Ria

 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.

 The

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

 This

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



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 of radioisotopes in the measurement of insulin. • The RIA is the predecessor of modern immunoassays.

Dr. Rosalyn Yalow became the first female 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

› 3H

14

C

125

[Tagging procedure]

I are used as radioactive

tags › Antigens are tagged to 3 H 14 C › Tagging should NOT affect Antigenic specificity & Antigenic activity!!!

125

I

 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

› Dialysis

Chromatography 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,

 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  The

special counting equipment

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

- competitive, heterogeneous EIA  Reaction

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

 Absorbance

plate reader

 Sample

is measured using a micro-

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

 Pharmaceutical

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