RadioImmunoAssay By Saranvijay G K .pptx

It says history, nuclear reaction & radiation, introduction, Geiger Muller Counter, tagging compounds, spinthariscope, analysis and procedure of RIA & Instrumentation & Applications...

1. RADIOIMMUNOASSAY BY G.K.SARANVIJAY, IV – B.PHARMACY, VMCP.

2. HISTORY…

3. INTRODUCTION: DEFINITIONS: RADIOACTIVITY – The disintegration of substance that emits radioactive rays are known as radioactivity. RADIOACTIVE – The substance that emits radioactive rays are called as Radioactive substance. Electrons govern the atom’s chemical activity whereas the central nucleus governs the arrangement of electrons. NUCLEAR CHEMISTRY – The branch of chemistry dealing with nuclear changes. It’s discovery leads to discover various War weapons like nuclear bombs and hydrogen bomb due to its tremendous energy liberation. But controlled release energy of nuclear can be used for social welfare too in the field of medicine, agriculture and archeology and etc. At present over 1000 radioactive elements are discovered. Mostly used for tracer studies, even analyzing concentration at low 10 femto mol/ml.

4. NUCLEAR REACTIONS & RADIATIONS: Radionuclide – characterized by its half-life time, the type of transition involved when its decays, the type and energy of the radiation emitted. 1904 – Rutherford separated radiations emitted by radioactive elements into three types, under the influence of a strong electric field.

5. NUCLEAR REACTIONS & RADIATIONS: Three rays have been emitted namely Alpha ray, Beta ray and Gamma ray. Alpha ray:- Attracts toward the - vely charged plate, carrying 2 units of positive charge and 4 times the mass of the H. Velocity:- Ten thousand miles / sec. Penetrating Power:- Produce greater momentum but penetrates lesser. Maybe stopped or absorbed by the Al foil less than 1/10 of a millimeter thick. Luminescence:- Luminate on ZnS screen. Studied by Spinthariscope.

6. NUCLEAR REACTIONS & RADIATIONS: BETA RAYS:- High speed electrons. Attracts toward the + vely charged plate. Velocity:- 1 x 10 5 – 1.5 x 10 5 miles / sec. Penetrating Power:- Being lighter & faster. Stopped and absorbed by a layer of Al 5 mm thick or a layer of Pb 1 mm thick. Luminescence:- Luminescent lightly on ZnS screen. GAMMA RAYS:- Always released in the high excited state. They are similar to X-ray and light ray but have extremely small wavelength except cosmic rays. Do not carry any charges and not deflect the + or - vely charged plate. Velocity:- 3x10 8 m / s (Speed of light). Penetrating Power:- Extremely high penetrating power even greater than that of X-rays. They can penetrate 25 cm thick iron sheet & 8 cm thick Pb sheet. Toxicity:- Harmful to living tissues.

7. UNITS OF RADIOACTIVITY: Specific activity – the activity per unit quantity of radioactive sample, is expressed in variety of ways. - dps /weight or dps /volume. - microcurie/ml or g or mmol. or millicurie/ml or g or mmol. - curie, where 1 Ci = 3.700 x 10 10 disintegrations per second ( dps ). MEASUREMENT OF RADIOACTIVITY: The radioactivity of a substance can be measured by the instrument Geiger Muller counter. Cathode - It consists of a copper (cathode) cylinder closed with a thin mica window at one end. Anode - In the middle of the cylinder insulated with W (Tungsten) wires act as anode which has a potential of about 1500 V. The cylinder filled with a noble gas ( Ar and alcohol vapour ) at 10mmHg pressure.

8. MEASUREMENT OF RADIOACTIVITY: The usage of noble gas is for stability purposes and also to avoid the false signal generation. MECHANISM OF GEIGER MULLER COUNTER: The radioactive substance is placed outside the tube close to mica window. As a single alpha or beta particle (gamma ray) enters the tube, it causes ionization of the gaseous molecules. Which in turn can be amplified and current causes a flash of light in a neon tube measured in the units of Roentgen (exposure in the presence of a photon source).

9. TAGGING COMPOUNDS: Radioactive isotopes are chemically identical with their stable isotope, this may be used as “tag” compound. The tagged compound can be used for analytical purpose, industrial system & biological process. The compound has to be tagged by radioactive isotope but it is not possible widely or readily exchangeable with similar atom in compound with isotope. It is readily exchanged by ionization with the solvent. However, the radionuclide (radicle) will not resemble exactly as isotope. Difference weight may cause variation in reactivity of molecules. But negligible in case of H isotopes (Deuterium), (Tritium). At the same time if Ba-140 is used as a tracer for Ba, then the sample must be freed from the chemically of the daughter La-140 and counted without delay. In biological investigations, isotope must be from the compound itself i.e., which is under analyze. Even minor contamination affects the reports of the investigation.

10. TAGGING COMPOUNDS: In ordinary analytical work, radionuclides have been used to study errors resulting from adsorption, occlusion in gravimetric methods. COMPONENTS OF RIA: Pure antigen. Antiserum (Antibody). Radio-labeled antigen. REQUIREMENTS FOR DEVELOPMENT OF RIA: PURE ANTIGEN – for standards(microgram), tracer production (10 -5 gram), Ab production (10 -4 gram). TRACER - Radio-labeled antigen (self-made or commercial). Specific, high-affinity Antibody – self-made or commercial. A method to separate bound and free antigen and a system to extract Ag from sample.

11. ANALYSIS BY ISOTOPICALLY LABELLED COMPOUNDS: RIA is a competitive binding assay employing the principle of reversible binding of a labelled Ag to its specific Ab. CRITERIA FOR THE ANALYSIS: The non-radioactive Ag (A) and radioactive Ag (A*) are indistinguishable chemically. The two reactions ultimately go to completion i.e., reaches the equilibrium constants of the binding of labelled Ag (Ag*) and unlabelled Ag to antibody this reaction might be infinite. The Ag and Ab reacts in 1:1 ratio. There is no cross reactions observed in the medium. OBJECTIVE: To determine the Concentration ‘C’ of non-radioactive Ag.

12. PRINCIPLE OF RIA: Radioimmunoassay is one of the sensitive immunoassay techniques which helps in the determination of Ag or Ab in a sample with the use of radioisotopes. It is an in-vitro type of Ag-Ab interaction. RIA works on basic principle of biochemistry that competitive binding between Ag for same Ab binding site. The competition of an analyte with its radio isotopically labeled counterpart for a limited amount of Ab. PROCEDURE FOR RIA: In this method, a pure Ag competes with radio-labeled Ag for binding to an Ab with the appropriate specificity. First, known amount of labeled Ag is allowed to bind with the Ab. Then, followed by pure Ag introduced in the mixture of reaction. Finally, determining the amount of labeled Ag that is being separated from binding site of the Ab. Thus, labeled Ag is directly proportional to the pure Ag in the mixture.

13. PROCEDURE FOR RIA:

14. INSTRUMENTATION: The vital equipments essentially required for RIA are: Centrifuge. Radioactive counters. CENTRIFUGE: It has capability of 1200-2500 rpm using switch-bucket-rotor 3500-4000 rpm using fixed-angle-hand rotor. In the former pellet is formed at the bottom of the test tube and the supernatant layer is more easily removed in comparison to the latter one which forms at the angle. RADIOACTIVE COUNTERS: Two types of radioactive counters are mainly employed depending on the type of radioactive substance used, i ) Gamma counters ii) Scintillation counters.

15. RADIOACTIVE COUNTERS: Gamma Counters: Consists of a scintillation detector, with a hole in the center, for a sample to be placed inside holder where scintillation crystals are placed surround the sample and detect the gamma rays using detector photomultiplier tube converts the visible light to electrical signal. PHA – pulse-height analyzer.

16. SCINTILLATION COUNTERS: A scintillation counter is an instrument for detecting and measuring ionizing radiation by using the excitation effect of the incident radiation on scintillating material. Scintillator which generates photons in response to incident radiation, a sensitive detector photomultiplier tube, a charge-coupled device, which converts photon to electrical signals and electronics to process this signals.

17. ADVANTAGES OF RIA: It is very sensitive technique used to measure concentration of antigen without the need to use a bioassay. It can measure one trillionth (10-12) of gram of material per milliliter of blood. It is structurally specific an antigen : antibody reactions 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. LIMITATIONS OF RIA: Working with radioactive substances makes it a bit risky. Disposal of radioactive substances can be problematic. Equipment and reagents are expensive. Radiolabeled substances used have a short shelf-time.

18. APPLICATIONS OF Ria: It was first used for the detection of peptide hormones. Detection of different viral antigens. Detection of many hormones and drugs. Detection of Hepatitis B surface antigens. Detection of mycotoxins. Detection of the early stage of cancer.

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