Radioimmunoassay

This document describes detailed information about Radio immuno assay (RIA) including its principle, procedure, advantages, disadvantages, application etc

1. RADIOIMMUNOASSAY PRESENTED BY- SK.AZIZ UDDIN PRESENTED TO- DR.RIKESHWAR PRASAD DEWANGAN M.PHARM,PHARAMACEUTICAL ANALYSIS BATCH-2020-2022. COLLEGE NAME-JAMIA HAMDARD

2. CONTENTS Introduction History of RIA Theory Principle Requirements Procedure Applications Advantages Disadvantages References

3. introduction Radio immuno assay is an immunological assay to analyse antigens present in given biological samples. It is used as most sensitive and specific method of immuno assay. Sensitivity ranges 0.0006-0.001 µg/ml. If substance to be analysed is in very low quantities in the orders of micrograms,nanograms, conventional methods like gravimetric and colorimetric method fail but RIA has extensive application in this perspective.

4. History of ria Developed by 1959 by Rosalyn Yalow and Solomon Berson for measurement of Insulin in plasma. It represented the first time that hormone levels in the blood could be detected by an in vitro assay.. In 1977 Yalow received the Nobel Prize for her and Barson’s development for RIA.

5. principle It is based on competitive binding between radio labelled antigen(hot Ag) and unlabelled Ag (Cold Ag) with selected antibody and ultimately radio activity is determined. It basically involves three reaction i.e. antigen, antibody binding. A competitive binding or competitive displacement reaction(it gives specificity). Measurement of radio emission(it gives sensitivity).

6. Principle of ria RIA is performed by using antibody-antigen binding and radioactive antigen. The basic principle of RIA is competitive binding reaction, where the analyte(for example antigen) competes with radio-labelled antigen for binding to the fixed antibody or binding sites of receptor.

7. requirements Micro titre plate/ Test tubes. Pure antigen Radio labelled antigen Antibodies Standard’s Centrifuge Radioactive counter

8. Micro titre plate A microtitre plate is mostly used for this assay. A microtitre plate could have 60,24,96,384, or even sometimes 1536 wells arranged in rows. Each well of a microtitre can only hold very small amounts of liquid

9. Antigen Pure Antigen Antigen may be obtained from biological sample or by synthetic form. It should be pure. It is used as standard or calibrator. Radio labelling of antigen The most commonly used radio labelles in RIA are tritium and iodine. They have adequate activity and have long enough half lifes .

10. antibody Specific antibodies are obtained by injecting the Ag to animals. Ag I.e. Drug molecule + bovine serum albumin.

11. Standard Draw a standard curve by taking conc. Of Ag in x- axis and radio activity in y-axis. It will help to determine the unknown conc. by using radio activity .

12. Centrifuge Uses for the separation of precipitated form and supernatant liquid form. Range: 1200-2500

13. Radio active counters There are two types of counters are used they are: Gamma counters- These are used for the gamma energy emitting isotopes. E.g.- common iodine isotopes Scintillation counters These are used for counting beta energy emitting isotopes. E.g. – tritium, carbon 14 isotopes

14. Gamma counter vs scintillation counter

15. Procedure Step 1- In micro titre plate add antibody and add radiolabelled Ag to it so all radiolabelled Ab will bind to the Ag and forms Ag-Ab complex. Step 2- In this step add unlabelled Ag or cold Ag and this cold Ag will replace the radio labelled Ag and bind with Ab Step 3- Then wash with buffer solution and washed solution will be collected in test tube and do centrifugation process and supernatant liquid will be analyzed for radio activity.

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

17. Applications Narcotics drug detection Early cancer detection Measurement of growth hormone levels. Tracking of leukemia virus. Diagnosis and treatment of peptic ulcers. Research with brain chemicals called neurotransmitters. Estradiol measurement in translational studies of breast cancer.

18. Advantages of ria Radio immuno assay is very sensitive technique used to measure concentration of Antigen without the need to use a bioassay. 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 .

19. Disadvantages of ria Radio active iodine used in is not a cheap reagent. Possible health hazards due to handling of radio isotopes. All the reagents must be added precisely. Limited assay range. Difficulty of automation Lengthy counting time.

20. references Google slide share You tube- Dr. Pushpendra classes Immuno assay books. Thank you