This document provides an overview of immunoassays including radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). It discusses the principles, types, and applications of immunoassays. Key points include: immunoassays use the specific binding of antibodies and antigens to detect analytes, RIA uses radioisotopes providing high sensitivity but potential hazards, ELISA uses enzyme reactions and is safer and more versatile than RIA, and common applications of immunoassays include detecting hormones, drugs, and infectious agents.

1. ADITYA BANGALORE
INSTITUTE OF PHARMACY
DEPARTMENT OF PHARMACOLOGY
Modern Pharmaceutical Analysis
TOPIC ON: Immunoassays, RIA and ELISA
Submitted by: RajeshYadav
(M Pharm)

2. contents
• Introduction
• Principle
• Types
• Applications
• Reference

3. Introduction
• Analytical method or Biochemical test
• An antibody: antigen complex is also known as
an immuno-complex
• Highly specific “lock and key” system:
antibody –antigen reaction

4. “Immuno”& “assay”
• “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 immunocomplexing when
antibodies and antigens are brought together

5. • Immunoassays are different from other types
of laboratory tests, such as colorimetric tests,
because they use antibody:antigen complexes
to generate a signal that can be measured.

6. Immunoassay: Antibodies, Antigens
and Analytes
• An antibody is a protein that is produced by the
body in response to an “invading” (foreign)
substance.
• An Antigen is the substance that the body is
trying to fight off (eliminate or reduce) by
mounting an immune response.
• An analyte is anything measured by a laboratory
test. In immunoassay testing, the analyte may be
either an antibody, or an antigen.

7. Immunoassays
• Immunoassays utilize one or more select
antibodies to detect analytes of interest.
• The analytes being measured may be those that
are naturally present in the body (such as a
thyroid hormone), those that the body produces
but are not typically present (such as a cancer
antigen), or
• Those that do not naturally occur in the body
(such as an abused drug).

8. Antibodies
• Antibodies possess high
a) specificity
and
b) affinity
for a specific antigen. It is the specific
binding of an antibody to an antigen that
allows the detection of analytes by a variety of
immunoassay methods.

9. Structure of Antibodies
• Antibodies (Ab) are a type of protein called
immunoglobulins.
• The most common one is immunoglobulin G
(IgG).
• IgG is a protein composed of two main
structural and functional regions

11. Principle
• Technique which incorporates the binding
reaction of a target substance (antigen) with
an antibody
• Antibodies bind to different natural and
synthetic antigens in the body such as
carbohydrates, lipids, proteins and nucleic
acids.

12. TYPES:-
• Competative immunoassays.
• Non-competative immunoassays.
• Homogenous immunoassays.
• Heterogenous immunoassays

14. Homogeneous vs Heterogeneous
Immunoassay Methods
• Immunoassay methods that require
separation of bound Ab-Ag*complex are
referred to as heterogeneous immunoassays.
• Those that do not require separation are
referred to as homogeneous immunoassays

16. Radioimmunoassay
• Oldest type of immunoassay
• Sensitive method (ng to pg concentration)
• Less specific
• Qualitative as well as Quantitative analysis
• Radioisotope

17. Radioimmunoassay can detect substance like :
• Hormones
• Vitamins
• Serum Protein
• Drugs
• Infective Agent

18. Principle
I. An immune reaction i.e. antigen, antibody
binding.
II. A competitive binding or competitive
displacement reaction. (It gives specificity)
III. Measurement of radio emission. (It gives
sensitivity)

20. Advantages of Radioimmunoassay
• Specific
• Sensitive
• Convenient
• Reliable
• Reproducible

21. Disadvantages of Radioimmunoassay
• Prolonged reaction time (in days)
• Radioisotopes are costly.
• Possible health hazards due to handling of
radioisotopes.
• Limited assay range.
• Lack of direct linear relationship between
analyte concentration and signal response.
• Lengthy counting time.

22. Application Of Radioimmunoassay
i)Detection of Narcotic Drugs
ii)Radioimmunoassay of Hydromorphone &
Hydrocodone in Human Plasma
iii) Radioimmunoassay of Flunisolide in human
plasma
iv) Detection of Digoxin
v) Thyroid Testing

23. ELISA
• Linked to an enzyme
• A test that uses antibodies and color change to
identify a substance
• Involves at least one antibody with specificity
for a particular antigen

24. Principle
• The basic principle of an ELISA is to use an
enzyme to detect the Ag-Ab binding. The
enzyme converts a colorless substrate
(chromogen) to a colored product, indicating
the presence of Ag-Ab binding

25. Types: i)Direct ELISA

26. ADVANTAGES OF DIRECT DETECTION
 Quick methodology since only one antibody is used.
 Cross-reactivity of secondary antibody is eliminated
DISADVANTAGES OF DIRECT DETECTION
 Immunoreactivity of the primary antibody may be
reduced as a result of labeling.
 Labeling of every primary antibody is time-consuming
and expensive.
 Little signal amplification.

27. ii) Indirect ELISA

28. ADVANTAGES OF INDIRECT DETECTION
 Wide variety of labeled secondary antibodies are
available commercially.
 Immunoreactivity of the primary antibody is not
affected by labeling.
 Sensitivity is increased because each primary antibody
contains several epitopes that can be bound by the
labeled secondary antibody, allowing for signal
amplification
DISADVANTAGES OF INDIRECT DETECTION
 Cross-reactivity may occur with the secondary
antibody, resulting in nonspecific signal.
 An extra incubation step is required in the procedure

29. iii) Sandwich ELISA

30. Advantages
Assay is quantitative, amount of viral antigen can
be detected
Assay has high sensitivity and specificity
More samples can be tested at the same time
Disadvantages
Need ELISA reader for result interpretation; not
possible under field conditions
The method is time consuming and labourious.

31. iv) Competitive ELISA

32. ADVANTAGES
Suitable for complex (crude or impure)
samples, since the antigen does not require
purification prior to measurement.
DISADVANTAGES
Each antigen may require a different method
to couple it to the enzyme

33. COMPARISON BETWEEN VARIOUS
TYPES OF ELISA

34. Applications
• HIV test
• Detection of Mycobacterium antibodies in
tuberculosis
• Detection of rotavirus in feces
• Detection of hepatitis B markers in serum
• Detection of enterotoxin of E. coli in feces

35. Reference
• Pharmaceutical Drug Analysis by Ashutosh Kar; 2005 P .485 – 504
• Yalow R, Berson S - Immunoassay of endogenous plasma insulin in
man;1960 P .39 – 115
• The journal of nuclear medicine; 1979 P .748- 752
• Ibrahim A. Darvish. Review on Immunoassay methods and their
application in pharmaceutical analysis –International journal of biomedical
science; 2006 P .217 - 220