M.Pharm 1st Semester SeminarSubject : Modern Analytical TechniqueTopic : Application Of Radioimmunoassay (RIA)

By:Anindya JanaM.Pharm 1st Year (Pharmaceutics)Regd. No. : 1661611006

• Radioimmunoassay (RIA) is a sensitive method for measuring very small amounts of antigen, antibody, or antigen-antibody complex in the blood.

• The radioimmunoassay technique, as the name implies, achieves sensitivity through the use of radionuclides and specificity that is uniquely associated with immunochemical reaction.

Introduction

Before RIA Many methods like TLC, GLC, Spectrofluorometry & ordinary radiolabelling assay were employed for the analysis of ‘drug’ in the plasma but they have many disadvantage like:

Less specific.Less sensitive.Involment of the processes of extraction, purification &

concentration of the specimen under investigation.Heat treatment of the specimen resulted invariably in

degradation and destruction of the substances.Many process involved make the analysis rigorous and

unnecessarily sluggish.

History

• Developed in 1959 by Rosalyn Yalow & Soloman Berson for measurement of insulin in plasma.

• It represented the first time that hormone level in the blood could be detected by an in vitro assay.

• In 1977 Yalow received the Nobel Prize for her & Berson’s development of RIA.

Radioimmunoassay can detect substance like :• Hormones• Vitamins• Serum Protein• Drugs• Infective Agent

Sensitivity of Radioimmunoassay :It can detect substance from a range of Nano gram(ng) to Pico gram(pg) concentration.

Qualitative as well as Quantitative analysis.

Advantages of Radioimmunoassay

• Specific

• Sensitive

• Convenient

• Reliable

• Reproducible

Application Of Radioimmunoassay

1)Detection of Narcotic Drugs

Heroin & Morphine can be detected in hair with the use of Radioimmunoassay (RIA).

In a research hair samples obtained from morphine treated mice and heroin user contained Nano gram levels of drug per milligram of hair .The result of the hair analysis for all subject admitting the use of heroin were positive where as the result of only 30% of thin layer chromatographic urine analysis of these same subjects were positive.

2) Radioimmunoassay of Hydromorphone & Hydrocodone in Human PlasmaHydromorphone & Hydrocodone belongs to morphine group of drugs and are used in combination with antitussive & analgesic antipyretic mixture.

The RIA method is capable of estimating the above drug within a range of 2.5 to 20 ng/mL using standard 100 µl plasma sample.

RIA is carried out using morphine-6-antiserum & dihydromorphine.Free drug is separated from the bound drug using dextran coated charcoal & an aliquot of the supinate containing the antiserum bound drug is subsequently counted for radioactivity.

3) Radioimmunoassay of Flunisolide in human plasmaFlunisolide is a fast-acting corticoid designed for the treatment of allergic rhinitis, asthma, and other allied respiratory disorders in humans. As the quantum of drug delivered by inhalation (i.e., the usual route of administration of the drug), is invariably small, the plasma-levels attained can also be fairly small. Hence, there is a need for a sensitive method of plasma concentration evaluation which is satisfied by radioimmunoassay.

4) Measurement of FerritinSerum ferritin levels are indicative of iron stores present in a patient. Levels are useful in differentiating true iron deficiency from the body's failure to utilize these stores.

5) Detection of DigoxinThis allows direct measurement of serum digoxin levels quickly and accurately. It is important to rule out Digi toxicity quickly and accurately. We are also able to filter out Digi bind to let the physician know how much the level has dropped after Digi bind has been administered.

6) Thyroid TestingThis is used to determine the patient's thyroid status and to follow patients after iodine-131 therapy to see if the dose was indeed effective.

Disadvantages of Radioimmunoassay

• Prolonged reaction time (in days) as a consequence highly diluted reagent is used.

• Radioisotopes are costly.

• Possible health hazards due to handling of radioisotopes.

• Limited assay range.

• Lack of direct linear relationship between analyte concentration and signal response.

• Difficult of automation.

• Lengthy counting time.

Reference Annett M. Baumgartner, Peter F. Jones, Warner A. Baumgartner & Charles T. Black. Review on

Radioimmunoassay of hair for determining opiate-abuse histories – The journal of nuclear medicine; 1979 P.748 - 752

Ibrahim A. Darvish. Review on Immunoassay methods and their application in pharmaceutical analysis – International journal of biomedical science; 2006 P.217 - 220

Yalow R, Berson S - Immunoassay of endogenous plasma insulin in man;1960 P.39 – 115

Pharmaceutical Drug Analysis by Ashutosh Kar; 2005 P.485 - 504

Thank You