1. RADIOIMMUNOASSAY SUMITTED TO: SUMITTED BY: S.SUBBARAO SIR T.HARIPRIYA
ADVANCED INSTRUMENTAL METHODS OF ANALYSIS
MPHARM (DEPARTMENT OF PHARMACEUTICS)
MALINENI LAKSHMAIAH COLLEGE OF PHARMACY

2. CONTENTS Definition Introduction Principle RIA Methods Application
Reference

3. DEFINITION:
RIA is a radio analytical technique with high degree of sensitivity and specificity in tracing variety of molecules present in complex matrices
INVITRO DIAGNOSIS TEST FOR ANTIGENS USING RADIO ISOTOPES

4. Radio Immuno Assay Use of radio isotopes like
u3 H, 125 I, 75 Se, 32P, 57Co
gh Se, P, Co
Immuno
Antigen antibody competitive binding
Assay
Detection of compound
This technique is used over a wide spectra of substances such as hormones, steroids, vitamins, drugs, tumor markers and viral antigens.

5. Introduction :
This isotopic measuring method was developed in 1959 by two Americans,biophysicist Rosalyn Yalow and physician Solomon A. Berson.
RIA combines the specificity of an antigen-antibody reaction with sensitivity of radioactivity measurements.
It can also be used for detection of pictogram quantities (10−12 g) of biological constituents present in biological fluid.

6. Principle:
The basic principle involved are either competitve binding and specifity of particular antigen for particular antibody
1. – Ratio in unknown and
2. - Antigen in unknown
Red line – binding line
Green line – free labeled antigen

7. From graph we can also calculate %F (fraction of free labeled antigen) and %B (fraction of bound labeled antigen).
F – amount of free labeled antigen
B -amount of bound labeled antigen

8. RIA Methods: There are mainly two types:
Competitive binding method: The labeled and unlabeled antigens(Ag) compete with limited number of binding antibody sites
Ag + Ag* + Ab  AgAb + Ag*Ab + Ag
Unbound Ag* and Ag washed out
Ag : ligand to be measured
Ag*: radio labelled ligand

9. Non Competitive binding:
The concentration of antigen is directly proportional to bound labeled antibody concentration
Ag + Ab* + Ab  Ab*Ag + Ag
Unbound Ag washed out
No competition exist because concetration of antibody is more

10. Procedure for methods
Radio labelling of the Antigen or radio labelled production
Preparation & characterisation of the Antigen [Ligand to be analysed]
Preparation of the Specific Antibody
Development of Assay System or separation techniques

11. Flow Chart of Technique

12. The instrument usedfor RIA is GAMMA COUNTER

13. Advantages:
Highly specific: Immune reactions are specific,the greater the specificity of the antiserum, the greater the specificity of the assay.
High sensitivity : Immune reactions are sensitive, Using antibodies of high affinity it is possible to detect a few picograms (10−12 g) of antigen in the tube.
Accuracy and Precision

14. Disadvantages: Radiation hazards: Uses radio labelled reagents
Requires specially trained persons
Labs require special license to handle radioactive material
Requires special arrangements for
Requisition, storage of radioactive material
radioactive waste disposal.

15. Analysis of hormones, vitamins, metabolites, diagnostic markers:
Application of RIA:
Analysis of hormones, vitamins, metabolites, diagnostic markers:
E.g. ACTH, FSH, triiodothyronine (T3) and thyroxine (T4), Glucagons, Insulin, Testosterone, vitamin B12, prostaglandins, glucocorticoids,
Therapeutic drug monitoring:
Barbiturates, morphine, digoxin,
Diagnostic procedures for detecting infection :
HIV, Hepatitis A, B etc

16. Tumour markers: RIA of tumour markers such as alpha-fetoprotein (AFP), carcinoembrionic antigen (CEA), b-HCG for choroid -carcinoma, prostate specific antigen (PSA) for prostate cancer, are available for detection and management of cancer
Non clinical application: such as veterinary science, food processing industry, drug industry, forensic science and environmental monitoring.
RIA is used in place of bioassay in various branches of science like Biochemistry, Microbiology, and Hematology and Clinical pharmacology.

17. References :
www. Innovabioscience.com
www. Wikipedia free encyclopedia
Pharmaceutical drug analysis by Ashuthoshkar

18. THANK YOU