Introduction Principle Types Applications

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