Radioimmune assay

Radioimmuno assay/instruments/elements used/application

1. Sam Higginbottom University of Agriculture Technology and Sciences Prayagraj, India Radioimmune Assay Submitted to: Prof. (Dr). P.malai rajan (Hod, Department of Pharmaceutical Sciences, SIHAS, SHUATS, Prayagraj, U.P, India) Date of Submission: 08/03/2021 Submitted By: Name: Imdad H. Mukeri Bachelor of Pharmacy (8 th sem) Id No. 17BPH084 Subject: Advance Instrumentation technique (801T) 4/25/2021 1 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

2. Contents History and Introduction Of RIA Importance And Various components of RIA Principle of RIA And its Reagents used Method of separation and its procedure Limitation and its Application Conclusion And References 4/25/2021 2 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

3. History Developed in 1959 by Rosalyn Yalow and Solomon Berson for measurement of insulin in plasma. It represented the first time that hormone levels in the blood could be detected by an in vitro assay. In 1977 Yalow received the Nobel Prize for her and Berson’s development of RIA Rosalyn yalow 4/25/2021 3 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

4. Immunoassay An immunoassay is a test that uses antibody and antigen complexes An antibody: antigen complex is also known as an immune complex “Immune” refers to an immune response that causes the body to generate antibodies “Assay” refers to a test Immunoassay is a test that utilizes immune complexing when antibodies and antigens are brought together 4/25/2021 4 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

5. Antibodies, Antigens and Analytes An antibody is a protein that is produced by the body in response to an “invading” (foreign) substance. Antibodies are produced as part of the body’s immune response to protect itself. An antigen is the substance that the body is trying to “fight off” by mounting an immune response. for example, the drug is the antigen that binds to the antibody. An immunogen is a substance that elicits immune response. E.g. drug-protein conjugate. An analyte is anything measured by a laboratory test. 4/25/2021 5 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

6. In immunoassay testing, the analyte may be either an antibody, or an antigen. Immunoassays utilize one or more selected antibodies to detect analytes of interest. The analytes being measured may be:- That are naturally present in the body (such as a thyroid hormone) The body produces but are not typically present (such as a cancer antigen) Do not naturally occur in the body (such as an abused drug) 4/25/2021 6 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

7. Radioimmunoassay Radio Immuno Assay (RIA) is an elegant tech. in analytical chemistry. If substance to be analyzed is in very low quantities, in the orders of micrograms, nanograms, conventional methods like gravimetric and colorimetric method fail. RIA finds extensive application in the assay of many substances which are present in trace amount in blood. 4/25/2021 7 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

8. Importance of radio immune assay The investigation was conducted to study the role of the determination of tumor markers (CEA, β 2 -microglobulin, IgE and ferritin) in patients with malignant lymphomas. Radio immuno assay is very sensitive technique used to measure concentrations of antigen without the need to use a bioassay. It can measure one trillionth (10-12) of a gram of material per milliliter of blood. It is structurally specific as antigen: antibody reaction 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. 4/25/2021 8 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

9. Principle of RIA The amount of Ab per tube is kept constant, the amount of antigen added (known or unknown) is the variable parameter. The added antigen will be distributed between a bound (B) and a free (F) fraction. This distribution is governed by the association constant (KA ) of the Ab: Ab + Ag AgAb K = [AbAg] /[Ab][Ag] Competitive binding of radiolabelled antigen and unlabelled antigen to a high affinity antibody. 4/25/2021 9 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

10. Principle of RIA The labelled antigen is mixed with the antibody at a concentration that saturates the antigen –binding sites of the antibody. As the concentration of the unlabelled antigen increases more labelled antigen will be replaced from the binding site. The decrease in the amount of radiolabelled antigen bound specific antibody in the presence of the test samples is measured to determine the amount of antigen Present in the test sample. In Standard Condition, amount of labelled antigen bound to the antibody decreases as the amount of unlabelled antigen increases in sample. 4/25/2021 10 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

11. Reagents used in RIA A tracer i.e. a labeled ligand. A binder (Antibody) which is the specific antiserum. A separation system to separate to separate the ‘bound’ and ‘free’ phases. A standard (in highly pure form) A free human antiserum. The radioisotopes used are Beta emitters3H and 14C Gamma emitters125I 4/25/2021 11 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

12. Reagents used in RIA Tritium – 3 H: Weak β− ray emitter Significantly lower energy than 14C Long physical half-life of 12.3 yrs Biological half-life: 10 – 12 days Produced by neutron bombardment of a lower hydrogen isotope Used for drugs like proteins and amino acids 4/25/2021 12 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

13. Reagents used in RIA Carbon-14: Weak β− ray emitter Long physical half-life (~ 5.7 x 103 yrs) Biological half-life: Bound – 12days; Unbound – 40days Commercially available as Barium carbonate14C Iodine-125: Low γ−emission: 35.4 keV High specific activity Short physical half-life: 60 days It can be obtained with specific activity and almost 100% isotopic abundance, thus reducing counting time and being economic. • Convenient half-life (60.2 days) hence shelf life for labeled antigen is long. • Iodine is natural constituent of thyroxin and triiodothyronine. • It can be easily introduced into peptide molecules, steroids. • Gamma emission permits the use of simple inexpensive equipment for counting radioactivity. 4/25/2021 13 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

14. Reagents used in RIA Positron vs. Gamma Isotopes: The positron ( β- emitting) radionuclides are mainly restricted for in-vitro experiments. The γ- emitting radionuclides are useful for in-vivo imaging Other commonly used isotopes: Positron: 11C, 13N, 15O, and 18F. Gamma : 111In(indium), 123I, 131I, 153Sm(Samarium), 75Se. 4/25/2021 14 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

15. Specific antiserum(binder/antibody) It is prepared by injecting (repeatedly) the antigen together with Freund’s adjuvant into suitable animal such as guinea pig, rabbit or goat. Molecule like thyroid hormone, steroids, drug are not immunogenic. So they are conjugated to carrier proteins and polymer to make them immunogenic 4/25/2021 15 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

16. SEPERATION SYSTEM It is required because the bound fraction does not precipitate spontaneously at the low concentration. Variety of procedure are available a)Physical method Filtration,chromatography,electrophoresis,charcoal dextran adsorption b)Chemical method Organic solvent such as ethanol,dioxane,PEG or salts such as sodium ,zinc and ammonium sulphate. C)Solid phase system 4/25/2021 16 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

17. A STANDARD i.e. LIGAND (ANALYTE) IN HIGHLY PURE FORM Drugs , protein, hormone etc must be in pure form so they can be diluted . Standard are prepared in ligand free serum. In case of protein ,hormones the standard should be prepared preferably with the hormone from the species from which the serum is going to be analysed. LIGAND FREE HUMAN SERUM It is prepared by treating human serum with charcoal . It can also be prepared by collecting serum from volunteers in whom production of that ligand or hormone has been inhibited by treatment with an appropriate drug 4/25/2021 17 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

18. General Procedure for Performing a RIA Analysis A known quantity of an antigen is made radioactive This radio labeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two chemically bind to one another. 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 radio labeled antigen for antibody binding sites As the concentration of "cold" antigen is increase, more of it binds to the antibody 4/25/2021 18 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

19. General Procedure for Performing a RIA Analysis And by displacing the radio labelled variant and reduces the ratio of antibody-bound radio labelled antigen to free radio labelled antigen. The bound antigens are then separated from the unbound ones The radioactivity of the free antigen remaining in the supernatant is measured. Separating bound from unbound antigen is crucial Initially, the method of separation employed was the use of a second "anti-antibody“. 4/25/2021 19 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

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21. The limitations of the RIA include: The main drawback of RIA are the expensive and hazards of preparing and handling the Radioactive antigen. The gamma emitting isotope such as I 125 Requires special counting equipment. Require special license to handle Radioactive materials. The cost of equipment and reagents Short shelf-life of radiolabeled compounds The problems associated with the disposal of radioactive waste. 4/25/2021 21 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

22. Application of Radio Immuno Assay The test can be used to determine very small quantities (e.g. monogram) of antigens and antibodies in the serum. The test is used for quantitation of hormones, drugs, HBsAg, and other viral antigens. Analyze nanomolar and picomolar concentrations of hormones in biological fluids 4/25/2021 22 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

23. Conclusion In above we study about Radio Immuno Assay (RIA) is an elegant tech. in analytical chemistry. RIA used to be analyzed is in very low quantities, in the orders of micrograms, nanograms. 4/25/2021 23 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

24. References https://microbenotes.com/radioimmunoassay-principle-uses-and-limitations/ (accessed on 07/03/2021) https://www.slideshare.net/justinsolin/radioimmunoassay-56112157 (accessed on 06/03/2021 ) http://mcb.berkeley.edu/courses/mcb150/lecture5/Lecture5(6).pdf (accessed on 06/03/2021) 4/25/2021 24 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India

25. Thank you 4/25/2021 25 DOPs, SIHAS, SHUATS, Prayajraj, U.P,India