2. An assay based on the reversible and non-covalent binding of an antigen by a
specific antibody employing radioactivity labeled antigen to measure the
fraction of the antigen bound to a substoichiometric amount of antibody.
The technique of radioimmunoassay has revolutionized research and clinical
practice in many areas, e.g.,
blood banking
diagnosis of allergies
endocrinology
3. It is based on the competition between unlabelled antigen and a fixed amount of
the corresponding radio labelled antigen for limited number of antibody
binding sites in fixed amount of antiserum.
At equilibrium, the unbound antigen (F)+Antigen-antibody complex(Ag-Ab)
are separated and quantified.
Ag(larger) + Ab → Ag-Ab
Ag*(smaller) + Ab → Ag*-Ab
In std. Condition, amount of labeled antigen bound to the antibody decreases as
the amount of unlabelled antigen increases in sample.
The reaction between Ab and Ag* is quantified by counting either the bound
(B) or the free labeled compound (F).
4. The amount of labeled compound bound to antibody is dependent on
The amount of ligand (Ag +Ag *) present
The amount of antibody (Ab) present
The equilibrium constant/ affinity constants k1’ and k2’ where, k1’ = k1/k2
and k2’=k3/k4.
Ag* + Ab Ag*-ab and ,
Ag + Ab Ag-Ab
In assay ag* and Ab are constant as binding of Ag* solely depends on amount
of Ag present.
% F = [F/ (F+B)]*100. % B = [B/ (F+B)]*100
5. A known quantity of an antigen is made radioactive, frequently by labeling it
with gamma-radioactive isotopes of iodine attached to tyrosine.
This radio labeled antigen is then mixed with a known amount of antibody for
that antigen, and as a result, the two chemically bind to one another.
Then, a sample of serum from a patient containing an unknown quantity of that
same antigen is added. This causes the unlabeled (or "cold") antigen from the
serum to compete with the radio labeled antigen for antibody binding sites.
As the concentration of "cold" antigen is increased, more of it binds to the
antibody, displacing the radio labeled variant, and reducing the ratio of
antibody-bound radio labeled antigen to free radio labeled antigen.
The bound antigens are then separated from the unbound ones, and the
radioactivity of the free antigen remaining in the supernatant is measured.
6. With this technique, separating bound from unbound antigen is crucial.
Initially, the method of separation employed was the use of a second "anti-
antibody" directed against the first for precipitation and centrifugation.
Using known standards, a binding curve can then be generated which allows
the amount of antigen in the patient's serum to be derived.
Determine standard curve
Non-linear plot of radioactivity versus concentration.
7. Radioimmunoassay (RIA) is a very sensitive technique used to measure
concentrations of antigens (for example, hormone levels in the blood) without
the need to use a bioassay. It can measure one trillionth of a gram of material
per milliliter of blood. Because of the small sample required for measurement,
RIA quickly became a standard laboratory tool.
It is structurally specific as Antigen: Antibody reaction are highly specific.
Therefore, RIA includes specificity of antigen antibody reaction and sensitivity
of radioactivity measurement.
It is an indirect method of analysis since neither the radioactive standard nor the
antibody is present in the original sample.
It is a saturation analysis as active reagent (antibody) is added in smaller
quantity than that of analyte (antigen).
8. Prolonged reaction time (in days) as a consequence highly diluted reagent is
used.
Radioactive Iodine used in is not a cheap reagent.
Possible health hazards due to handling of radioisotopes.
All the reagents must be added precisely.
Limited assay range.
Lack of direct linear relationship between analyte concentration and signal
response.
Difficulty of automation.
Lengthy counting time.
9. 1)A tracer i.e. a labeled ligand.
Carbon-14
Tritium-311
Iodine-125
13’I
13N
15O
2) A binder (Antibody) which is the specific antiserum.
3)A separation system to separate to separate the ‘bound’ and ‘free’ phases.
4)A standard i.e. the ligand (analyte) in highly pure form.
5)A ligand (analyte) - free human antiserum.
10. RIA requires a separation procedure because the bound fraction does not
precipitate spontaneously at the low concentration employed.
Variety of procedures are available that exploit physicochemical or
immunological differences between the two fractions. They are:
Physical methods include: filtration, chromatography, electrophoresis,
charcoal-dextran adsorption, adsorption on ion-exchange resin.
Chemical methods include: organic solvents, such as ethanol, dioxane and
PEG, or salts, such as sodium, zinc and ammonium sulphate, to precipitate
antibody bound heptan.
Solid phase system.
No one is ideal in establishing a new assay as two or more principles employed
for separation.
11. RIA has many uses,
Narcotics (drug) detection,
Blood bank screening for the hepatitis (a highly contagious condition) virus,
Early cancer detection,
Measurement of growth hormone levels,
Tracking of the leukemia virus,
Diagnosis and treatment of peptic ulcers, and
Research with brain chemicals called neurotransmitters
12. Endocrinology
Insulin, HCG, Vasopressin
Detects Endocrine Disorders
Physiology of Endocrine Function
Pharmacology
Morphine
Detect Drug Abuse or Drug Poisoning
Study Drug Kinetics
13. Epidemiology
Hepatitis B
Clinical Immunology
Antibodies for Inhalant Allergens
Allergy Diagnosis
Oncology
Carcinoembryonic Antigen
Early Cancer Detection and Diagnosis
14. INTRODUCTION:
ELISA is an important immunological technique for detecting and
measuring the amount of antigen or antibody in the sample using various
enzymes.
PRINCIPLE:
It is based on the measurements of the enzyme labeled antibody. In this
antigen or antibody are linked to the enzymes and forms the complex with
enzymes which is measured by colorless substances to coloured compound.
15.
16. It is useful technique for determining serum antibody concentration.
It has also found application in the food industry in detecting potential food
allergens such as milk, peanuts, walnuts, almonds, and eggs.
It is used to assay of hepatitis B-antigen.
It is used for detection of anti-HIV-1 and anti-HIV-2 anti body in the patient
serum.
An out off point may be determined by comparing it with a known
standard.
18. Principle:
competition between endogenous insulin in body fluids i.e. blood
plasma and exogenous radioactive insulin for the binding sites on anti insulin
antibody.
Formerly developed techniques:
1) A Laborious techniques developed by yallow et all in which paper
electrophoresis was used to separate antibody bound insulin from free insulin.
2) Morgan et all utilized the double antibody techniques wherein antiguinea pig
gamma-globulin is used to precipitate the soluble complex formed between
insulin and guinea pig anti insulin.
19. Components used in the assay:
Radio labeled tracer: insulin
Analyte: Immunoreactive insulin
Buffer: phosphate buffer ph 7.4
Adsorbent: Zirconyl phosphate
Separation system: by centrifugation
20. Here we are going to describe RIA of digitoxin-3-hemisuccinyl-leucine using
antiserum elicited by digoxin 3-hemisuccinate – BSA conjugate and then
measuring concentration of digoxin in digitalized patients.
REQUIREMENTS:
Specific antiserum: Immunization of rabbit with digoxin 3-hemisuccinate-BSA
conjugate.
Tracer: digoxin 3 hemisuccinyl-[3H] leucine
Analyte: serum digoxin in digitalized patient
Buffer: phosphate saline buffer(ph-7.4)
Adsorbent: dextran coated charcoal suspension
Separation system: centrifugation
21. Digoxin free serum + digoxin 3-hemisuccinyl [3H] leucine+ assay buffer
+diluted antiserum
Add 0.1ml non labeled digoxin in test tubes.
All tubes are shaken in a vortex mixture and incubated at 4c.
A dextran coated charcoal suspension is added to each test tube. which was
then vortexed, incubated for 10min in an ice bath and centrifuged at 1700*g for
10 min at 4°c.
The supernatant is then transferred to a counting vial, scintillation solution is
added and the radioactivity is counted in liquid scintillation counter.
The radioactivity of the bound antibody is calculated after correction for the
blank value.
The dose-response curve is constructed using duplicate samples.
22. (1)James w. Munson ”Pharmaceutical Analysis modern method” , part A, page
no:412-422
(2) Dr. Chandrakant R.kokare “Pharmaceutical Microbiology principle &
application”, 6th edition,Nirali publication, page no:23.16-23.17