This document discusses immunoassays and two common types - radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). RIA involves labeling an antigen or antibody with a radioactive material to measure it in a mixture. It is very sensitive but involves radiation hazards. ELISA uses an enzyme-linked antibody or antigen to detect the presence of a substance. It is a plate-based assay that is sensitive, reproducible, and does not use radiation. Both methods are used for applications like disease detection, drug monitoring, and analyzing hormones and metabolites.

1. Immunological
Assay
Dr. Ashwani K. Dhingra
Professor
Guru Gobind Singh College of
Pharmacy, Yamuna Nagar

3. Immunoassay
• Immunoassay means a method to measure
any particular substance in a mixture using
its specific-binding antibody.
• One of the merits of immunoassay is that we
can measure a substance that is present in a
mixture of various contaminants.
• Immunoassays have become very popular in
view of their high sensitivity, safety, economy
and simple instrument requirements.
Types :
1. RIA (Radio Immune Assay)
2. ELISA (Enzyme Linked Immunosorbent
Assay)

4. RIA
(Radio
Immune
Assay)
The technique was introduced in 1960 by
Berson and Yalow as an assay for the
concentration of insulin in plasma.
It represented the first time that hormone
levels in the blood could be detected by an in
vitro assay.
The sensitivity range is 0.0006–0.006 μg
antibody/ml.
It is a very sensitive in-vitro assay technique in
which antibody or antigen is labelled with a
radioactive material (like ¹²⁵I, ³H)

5. Principle
It involves three principles:
• An immune reaction i.e., antigen,
antibody
binding.
• A competitive binding or competitive
displacement reaction. (It gives
specificity)
• Measurement of radio emission. (It
gives sensitivity)

7. Procedure
 Known quantity of an antigen is made radioactive, frequently by labelling it
with γ-radioactive isotopes of iodine.
This radio labelled antigen is then mixed with a known amount of antibody
for that antigen.
 A sample of serum from a patient containing an unknown quantity of same
antigen is added.
This causes the unlabelled (cold) antigen from serum to compete with radio
labelled antigen (hot) for antibody binding site.
 As the concentration of cold antigen is increased, more of it binds to antibody,
displacing the radio labelled variant and reducing the ratio of antibody bound
radio labelled antigen to free radio labelled antigen.

8. The bound antigens are then separated from the unbound
ones, and the radioactivity of the free antigen remaining in
the supernatant is measured using a γ counter.
The concentration of the test antigen can be calculated
from the ratio of the bound and total antigen labels, using a
standard dose response curve.
From these data, a standard binding curve can be drawn.
After determining the ratio of bound to free antigen in each
unknown, the antigen concentrations can be read directly.

9. Advantages
•Highly specific: Immune reactions are specific
•High sensitivity : Immune reactions are sensitive
Disadvantages
•Radiation hazards: Uses radiolabeled reagents
•Requires specially trained persons
•Labs require special license to handle radioactive material
•Requires special arrangements for requisition, storage of radioactive
material, radioactive waste disposal.
APPLICATIONS
•Detection of narcotic drugs
•Blood bank screening for the hepatitis (a highly contagious
condition) virus

10.  Measurement of growth hormone levels, immunoglobulin's, tumour
markers
 Tracking of the leukaemia virus.
 Diagnosis and treatment of peptic ulcers
 Endocrinology
– Insulin, HCG, Vasopressin
– Detects Endocrine Disorders
– Physiology of Endocrine Function
 Pharmacology – Morphine – Detect Drug Abuse or Drug Poisoning
 Study Drug Kinetics.
 Clinical Immunology
– Antibodies for Inhalant Allergens
– Allergy Diagnosis
 Oncology
– Carcino-embryonic Antigen
– Early Cancer Detection and Diagnosis
APPLICATION
S

11. ELISA
(Enzyme
Linked
Immunosorbent
Assay)
ELISA is a biochemical technique used mainly in immunology
to detect the presence of an antibody or an antigen in a
sample.
In this method the antigen or antibody is conjugated to an
enzyme.
It is a plate based assays designed for detecting and
quantifying substances such as peptides, proteins,
antibodies, antigens and hormones.
It involves detection of ‘analyte’ in a liquid sample using
liquid reagent (wet lab) or dry strips (dry lab).
The test can be done in polystyrene tubes (macro-ELISA) or
polyvinyl micro titre plates (micro-ELISA).

12. Basic
Principle
Based on Basic Immunology Response.
• Lock and Key Concept
Antigen (key): substance when introduced into
the body produces antibodies.
Antibody (lock): protein in the body that is used
by immune system to identify and neutralize
foreign targets (referred to as antigens).
Key fits into the lock.
• Enzyme conjugate substrates
 Enzyme that converts colourless substrates to a
colored product –Bound to the antibody that is
part of the antibody-antigen complex.
 Bound to a secondary antibody that binds with
the antibody- antigen complex.

13. Procedure

14. Types of
ELISA
Direct ELISA
Indirect ELISA
Sandwich ELISA
Competitive ELISA

19. Competitive
ELISA

20. • Sensitive: nanogram levels or lower
• Reproducible
• Minimal reagents
• Qualitative & Quantitative
Qualitative – HIV testing
Quantitative assays – Therapeutic drug
monitoring (TDM)
Greater scope: Wells can be coated with
Antigens OR Antibodies
• NO radiation hazards
• Fast - 90 samples tested in 2-3 hr.
• Sensitivity (up to 10 pg/mL).
ADVANTAGE
S

21. Detection of HIV antibodies in serum.
Detection of mycobacterial antibodies in
tuberculosis.
Detection of rotavirus in faeces.
Detection of hepatitis B markers in serum.
Detection of enterotoxin of E coli in faeces.
Detection of potential food allergens.
Analysis of hormones, vitamins, metabolites,
diagnostic markers • like ACTH, FSH, T3, T4,
Glucagon, Insulin, Testosterone, vitamin B12,
prostaglandins, glucocorticoids,
Therapeutic drug monitoring: like Barbiturates,
morphine, digoxin.
APPLICATIONS