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Radio immuno assay, RIA, by kk sahu
- 1. RADIO IMMUNO ASSAY By KAUSHALKUMAR SAHU Assistant Professor (Ad Hoc) Department of Biotechnology Govt. Digvijay Autonomous P. G. College Raj-Nandgaon ( C. G. )
- 2. SYNOPSIS INTRODUCTION PRINCIPLE HISTORY HOW TO RIA WORK METHOD APPLICATION OF RIA ADVANTAGE DISADVANTAGE CONCLUSION REFERENCES
- 3. INTRODUCTION The techniquein which a radioisotope is used as a tag or label (i.e. radioisotope covalently linked to antigen or antibody) for the detection of antigen-antibody complex is known as RIA. RIA involves the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantifitation using radioactivity. RIAs utilize a radioactive label (usually 125I, 3H or 14C), which emits radiation that can be measured with a beta or gamma counter.
- 4. The techniqueof radioimmunoassay has revolutionized research and clinical practice in many areas, e.g., ◦blood banking ◦diagnosis of allergies ◦endocrinology
- 5. RRINCIPLE RIA involvesthe competitive binding of a radiolabelled antigen to a high affinity antibody. The antigen is generally labelled with Gama ray.
- 7. HISTORY The techniquewas introduced in 1960 by Berson and Yalow as an assay for the concentration of insulin in plasma. It represented the first time that hormone levels in the blood could be detected by an invitro assay.
- 8. LABELS IN IMMUNOASSAYS-Immunoassays require the use of labeled materials in order to measure the amount of antigen or antibody present. A label is a molecule that will react as part of the assay, and in doing so produce a signal that can be measured in the solution. Examples of a label include a radioactive compound, or an enzyme that causes a change of color in a solution or its fluorescence . HOW RIA WORK
- 9. METHOD Classically, toperform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine, such as 125-I, attached to tyrosine. This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two specifically bind to one another. Then, a sample of serum from a patient containing an unknown quantity of that same antigen is added. This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled antigen ("hot") for antibody binding sites. As the concentration of "cold" antigen is increased, more of it binds to the antibody, displacing the radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled
- 10. -Take a microtiterplate. -Coated with antibody and specific to the Ag. -Washing to remove unbound Ag.
- 11. •After small amountof antigen added to the well •Effect that competition b/w Ab and Ag. •Removed hot Ab by cold Ag. •After washing the unbound for unbound Ag removed. •Radioactivity is not 100%
- 12. •If 0 ngadded to the well radioactivity is 100% •When 1 ng unlabeled Ag will added it decreased by 90% •And the process is repeated all the process again by increasing the •It will be proportional to the concentration of cold Ag.
- 13. •We can drawlinear graph to the data correct •And then to now determine concentration of A Ag present in sample. •Take microtiter plate and coated with monoclonal Ab and again add hot Ab. •Instead we add sample there are Ag present in the sample
- 14. •According to theconcentration unknown ag the will compete to the radiolabel Ag, and remove them from the antibody. •If radioactivity of the antibody is now 40%.we have to extrapolate this value on standard graph. •It will tell as concentration it 6 ng so our sample contain 6 ng of Ag.
- 15. The bound antigensare then separated from the unbound ones, and the radioactivity of the free(unbound) antigen remaining in the supernatant is measured using a gamma counter.
- 16. APPLICATION OF RIA- RIA is used in the assay drugs like digitoxin, morphin, barbifurate, amphetonine. Analysis of vitamins like riboflavin, folic acid. Analysis of hormone like aldosteron, insulin, growth hormone, thyroxin.
- 17. ADVANTAGE Radioimmunoassayis widely-used because of its great sensitivity. Using antibodies of high affinity, it is possible to detect a few pictograms (10−12 g) of antigen in the tube. The greater the specificity of the antiserum, the greater the specificity of the assay RIA has become a major tool in the clinical laboratory where it is used to assay . Plasma levels of: ◦ most of our hormones; ◦ digitoxin or digoxin in patients receiving these drugs; ◦ certain abused drugs.
- 18. DISADVANTAGE The maindrawbacks to radioimmunoassay are the expense and hazards if preparing and handling the radioactive antigen. Both 125I or 131I emit gamma radiation that requires special counting equipment; expensive instrumentation.
- 19. CONCLUSION Radio immuneassay (RIA) is an elegant tech. in analytical chemistry. If substance to be analysed is very low quantities, in the order of microgram, conventional methods like gravimetric and colorimetric method fail. RIA finds extensive application in the assay of many substance which are present in trace amount in blood.
- 20. REFERENCES BOOK- Textbookof microbiology by Prescott, Harley. INTERNET SOURCE- http://www.antibodies- online.com/resources/17/1215/radioimmunoassa y-ria/ http://www.biologynoteshelp.com/radio- immuno-assaytypes-of-elisa/ http://en.wikipedia.org/wiki/Radioimmunoassay http://en.wikipedia.org/wiki/RIA