Immunoassays – RIA and ELISA

By SUNILBOREDDY

IMMUNOASSAYIMMUNOASSAYBiochemical test that measures the concentration of a Biochemical test that measures the concentration of a

substance in a biological liquid, typically serum or urine, using the substance in a biological liquid, typically serum or urine, using the reaction of an antibody or antibodies to its antigen.reaction of an antibody or antibodies to its antigen.

PURPOSEPURPOSE• The purpose of immunoassay is to measure (or, in a qualitative The purpose of immunoassay is to measure (or, in a qualitative assay to detect) an analyte.assay to detect) an analyte.• Immunoassay is a method of choice for measuring analytes Immunoassay is a method of choice for measuring analytes normally present at very low concentrations that cannot be normally present at very low concentrations that cannot be determined accurately by other less expensive tests. determined accurately by other less expensive tests. Common uses include measurement of drugs, hormones, specific Common uses include measurement of drugs, hormones, specific proteins, tumor markers and markers of cardiac injuryproteins, tumor markers and markers of cardiac injury

Types of Immunoassay:

Radioimmunoassay (RIA)Radioimmunoassay (RIA)A Remarkably Sensitive Bioassay

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 It combines the principle of radio activity of isotopes and immunological reaction hence the name radio immunoassay. immunological 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 H and H33(Beta emitting isotopes)(Beta emitting isotopes)

PRINCIPLEPRINCIPLE

The principle of RIA involves competitive binding of The principle of RIA involves competitive binding of radiolabeled antigen and unlabeled antigen to high affinity radiolabeled antigen and unlabeled antigen to high affinity antibody.antibody.

Since the antibody cannot distinguish between Since the antibody cannot distinguish between unlabeled unlabeled antigenantigen both antigens compete for antigen binding site on both antigens compete for antigen binding site on antibody with the increasing concentration of antibody with the increasing concentration of unlabeled antigenunlabeled antigen more and more more and more labeled antigen labeled antigen will get displaced from the will get displaced from the binding site by measuring the free binding site by measuring the free labeled antigenlabeled antigen in the in the solution, it is possible to determine the concentration of solution, it is possible to determine the concentration of unlabelled antigenunlabelled antigen..

The The label antigenlabel antigen is mixed with antibody at a concentration is mixed with antibody at a concentration sufficient enough to saturate the antigen binding sites of the sufficient enough to saturate the antigen binding sites of the antibody molecule, then the sample containing known antibody molecule, then the sample containing known concentration of concentration of unlabeled antigenunlabeled antigen is added in increasing is added in increasing amounts.amounts.

General Procedure:General Procedure:

Two methods of estimating antigen-antibody capacity…Two methods of estimating antigen-antibody capacity…1.1.Farr techniqueFarr technique2.2.Antiglobulin coprecipitation techiqueAntiglobulin coprecipitation techique

Estimation of Antigen-Antibody Binding Estimation of Antigen-Antibody Binding CapacityCapacity

Farr Technique:Farr Technique:In this method, the complexed antigen antibody is separated out In this method, the complexed antigen antibody is separated out from the solution by precipitation with from the solution by precipitation with 50% ammonium sulfate50% ammonium sulfate. . This technique is applicable only to those antigens, which are This technique is applicable only to those antigens, which are soluble at this salt concentration.soluble at this salt concentration.

Antigen bound to antibody together with free antibody is precipitated by Antigen bound to antibody together with free antibody is precipitated by antiglobulin.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 From both these, amount of bound radiolabeled antigen is

measured out at various concentrations of unlabelled antigen and a curve measured out at various concentrations of unlabelled antigen and a curve is prepared. is prepared. Curve is linear over a limited rangeCurve is linear over a limited range unknown sample needs to be unknown sample needs to be diluted such that the readings fall within the linear range of the curve. diluted such that the readings fall within the linear range of the curve. After standardization, the experiment is carried out with unknown sample After standardization, the experiment is carried out with unknown sample of patient’s serum. of patient’s serum. Using the standard graph, the concentration of the hormone or unlabelled Using the standard graph, the concentration of the hormone or unlabelled antigen in patient’s serum is found out.antigen in patient’s serum is found out.

Antiglobulin Co-Precipitation TechniqueAntiglobulin Co-Precipitation Technique

RIA - Technique

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

ENZYME LINKED IMMUNOSORBENT ASSAY(ELISA)

Antibody is immobilized on micro-plate wells Antibody is immobilized on micro-plate wells Competition between in sample and labeled enzyme for Competition between in sample and labeled enzyme for

antibody binding sites antibody binding sites The unbound material is washed out The unbound material is washed out Chromogenic substrate added to develop color Chromogenic substrate added to develop color Resulting color is read in a spectrophotometerResulting color is read in a spectrophotometer

Principle

ELISA:

ELISAELISA, or , or enzyme-linked immunosorbent assayenzyme-linked immunosorbent assay, is an , is an immunoassay technique involving the reaction of antigen and immunoassay technique involving the reaction of antigen and antibody in vitro. antibody in vitro.

ELISA is a sensitive and specific assay for the detection and ELISA is a sensitive and specific assay for the detection and quantitation of antigens or antibodies. quantitation of antigens or antibodies.

ELISA tests are usually performed in ELISA tests are usually performed in microwell platesmicrowell plates. . The ELISA test, or the enzyme immunoassay (EIA), was the first The 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 Engval and Pearlman in 1970 introduced this technique. It was

later developed by Clark and Adam in 1977.later developed by Clark and Adam in 1977.

A 96-WELL MICROTITER PLATE USED FOR ELISA

Types of ELISA:

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

Indirect ELISA:

In this technique, the antibody (rather than the antigen) is immobilized on a microtiter well.

Sandwich ELISA:

competitive ELISA:

In this technique, antibody is first incubated in solution with a In this technique, antibody is first incubated in solution with a sample containing antigen.sample containing antigen.

Modified ELISA:

Elispot ELISA:

Applications:

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