1. Immunoassays – RIA and ELISA
By
SUNILBOREDDY

2. IMMUNOASSAYIMMUNOASSAY
Biochemical test that measures the concentration of a substance in aBiochemical test that measures the concentration of a substance in a
biological liquid, typically serum or urine, using the reaction of an antibody orbiological liquid, typically serum or urine, using the reaction of an antibody or
antibodies to its antigen.antibodies to its antigen.
PURPOSEPURPOSE
• The purpose of immunoassay is to measure (or, in a qualitative assay toThe purpose of immunoassay is to measure (or, in a qualitative assay to
detect) an analyte.detect) an analyte.
• Immunoassay is a method of choice for measuring analytes normally presentImmunoassay is a method of choice for measuring analytes normally present
at very low concentrations that cannot be determined accurately by other lessat very low concentrations that cannot be determined accurately by other less
expensive tests.expensive tests.
Common uses include measurement of drugs, hormones, specific proteins,Common uses include measurement of drugs, hormones, specific proteins,
tumor markers and markers of cardiac injurytumor markers and markers of cardiac injury

3. Types of
Immunoassay:

4. RadIoImmunoassay (RIa)RadIoImmunoassay (RIa)
A Remarkably Sensitive Bioassay

5. Radioimmunoassay (RIA)Radioimmunoassay (RIA)
Most sensitive test for detecting antigen/antibody.Most sensitive test for detecting antigen/antibody.
It combines the principle of radio activity of isotopes and immunologicalIt combines the principle of radio activity of isotopes and immunological
reaction hence the name radio immunoassay.reaction hence the name radio immunoassay.
It is highly sensitive and specific analytical tool.It is highly sensitive and specific analytical tool.
Commonly used radio isotopes…Commonly used radio isotopes…
II125125(gamma emitting isotopes)(gamma emitting isotopes)
CC1414 and Hand H33(Beta emitting isotopes)(Beta emitting isotopes)
PRINCIPLEPRINCIPLE
The principle of RIA involves competitive binding of radiolabeledThe principle of RIA involves competitive binding of radiolabeled
antigen and unlabeled antigen to high affinity antibody.antigen and unlabeled antigen to high affinity antibody.

6. Since the antibody cannot distinguish betweenSince the antibody cannot distinguish between unlabeled antigenunlabeled antigen bothboth
antigens compete for antigen binding site on antibody with the increasingantigens compete for antigen binding site on antibody with the increasing
concentration ofconcentration of unlabeled antigenunlabeled antigen more and moremore and more labeled antigenlabeled antigen will getwill get
displaced from the binding site by measuring the freedisplaced from the binding site by measuring the free labeled antigenlabeled antigen inin
the solution, it is possible to determine the concentration ofthe solution, it is possible to determine the concentration of unlabelledunlabelled
antigenantigen..
TheThe label antigenlabel antigen is mixed with antibody at a concentration sufficientis mixed with antibody at a concentration sufficient
enough to saturate the antigen binding sites of the antibody molecule, thenenough to saturate the antigen binding sites of the antibody molecule, then
the sample containing known concentration ofthe sample containing known concentration of unlabeled antigenunlabeled antigen is added inis added in
increasing amounts.increasing amounts.
General Procedure:General Procedure:

7. Two methods of estimating antigen-antibody capacity…Two methods of estimating antigen-antibody capacity…
1.1.Farr techniqueFarr technique
2.2.Antiglobulin coprecipitation techiqueAntiglobulin coprecipitation techique
Estimation of Antigen-Antibody Binding CapacityEstimation of Antigen-Antibody Binding Capacity
FarrTechnique:FarrTechnique:
In this method, the complexed antigen antibody is separated out from theIn this method, the complexed antigen antibody is separated out from the
solution by precipitation withsolution by precipitation with 50% ammonium sulfate50% ammonium sulfate..
This technique is applicable only to those antigens, which are soluble at thisThis technique is applicable only to those antigens, which are soluble at this
salt concentration.salt concentration.

8. Antigen bound to antibody together with free antibody is precipitated by antiglobulin.Antigen bound to antibody together with free antibody is precipitated by antiglobulin.
 Antiglobulin is antibody against antibody raised in rabbit or sheep.Antiglobulin is antibody against antibody raised in rabbit or sheep.
Free antigen will be left behind in the supernatant.Free antigen will be left behind in the supernatant.
From both these, amount of bound radiolabeled antigen is measured out atFrom both these, amount of bound radiolabeled antigen is measured out at
various concentrations of unlabelled antigen and a curve is prepared.various concentrations of unlabelled antigen and a curve is prepared.
Curve is linear over a limited rangeCurve is linear over a limited range  unknown sample needs to be diluted such thatunknown sample needs to be diluted such that
the readings fall within the linear range of the curve.the readings fall within the linear range of the curve.
After standardization, the experiment is carried out with unknown sample of patient’sAfter standardization, the experiment is carried out with unknown sample of patient’s
serum.serum.
Using the standard graph, the concentration of the hormone or unlabelled antigen inUsing the standard graph, the concentration of the hormone or unlabelled antigen in
patient’s serum is found out.patient’s serum is found out.
Antiglobulin Co-Precipitation TechniqueAntiglobulin Co-Precipitation Technique

10. RIA - Technique

11. Applications of RadioimmunoassaysApplications of Radioimmunoassays
 EndocrinologyEndocrinology
 Insulin, HCG, VasopressinInsulin, HCG, Vasopressin
 Detects Endocrine DisordersDetects Endocrine Disorders
 Physiology of Endocrine FunctionPhysiology of Endocrine Function
 PharmacologyPharmacology
 MorphineMorphine
 Detect Drug Abuse or Drug PoisoningDetect Drug Abuse or Drug Poisoning
 Study Drug KineticsStudy Drug Kinetics
 EpidemiologyEpidemiology
 Hepatitis BHepatitis B
 Clinical ImmunologyClinical Immunology
 Antibodies for Inhalant AllergensAntibodies for Inhalant Allergens
 Allergy DiagnosisAllergy Diagnosis
 OncologyOncology
 Carcinoembryonic AntigenCarcinoembryonic Antigen
 Early Cancer Detection and DiagnosisEarly Cancer Detection and Diagnosis

12. ENZYME LINKED IMMUNOSORBENT ASSAY
(ELISA)

13.  Antibody is immobilized on micro-plate wellsAntibody is immobilized on micro-plate wells
 Competition between in sample and labeled enzyme for antibody bindingCompetition between in sample and labeled enzyme for antibody binding
sitessites
 The unbound material is washed outThe unbound material is washed out
 Chromogenic substrate added to develop colorChromogenic substrate added to develop color
 Resulting color is read in a spectrophotometerResulting color is read in a spectrophotometer
Principle

14. ELISA:
 ELISAELISA, or, or enzyme-linked immunosorbent assayenzyme-linked immunosorbent assay, is an immunoassay, is an immunoassay
technique involving the reaction of antigen and antibody in vitro.technique involving the reaction of antigen and antibody in vitro.
 ELISA is a sensitive and specific assay for the detection and quantitationELISA is a sensitive and specific assay for the detection and quantitation
of antigens or antibodies.of antigens or antibodies.
 ELISA tests are usually performed inELISA tests are usually performed in microwell platesmicrowell plates..
 The ELISA test, or the enzyme immunoassay (EIA), was the firstThe ELISA test, or the enzyme immunoassay (EIA), was the first
screening test commonly employed for HIV.screening test commonly employed for HIV.
 Engval and Pearlman in 1970 introduced this technique. It was laterEngval and Pearlman in 1970 introduced this technique. It was later
developed by Clark and Adam in 1977.developed by Clark and Adam in 1977.

15. A 96-WELL MICROTITER PLATE USED FOR ELISA

16. Types of
eLIsA:

17.  Indirect ELISA is the method of choice to detect the presence of serum antibodiesIndirect ELISA is the method of choice to detect the presence of serum antibodies
against human immunodeficiency virus (HIV), the causative agent of AIDS.against human immunodeficiency virus (HIV), the causative agent of AIDS.
 In this assay, recombinant envelope and core proteins of HIV are adsorbed.In this assay, recombinant envelope and core proteins of HIV are adsorbed.
IndIrecT eLIsA:

18.  In this technique, the antibody (rather than the antigen) is immobilized on a
microtiter well.
sAndwIch eLIsA:

19. competitive eLiSA:
 In this technique, antibody is first incubated in solution with a sampleIn this technique, antibody is first incubated in solution with a sample
containing antigen.containing antigen.

20. modified eLiSA:
Elispot ELISA:

22. Applications:
 It is used for determining serum antibody concentrations (such as with theIt is used for determining serum antibody concentrations (such as with the
HIV test or west Nile virus)HIV test or west Nile virus)
 It has applications in the food industry in detecting potential food allergensIt has applications in the food industry in detecting potential food allergens
such as milk, peanuts, walnuts, almonds, and eggs.such as milk, peanuts, walnuts, almonds, and eggs.
 ELISA can also be used in toxicology as a rapid presumptive screen forELISA can also be used in toxicology as a rapid presumptive screen for
certain classes of drugscertain classes of drugs