RADIOIMMUNOASSAY (RIA):Principle, Procedure, Application and MCQ for GPAT, NET JRF and GATE exam

RADIOIMMUNOASSAY :-

• This techinque is used to determine concentration of antigen in given sample.
• This techinque is very sensitivity it can detected 0.001 μg/ml.
• This techinque was introduced in 1960 by berson & yalow.

PRINCIPLE OF RIA :- 

• It involves three principles which make it most specific & sensitive than other immune assays.
• An immune reaction i.e. antigen,antibody binding.
• A competitive binding or competitive displacement reaction
• Measurement of radio emission (it gives sensitivity)

WHAT IS A LABEL ?

All immunoassay require the use of labeled material in order to measure the amount of antigen or anti body present.

A label is a molecule that will react as part of the assay so a change in signal can be measured in the blood : reagent solution.

GENERAL PROCEDURE OF RIA :-

• A known quantity of an antigen is made radio active frequently by labeling it with gamma radioactive isotopes of iodine attached to tyrosine.
• This radio labeled antigen is then mixed with a known amount of antibody for that antigen and as a result the two chemically bind to one another.
• Then a sample of serum from a patient containing an unknown quantity of that same antigen is added.
• This causes the unlabeled anti gen from the serum to complet with the radio labeled antigen for antibody binding site.
• The concentration of cold antigen is increased more of it binds to the antibody displacing the radiolabled variant and reducing the ratio of antibody – bound radio labeled antigen to free radio labeled antigen.
• The radioactivity falls because unlabelled antigen dilute it.
•  The count obtained from the radioactivity are used to determine the hapten concentration in the sample the interpretetion being done on the standard curve.

• Test tube-1 blank	Ab +Ag
  Test tube-2  calibrator	Ab+pure Ag + Ag
  Test tube-3 sample	Ab + sample A g + Ag

• 

REQUIREMENTS :-

1. Micro titer plate/ test tube
2. Pure antigen
3. Radio labelled antigen
4. Anti body
5. Standards
6. Centrifuge
7. Radio active counter

1. Micro titer plate :- A micro titer plate is used mostly used for this assay. A microtiter plate could have 6,24,96,384 or even sometimes 1536 wells arranged in rows.Each well of a microplate can only hold very small amount of liquid.
2. Pure antigen :- Antigen may be obtained from biological sample or by synthetic form it should be pure. It is used as standard or calibrator.
3. Radio labelling of antigen :- The most commonly used radiolabels in RIA are tritum and iodine.They have adequate activity and have long enough half lifes.
4. Antibody :- sepcific antib0dies are obtained by injecting the Ag to animals.
5. Standards :-
6. CENTRIFUGE :- Used for the separation of precipitated from and supernatant liquid from. Range : 1200-2500 rpm
7. Radio active counters :- two types of counters are used.1.gamma counter 2. scintillation counters

• Gamma counter:- these are used for the gamma energy emitting isotopes
• Scintillation counter :- These are used for counting beta energy emitting isotopes

• Separation techniques :-

• After completion of reaction of reaction free from and bound forms are determined by separation techniques.
• Various technique include gel filtration, electrophoresis, solid phase adsorption of Ag,Ab & fractional precipitate.

APPLICATION OF RIA :-

• RIA is used in the assay of drug like amphetamine,barbiturates,digitoxin,morphine etc.
• In analysis of vitamine like riboflavin,folic acid.
• RIA IS USED TO ASSAY THE PLASMA LEVEL OF FOLLOWING:-

1. Digitoxin or digoxin in patients receiving these drugs.
2. Certain abused drug.
3. It is used to assay the presence of hepatitis B surface anti gen in donated blood.

MCQ

1. Which technique is used to assay drug concentration in plasma ?

A. IR sepctroscopy

B. UV sepctroscopy

C.Non aqueous titration

D. RIA

2.RIA was developed by

A.  Berson & Yalow

B. chals & wastone

C. vector&logan

D. lewis & bronstand

3.How many microgram antigen detected in sample by RIA ?

A.0.1 μg/ml

B.0.0001 μg/ml

C.0.001 μg/ml

D.0.01 μg/ml

4.Which sentence is not true about RIA?

A.The most commonly used radiolabels in RIA are tritum and iodine.

B.Centrifugation rpm is 1200-2500.

C.This techinque is very sensitivity it can detected 0.001 μg/ml

D.This techinque is very sensitivity it can detected 0.01 μg/ml

5.RIA standarded graph as

A. X-axis = % Radioactivity and Y.-axis = unlabled Ag(ng)

B.X-axis = unlabled Ag(ng) and Y.-axis = % Radioactivity

C.X-axis = % Radioactivity and Y.-axis = unlabled Ag(mg)

D.X-axis = Radioactivity and Y.-axis = unlabled Ag(ng)

6.Which is not application of RIA ?

A.It is used to assay the presence of hepatitis B surface anti gen in donated blood.

B.In analysis of vitamine like riboflavin

C. T4 &  T3 measurement

D.Digitoxin or digoxin in patients receiving these drugs and measurement its concentration.

ANSWER KEY :-

1. D

2.A

3.C

4.D

5.A

6.C

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REFERENCE :-

—Textbook of microbiology by Prescott, Harley.

—Practical analytical chemistry by BECKEET.