enzyme label.
• Antibody-bound enzyme conjugate & antibody-bound drug is left immobilized
to the wall of the antibody-coated microplate.
• The amount of enzyme-labelled drug bound to the solid-phase antibody, and
therefore available for colour development, is inversely proportional to the
amount of drug in the specimen.
• The colour produced at the final stage is therefore inversely proportional to the
amount of drug in specimen, which gives a calibration curve.
 Applications of Immunoassay
• Detection of HIV antibodies in serum
• Detection of Hepatitis B markers in serum
• Detection of narcotic drugs ( therapeutic drug monitoring)
• Analysis of hormones , vitamins  , metabolites , diagnostic markers:
E .g. ACTH , FSH , triiodothyronine (T3) and thyroxine (T4) ,
glucagon , insulin , testosterone , prostaglandins.
• Non clinical applications – such as veterinary science , food
processing industry , drug industry , forensic science and
environmental monitoring,
 Conclusion
• In conclusion, the number of analytical and clinical investigations relying on these
measurement procedures worldwide is exceedingly large. Thus, one can imagine that
the numbers of measurements and determinations using immunoassay for routine
patient care are astronomical. The impact of diagnostic immunoassays on patients,
clinicians, and the healthcare system in general is virtually overwhelming. Given the
impact that their inventions had on clinical diagnosis and healthcare in general, as
well as on the development of a well-established in vitro diagnostic industry .
 References
• Laboratory Techniques in Biochemistry and Molecular Biology
Volume 27, 1999, Pages 1-3

• BOYDEN, S. V. (1960). Antibody Production. Nature, 185(4715), 724–727.

• separation techniques. Principles and Practice of Immunoassay, 78–95. 

• Immunoassay Methods and their Applications in Pharmaceutical Analysis: Basic

Methodology and Recent Advances
Ibrahim A. Darwish, Int J Biomed Sci. 2006 Sep; 2(3): 217–235.
THANK YOU