Radioimmunoassay.pptx

Introduction
Principle, Requirements , Procedure and Application of Radioimmunoassay.

1. Ankit Rajput Gmail- [email protected] Radioimmunoassay.  Topic :- introduction, principle, requirements, procedure,  applications. Subject – Advanced Instrumentation Technique For B.pharm 4 th year.

2. INTRODUCTION Radio immuno assay is a immnunological assay to analyse  antigens present in given biological samples. It is most sensitive and specific method of  immuno assay. Senstivity ranges from 0.0006-0.006 ug/ml. It was developed by S.A. Berson and Rosalyn  yalow  in 1959 in received nobel  prize in 1977

3.           PRINCIPLES It involves 3 principles which make it most specific & sensitive than other immuno assays. An immuno reaction i.e. antigen, antibody binding. A competitive binding or competitive displacement reaction. Measurement of radio emission.

4. REQUIREMENTS Micro titer plates / Test tubes Pure antigen Radio labelled of antigen Antibodies Standard's Centrifuge Radioactive counter

5. MICRO TITER PLATE Micro titer plate is commonly used for this assay. It could have 6, 24, 96, 384 or even sometimes 1536 wells arranged in rows. Each well of a microtiter plate can only hold very small amounts of liquid.

6. PURE ANTIGENS Antigens may be obtained from biological sample or by synthetic form, it should be pure. It is used as standard or calibrator. RADIO LABELLING OF ANTIGENS   The most commonly used radiolabels are tritium and iodine. They have adequate activity and have long enough half lifes .

7. ANTIBODY   1. Specific antibodies are obtained by injecting          Ag to animals.  2.  Ag I.e. ,drug molecule + bovineserum albumin

8. CENTRIFUGE 1 . Use for the separation of precipitated form and supernatant liquid form. 2. Range :- 1200 – 2500 rpm. RADIO ACTIVE COUNTERS  2 types of counters are used :- Gamma counters  Scintillation counter s 

9. APPLICATIONS RIA of clonazepam RIA of barbiturates RIA of human plasma Determination of Ag concentration Estimation of hormones like LH, FSH, ACTH To detect hepatitis and HIV antigens Estimation of vitamins like folic acid , riboflavin etc.

10. ADVANTAGES It is structurally specific as antigen: antibody reaction are highly specific. It is indirect method of analysis. It is a saturation analysis as active reagent added in smaller quantity than that of analyte.

11. DISADVANTAGES Radioactive iodine is used in is not a cheap reagent. Limited assay range. Difficullty of automation. Lengthy counting time. All the reagents must be added precisely.