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Prepared By: Pradip Parikh Guided By: Mr.Ujjwal Sahoo RIA ( Radio Immuno Assay)Contents :: Contents : Definition Introduction Principle and Theory Methods in RIA Other Immuno Assays Application ReferenceDefinition :: Radioimmunoassay (RIA) is a scientific method used to test antigens (for example, hormone levels in the blood) without the need to use a bioassays. Radioimmunoassay (RIA) is a Radio-analytical technique with remarkable sensitivity and a high degree of specificity that is widely used for the estimation of a variety of molecules present in complex matrices. Also known as Radio tracer technique and best example of invitro diagnosis technique using radio isotopes. This technique is used over a wide spectra of substances such as hormones, steroids, vitamins, drugs, tumor markers and viral antigens. Radio Immuno Assay Definition : Use of radio active material Antigen antibody binding theory Detection of compoundIntroduction :: 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 . This is a technique used for detection of micro quantities of protein, viral antigens, antibodies, structural proteins, vitamins and drug and their metabolites. It can also be used for detection of pictogram quantities (10 −12 g) of biological constituents present in biological fluid. RIA is used in place of bioassay in various branches of science like Biochemistry, Microbiology, and Hematology and Clinical pharmacology .Principle and theory :: RIA works on basic principle of biochemistry that competitive binding between antigens for same antibody binding site. The competition of an analyte with its radioisotopically labeled counterpart for a limited amount of antibody, the specific reagent, is the underlying principle of this technique. Increasing the analyte concentration inhibits the binding of the labeled analyte to the antibody. Ag + Ag* + Ab Ag Ab + Ag* Ab + Ag Unbound Ag* and Ag washed out Radioactivity of bound residue measured Ligand conc. is inversely related to radioactivity Principle and theory : Ag : ligand to be measured ; Ag* : radio labelled ligandPowerPoint Presentation: A B C Antibody Unlabeled antigen Labeled antigen Now how the competition occur with increase the concentration of unlabeled antigen in the system of RIA in three different cases A, B and C. Here first antibodies bound with labeled antigen are put into the known concentration of analyte solution and it is observed how the labeled antigen free from antibody and unlabeled will bind in place of there.PowerPoint Presentation: The concentration of the unknown analyte is thus obtained by comparing its inhibitory Effect on the binding of the labeled analyte to that of a known standard. After determining the ratio of bound to free antigen in each unknown, the antigen concentrations can be read directly from the standard curve (as shown above After determining the ratio of bound to free antigen in each unknown, the antigen concentrations can be read directly from the standard curve (as shown above). 1 2 1. – Ratio in unknown and 2. - Antigen in unknown Red line – binding line Green line – free labeled antigenPowerPoint Presentation: 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 Here as the concentration of unlabeled increase it replaces the labeled bound antigen by competitive binding it inhibiting the binding of labeled one. Antigen- antibody complex depend on antibody affinity (Ka) – The affinity with which antibody binds antigen results from a balance between the attractive and repulsive forcesPowerPoint Presentation: 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.PowerPoint Presentation: 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 Methods in RIA :PowerPoint Presentation: Preparation and radio labeling of antigen Antigen preparation: Synthesis of the molecule Isolation from natural sources Radiolabelling [Tagging procedure] : Two most commonly used radio labels in RIA 3 H and 125 I although Se, P, Co 14 C and 131 I have also been used. but these have some limitations that are – 32 P and 57 Co = limited by stereo chemical aspects because drug naturally contain phosphorous and cobalt 14 C = low specific activity field 131 I = Short decay half life and radio degradation character on the other hand 3 H and 125 I have advantages as follows: 3 H = Direct incorporate into molecular structure, half lives(12 years) 125 I= High activity and ease of counting (half lives 60 days) 75 32 57PowerPoint Presentation: Technique of labeling of drug or antigen with 3 H and 125 I: Specific tritium labels are generally obtained by reducing an appropriate precursor in presence of 3 H . Labeling of drug with 125 I include chloramines-T, monochloride exchange and enzymatic iodination methods. The most suitable iodination procedure is depending on the stability of the drug and the specific activity that is sufficient to meet the sensitivity requirements of assy. Comparison of 3 H and 125 I is as follow: Sr no. Tritium ( 3 H) Iodine ( 125 I) 1 It is more efficient when relatively small numbers of samples are assayed. It is more efficient as the numbers of samples are increased. 2 Such type of problem does not occur. It’s having quality control problems such as damage during the reaction and radiation damage following synthesis. 3 Having advantages of long half life, higher affinity. It has short half life dictates frequent preparation.PowerPoint Presentation: it is possible to find the specific antibody for drug(antigen) as a result of sensitivity reaction Several drugs which have induced antibodies production due their inherent antigenicity like – penicillin, strychnine, tetracycline, sulphonamide and procainamide. However their low specificity and limited availability makes their use rather improbable. In most cases the drugs (analysed antigen) are bind with suitable carrier protein to make conjugated antigen immunogenic. There are several reactive groups on protein carrier which can used for the purpose of conjugation of standard drug(antigen).these groups include the terminal amino and carboxyl groups, ε-amino group of lysine, the carboxyl group of aspartic and glutamic acid, the phenolic group of tyrosine. Preparation purification of drug-protein conjugate(ligand-antigen) to be analyzed:PowerPoint Presentation: The most commonly used methods for conjugation are as follows: Carbodimide and glutaraldehyde reaction Carbony-diimidazole reaction Scotten-baumann reaction Mannich reaction Diazotization reaction The method chosen for conjugated will depend on the functional groups available for coupling the drug to protein and no. of drugs molecules which are to be coupled to protein.PowerPoint Presentation: Preparation : Once the pure antigen prepared then it is emulsified into equal volumes of saline and Freud’s adjuvant (contain alum ppts, natural detergents, mineral oils, killed mycobacterium) to get final concentration of 10 to 50mg/ml. One ml of the emulsion is injected intradermally, subcutaneously, and/or intramuscularly at weekly or monthly intervals into multiple sites of a suitable animal species such as rat, guinea pigs or rabbits . A suitable animal species is usually dictated by the size of the animal facilities. Animals are tested after 3-4 weeks and then blood is collected and separated. The resulting blood containing antibody called antiserum. It is directly used in assay and should be stored at 4°c. Characterization: Characterization of antiserum done by fractionation, immunoadsorption or immunosaturation technique. Preparation & characterization of the Specific and high affinity Antibodies:PowerPoint Presentation: Development of suitable separation techniques to separate free from bound standard drug: Several methods that employ physiochemical and immunological separation have been devised as follows – Physical methods: Filtration, Chromatography, Electrophoresis, charcoal- dextran adsorption, and ion exchange resin. Disadvantages: it is tend to be time dependent and harsh so they may remove bound drug from antibody during separation. Chemical method: organic solvents such as ethanol, dioxane and polyethylene glycol (PEG), and salts such as sodium, zinc, ammonium sulfate. Disadvantages: chemical precipitation may precipitate free as well as bound drug during separation depending on physicochemical nature of drug. Second antibody method: it is most physiologic procedure to precipitate bound antigen. This method employs, an antibody against gamma globulin of the animal species used to produce antidrug antibody to the plasma serum to precipitate drug-antibody complex. Disadvantages: This technique have required prolong incubation time.Flow Chart of Technique: Flow Chart of TechniquePowerPoint Presentation: Other related Immuno Assay: Related methods include the use of antibodies to specifically bind antigenic substances. They do not include competitive protein binding assays (CPBA) or receptor site binding assay (RSBA). The most commonly related method is Immunoradiometric (IRM ) – the technique which employs an excess of labeled antibody to quantify the drug in the system. The principle of the system displayed in figure as below. S + Ab ٭ SAb ٭ + Ab ٭ S Ab ٭ + SP Ab ٭ SP S is unknown or standard antigen, Ab ٭ is labeled antibody, and SP is the antigen coupled to a solid support. SAb ٭ fraction will remain in the supernatant, where as the SPAb ٭ will be coupled to the solid support.PowerPoint Presentation: Advantages: The antibody can be more readily labeled with high specific activity using 125 I. No need of any specific separation technique as in RIA. Instead of using radio tracers several other methods rely on alternate labeling procedures. They include hemaglutination-inhibition, fluorescence , and free radical or spin label and last Enzyme Immuno-Assays . Only Enzyme Immuno-Assays (EIA) – has gained wide acceptance. The theoretical concept of EIA is same as RIA, principle of EIA is as follows: P S ٭ + Ab S ٭ Ab + S SAb S and S ٭ are pure and enzyme linked drug, respectively; Ab is antibody; and P is the measurable reaction product that can result only when free S ٭ react with an appropriate antibody.PowerPoint Presentation: Advantages of EIA over RIA: No radiation hazard Economy of quantitation Homogenous EIA does not require separation of free and bound ligands (antigen) Disadvantages: Relatively insensitive due to the low catalytic activity of enzymes A linkage specificity problem when same linkage used for antibody conjugates and enzyme conjugates. Several drug EIA systems are currently in use in the clinical chemistry laboratory, primarily for the detection of drugs of abuse and quantification of Anticonvulsant and digoxin plasma concentration.PowerPoint Presentation: Application of RIA: Analysis of hormones, vitamins, metabolites, diagnostic markers: E.g. ACTH, FSH , triiodothyronine (T 3 ) and thyroxine (T 4 ) , 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.PowerPoint Presentation: References : www. Innovabioscience.com www. Wikipedia free encyclopedia You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.