Introduction Principle of RIA Theory Requirements Methods Merits & De-Merits Applications Related Techniques References

Radioimmunoassay (RIA) is a sensitive method for measuring very small amounts of a substance in the blood.

Radioactive versions of a substance, or isotopes of the substance, are mixed with antibodies and inserted in a sample of the patient's blood.

The same non-radioactive substance in the blood takes the place of the isotope in the antibodies, thus leaving the radioactive substance free.

This isotopic measuring method was developed in 1959 by two Americans, biophysicist Rosalyn Yalow (1921-) and physician Solomon A. Berson (1918-1972).

The amount of free isotope is then measured to see how much of the original substance was in the blood.

Yalow and Berson - first radioisotopic technique - to study blood volume and iodine metabolism. They later adapted the method to study how the body uses hormones, particularly insulin, which regulates sugar levels in the blood.

The researchers proved that Type II (adult onset) diabetes is caused by the inefficient use of insulin. Previously, it was thought that diabetes was caused only by a lack of insulin.

• In the year 1959, Drs. Rosalyn Yalow & Soloman

Berson invented the radioimmunoassay, which

applied the use ofradioisotopes in the

measurement ofinsulin.

• The RIA is the predecessor of modern

immunoassays.

Dr. Rosalyn Yalow became the firstfemale to win a Nobel Prize with

her work on the radioimmunoassay.

Radioimmunoassay (RIA) analysis - RIA involves the separation of the drug using the specificity of antibody - antigen binding and quantitation using radioactivity

Radioimmunoassay is based on the antigen-antibody reaction in which tracer amounts of the radio-labeled antigen competes with endogenous antigen for limited binding sites of the specific antibody against the same antigen.

In principle, radio-labeled antigen should be similar in bio-activity and/or immunoreactivity of the native antigen

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]

A fixed concentration of radio-labeled antigen in trace amounts - incubated with a constant amount of antiserum

- total antigen binding sites on the antibody are limited such that the only 30–50% of the total radio-labeled antigen may be bound in the absence of the antigen -

When unlabeled antigen, either as standard or test sample, is added to this system, there is competition between radio-labeled antigen and unlabeled antigen for the limited constant number of binding sites on the antibody.

The amount of radio-labeled antigen bound to antibody decreases as the concentration of unlabeled antigen increases.

The radioactivity in the labelled antigen-antibody complex is measured after separating the bound complex from the free antigens by suitable separation technique.

The counts obtained are used to determine the unknown antigen conc., by interpreting on the standard curve.

The method of assaying the radioactivity of the bound and/or unbound fraction solely depends on the nature of isotope & separation method.

The expt. Cond. such as – pH, ionic comp.- protein content or any other interfering factors should be identical for std and sample.

A highly purified labelled antigen A specific antiserum A method for separation A method for Quantitation

Radiolabelling [Tagging procedure] › 3 H 14 C 125 I are used as radioactive

tags› Antigens are tagged to 3 H 14 C 125I › Tagging should NOT affect

Antigenic specificity & Antigenic activity!!!

Usually high specific activity - radio-labeled (125-I) antigen is used - prepared by

- iodination of the pure antigen on its tyrosine residue(s)

- by chloramine-T or peroxidase methods and then separating the radio-labeled antigen from free-isotope by gel-filtration or HPLC.

Some ligands are not Antigenic › Hormones, Steroids, Drugs HAPTENS› Eg: Gastrin, Morphine,

Haptens conjugated to albumin antigenic

Antigen injected intradermally into rabbits or guinea pigs antibody production

Antibodies recovered from the serum, and stored in non-defrosting freezer at ≤ 20°C or in liquid nitrogen at -196°C and used.

Follow optimal incubation condition e.g. buffer, pH, time and temperature, while separation.

Various separation methods are used based on physical and biochemical characters of the bound complexes.

Precipitation of the antibody- Fractional Precipitation› Centrifugation› Filtration

Adsorption of the free Antigen Use of solid phase Antibody Gel Filtration Adsorption Chromatography Partition Chromatography

› Dialysis Electrophoresis Ultrafiltration

Precipitation of the antibody: Used if mol. Size of B & F forms of Ag differ

considerably

Chemically – ethanol, dioxan, isopropanol, PEG, Amm.sulfate etc.

Double Ab technique – A secondary antibody is used to precipate.

Adsorption of free antigen:

Charcoal coated with dextran is often used

Talc, cellulose and ion exchange resins also used.

Use of solid phase Ab:

Antibody is linked to solid phase – initial reaction – heterogenous system- after equilibrium – separation simple.

Dextran, acrylamide resins, inorg. Metal oxides (Si, Al, Ti) – controlled pore beads form – used.

Then,

Electrophoresis Gel Filtration Adsorption Chromatography Fractional Precipitation

› Centrifugation› Filtration

Partition Chromatography› Dialysis are used…

Various instruments are used – detection and measurement of radioactivity.

› Scintillation counter› Geiger-muller counter› Gas counters etc..

Both free(F) and bound(B) fractions are counted,

Various relationships are established between the variables and mathematical equations are defined for calculating required data and quantified.

Competitive Immunoassays

Non-Competitive Immunoassays

unlabelled analyte in the test sample is measured by its ability to compete with labeled antigen for a limited number of antibody binding sites.

- less label measured in the assay means more of the unlabeled (test sample) antigen is present.

The amount of antigen in the test sample is inversely related to the amount of label measured in the competitive format. As one increases, the other decreases.

Highest level of assay sensitivity and specificity.

This format is referred to as a “sandwich” assay because the analyte is bound (sandwiched) between two highly specific antibody reagents.

The reaction mixture typically includes an excess of labeled antibody, so that all drug/metabolite is bound. The amount of antibody-antigen complex is then measured to determine the amount of drug present in the sample. The measurement of labeled analyte, usually antibody, is directly proportional to the amount of antigen present in the sample.

Immunoassay methods that require separation of bound Ab-Ag* complex - heterogeneous immunoassays. Those that do not require separation - homogeneous immunoassays.

Homogeneous - generally applied - measurement of small analytes - abused and therapeutic drugs. Since - do not require the separation - much easier and faster to perform.

great sensitivity possible to detect a few picograms (10−12 g)

of antigen. greater specificity of the assay.

Expensive hazards in preparing & handling the radioactive

Ag. Requires special counting equipment The body concentrates iodine atoms —

radioactive or not — in the thyroid gland where they are incorporated in thyroxine.

Pharmacy› Quantification of drugs in serum › Detect Drug Abuse or Drug Poisoning› Study Drug Kinetics

Novel uses:

› In vitro hepatic metabolic activity› Stereospecific quantification – D & L forms.› Measuring – hormones, mediators, growth

factors, cytokines, prostanoids etc..

Endocrinology› Insulin, HCG, Vasopressin› Detects Endocrine Disorders› Physiology of Endocrine Function

Epidemiology› Hepatitis B

Clinical Immunology› Antibodies for Inhalant Allergens› Allergy Diagnosis

Oncology› Carcinoembryonic Antigen› Early Cancer Detection and Diagnosis

Enzyme Multiplied Immunoassay (EMIT )

Enzyme-linked immunosorbent assay (ELISA)

Fluorescent Polarized Immunoassay

the drug in the sample & the drug labeled with G6PD compete for antibody binding sites.

Binding inhibits enzyme activity, while free enzyme remains active to interact with.

Enzyme activity/absorbance is directly proportional to drug concentration.

- competitive, heterogeneous EIA

Reaction components are absorbed or bound to the surface of a solid phase, commonly a well of a microtiter plate

Absorbance is measured using a micro-plate reader

Sample absorbance is inversely proportional to drug concentration

the drug in the sample competes with fluorescein-labeled drug for antibody binding sites.

Reaction mixture is excited by planepolarized light.

As the tracer returns to a lower energy state, it emits light; polarization is measured.

The polarization value of the sample is inversely proportional to analyte concentration.

Pharmaceutical analysis – Ashutoshkar Biochemistry - Vasudevan www.abbottdiagnostics.com www.millipore.com www.boomer.org www.users.rcn.com www.answers.com