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

CONTENTS Definition Introduction Principle RIA Methods Application Reference

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

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.

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.

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

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

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

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

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

Flow Chart of Technique

The instrument usedfor RIA is GAMMA COUNTER

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

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.

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

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.

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

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