Immunoassay

Immunoassay is a biochemical test that estimate or asses the presence or concentration of a macromolecule (antigen) in a solution (eg-blood) through the use of an antibody or immunoglobulin(Ig). The macromolecule called "analyte". Analytes in biological liquids such as blooed serum, biological fuid and urine are frequently measured using immunoassays ( for medical and research purposes).

1. Radioimmunoassay(RIA)involvestheseparationofaprotein(fromamixture)
usingthespecificityofantibody-antigenbindingandquantitationusingradioactivity.
The technique of radioimmunoassay has revolutionized research and clinical practice in
many areas, e.g.,
Blood banking
Diagnosis of allergies
Endocrinology
The technique was introduced in 1960 by Bersonand Yalowas an assay for the
concentration of insulin in plasma.
Radioimmunoassay
Competitive binding assay.
Principle of Radioimmunoassay
Principle: Uses an immune reaction [Antigen –Antibody reaction] to estimate a ligand
Ag + Ag* + Ab AgAb + Ag*Ab + Ag + Ag*
Unbound Ag* and Ag washed out
Radioactivity of bound residue measured
Ligand conc is inversely related to radioactivity
[Ag : ligand to be measured ; Ag* radiolabelled ligand]

2. Requirements for RIA
1. Preparation and
characterisation of the Antigen
[Ligand to be analyzed]
2. Radiolabelling of the Antigen
3. Preparation of the Specific
Antibody
4. Development of Assay System
•Reagents
–Tracer: labeled antigen
–Antibody
–Standards: Known concentrations
of unlabeled antigen
–Unknown samples
Preparation and Radiolabelling of the Antigen
Antigens prepared by..
–Synthesis of the molecule
–Isolation from natural sources
Radiolabelling [Tagging procedure]
Internally Labeled Antigen
–
14
C and
3
H
Externally Labeled Antigen
–
131
I and
125
I
Tagging should NOT affectAntigenic specificity andAntigenic activity!

3. Preparation of the Specific Antibody
Antigen injected intradermallyinto rabbits
or guinea pigs antibody production
Antibodies recovered from the serum
Some ligandsare not Antigenic
–Hormones, Steroids, Drugs HAPTENS
e.g: Gastrin, Morphine,
–Haptensconjugated to albumin antigenic
Separation of Bound and Free Ligand
•Electrophoresis
•Gel Filtration
•Adsorption Chromatography
•Fractional Precipitation
–Centrifugation
–Filtration
•Partition Chromatography
–Dialysis
Assay Procedure
Add known amounts of the test sample +
labelled antigen into the microtitrewells
Incubate allow the reaction to reach
completion
Decant and wash contents of the well 
removes all unbound antigens
Radioactivity remaining in the Microtitrewells
measured by a Counter [GM counter ,
Scintillation counter etc]
Intensity of radioactivity is inversely correlated
with the concof antigens in the test sample
Sensitive to very low conc. of antigens
Gamma Counter

4. •From these data, a standard binding curve, like
theone shown in red, can be drawn.
•The samples to be assayed (the unknowns) are
run in parallel.
•After determining the ratio of bound to free
antigen in each unknown, the antigen
concentrations can be read directly from the
standard curve.
Applications of Radioimmunoassays
•Endocrinology
–Insulin, HCG, Vasopressin
–Detects Endocrine Disorders
–Physiology of Endocrine Function
•Pharmacology
–Morphine
–Detect Drug Abuse or Drug Poisoning
–Study Drug Kinetics
Advantages and Disadvantages of RIA
•Advantages
–Highly specific: Immune reactions are specific
–High sensitivity : Immune reactions are sensitive
•Disadvantages
–Radiation hazards: Uses radiolabelled 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.

5. Enzyme Linked ImmunosorbentAssay (ELISA)
ELISAis a widely-used method for measuring the
concentration of a particular molecule (e.g., a
hormone or drug) in a fluid such as serum or urine.
It is also known as enzyme immunoassay or EIA.
ELISA has many of the advantages (e.g., sensitivity,
ease of handling multiple samples) without the
disadvantages of dealing with radioactivity(like in
RIA).
Enzyme labels should have high
specific reactivity
Should be easily coupled to
ligands & the labelled complex
must be stable
The reactivity should be retained
after linking of the enzyme to the
antigen/antibody
The chosen enzymes should not
be normally present in the
patient samples
Examples of enzyme labels
Horse radish peroxidase,
Alkaline phosphatase,
Glucose oxidase
Enzyme labels
Assay procedure
–Titre wells coated with suitable antibody
–Add patient sample containing the antigen
–Incubate: till antigen antibody reaction is complete
–Washremove unbound antigen
–Add Antibody labelled with Enzyme
–Incubate till antigen binds labelled antibody
–Wash remove unbound labelled antibody
–Add substrate ; incubate
–Enzyme + Substrate Product measure
colour
–Colour proportional to antigen in patient sample

6. •Advantages of ELISA
•Sensitive: nanogramlevels or lower
•Reproducible
•Minimal reagents
•Qualitative and Quantitative
–Qualitative e.g. HIV testing
–quantitative assays e.g. Hormonal level
•Greater scope : Wells can be coated with Antigens OR Antibodies
•Suitable for automation high speed
•NO radiation hazards