Radio immuno assay

RIA

1. RADIO IMMUNO ASSAY A.KALYANI M.PHARMACY (PHARMACEUTICAL ANALYSIS)

2. IMMUNOLOGICAL ASSAY They are chemical tests used to detect or quantify a specific substance, the analyte, in a blood or body fluid samples, using an immunlogical reaction. Sensitive and specific Their higher sensitivity results from the use of antibodies and purified antigens as “reagents”. Immunoassays measure the formation of antibody-antigen complexes and detect them via an indicator reaction. They may be qualitative ( positive/ negative), quantitative( amount measured).

3. Purpose of IMMUNOASSAY: The purpose of an immunoassay is to measure ( or, in a qualitative assay , to detect ), an analyte. Immunoassay is the method of choice for measuring analytes normally present at very low concentrations that can not be determined accurately by other less expensive tests. Common uses include: measurement of drugs, hormones, specific proteins, tumor markers, markers of cardiac injury.

4. ANTIBODY: It is a protein ( immunoglobulin) produced by B- lymphocytes ( immune cells) in response to stimulation by an antigen. ANTIGEN: It is a substance that has the ability to induce an immunological response.

5. LABELS IN IMMUNO ASSAYS Immunoassays require the use of labelled 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. Example of a label include a : 1. Radioactive compound 2. An enzyme that causes a change of color in a solution 3. Substance that produce light labels may be applied to either to the antibody or to the antigen.

6. Categories of immunoassay tests: 1. Competitive Assays 2. Non competitive Assays 3. Homogenous Assays 4. Heterogeneous Assays. COMPETITIVE ASSAYS: Un labeled analyte ( antigen ) in the test sample is measured by its ability to compete with the labeled antigen in the immunoassay. In a competitive immunoassay, ↓ less label measured in the assay means, more↑ more unlabeled antigen is present.

7. 2.NON- COMPETITIVE ASSAYS: High sensitivity and specificity The analyte is bound between two highly specific Ab reagents- Sandwich assay. It contains excess of labeled Ab, so that all drug/ metabolite is bound. The amount of Ab-Ag complex is then measured to determine the amount of drug present in the sample. The labeled antibody is directly proportional to the amount of antigen present in the sample.

8. HETEROGENEOUS ASSAYS: Also called Separation assay. It requires multiple steps. Unbound analyte is separated or washed away, and the remaining labelled bound analyte is measured. HOMOGENOUS ASSAYS: Those that do not require separation are referred to as Homogenous Immunoassay. 2. Measurement of small analytes such as Ab used and therapeutic drug.

9. There are several methods used in Immunoassay tests: 1. RIA- RADIO IMMUNO ASSAY 2. ELISA- ENZYME LINKED IMMUNO SORBENT ASSAY 3. FPIA – FULORESCENCE POLARIZATION IMMUNO ASSAY.

10. RADIO IMMUNO ASSAY HISTORY The technique was 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. THEORY: RADIO IMMUNO ASSAY Use of radio Ag-Ab binding detection of active material theory compound

11. 1. RIA is a scientific method used to test antigens ( for ex: hormone levels in the body) without the need to used a bioassays. 2. RIA is a Radio-analytical technique with remarkable sensitivity and high degree of specificity that is widely used for the estimation of a variety of molecules present in complex matrices. 3. It is also known as “Radio tracer technique” and best example of invitro diagnosis technique using radio isotopes. 4. This technique is used over a wide spectra of substances such as hormones, steroids, vitamins, drugs, tumor markers, viral antigens, proteins, Ab. 5. RIA combines the specificity of an Ag-Abreaction with sensitivity of radio activity measurements. 6. Sensitivity ranges from 0.0006 - 0.006 µg/ml. 7. RIA used in place of bioassay in various branches of science like Biochemistry, Microbiology, and Hematology and Clinical Pharmacology.

12. PRINCIPLE : It involves 3 principles which make it most specific and sensitive than other Immunoassays. 1. An immune reaction i.e., Ag – Ab binding. 2. A competitive binding or competitive displacement reaction ( it gives specificity). 3. Measurement of radio emission ( it gives the sensitivity). AN IMMUNE REACTION:- When a foreign biological substance enters into the body blood stream through non oral route, body recognizes the specific chemistry on surface of foreign substance as antigen and produces specific antibodies against the antigen so as to nullify the effects and keep the body safe. The Antibodies are produced by body immune system, so , it is an immune reaction. A COMPETITIVE BINDING:- This a phenomenon where in, when there are two antigens which can bind to same antibody, the antigen with more conc. Binds extensively with the limited antibody displacing others. So, here in the experiment radio labeled antigen is allowed to bind to high affinity antibody. When patient serum is added, unlabeled antigen in it start binding to the Ab displacing the labeled antigen.

13. 3.MEASUREMENT OF RADIO EMISSION:- Once the incubation is over, then washings are done to remove any unbound antigens. Then radio emission of the Ag- Ab complex is taken, the gamma rays from radio labeled antigen are measured.

14. REQUIREMENTS OF RIA Micro titre plate or test tube A micro titre plate is mostly used for assay A micro titre plate could have 6,24,96,384 or even sometimes 1536 wells arranged in rows. Each well of a micro titre plate can only hold very small amounts of liquids. Preparation and characterization of antigen( ligand to be analyzed) Antigens are prepared by synthesis of the molecule Isolation from natural sources

15. Radio labelling or tagging procedure 3 H, 14 C, 125 I are used as radio active tags. Antigens are tagged to 3 H, 14 C, 125 I. Tagging should not affect the antigenic specificity and antigenic activity. Preparation of specific antibody Antigen injected intradermally into rabbits and guinea pigs. Antibody is produced Antibody removed from the serum

16. DEVELOPMENT OF THE ASSAY SYSTEM - Crucial step is separation of unbound antigens. - Antibodies bind to micro titre well surface[ Solid phase RIA] - Antigens bound to the fixed antibodies remain stuck to the inner surface - Decanting and washing the well removes unbound antigens - Other techniques of separation: Centrifugation, Precipitation and Electrophoresis Centrifuge Used for separation of precipitated form and supernatant liquid form. Range: 1200-2500 rpm

17. Radio active counters There are two types Gamma counter: used for gamma energy emitting isotopes. eg. Common iodine isotopes Scintillation counter: used for beta energy emitting isotopes. eg. Tritium, carbon 14 isotopes

18. Procedure: 1. The labeled antigen is mixed with antibody at a concentration that saturates the antigen-binding sites of the antibody. 2. Then test samples of unlabeled antigen of unknown concentration are added in progressively larger amounts. 3. The antibody does not distinguish labeled from unlabeled antigen, so the two kinds of antigen compete for available binding sites on the antibody. As the concentration of unlabeled antigen increases, more labeled antigen will be displaced from the binding sites. 4. the decrease in the amount of radiolabeled antigen bound to specific antibody in the presence of the test sample is measured in order to determine the amount of antigen present in the sample. 5. the antigen is generally labeled with a gamma- emitting isotope as 125 I but beta- emitting isotopes such as tritium (3H) are also routinely used as labels. 6. The radio labeled antigen is part of the assay mixture; the test sample may be a complex mixture; such as serum or other body fluids, that contains the unlabeled antigen.

19. ADVANTAGES: 1. Highly specific 2. High sensitive 3. It has become a major tool in clinical laboratory. 4. Using antigens of higher affinity, it is possible to detect few picograms (10 -12 ) of antigen in test tube. 5. Plasma levels of most of our hormones. DISADVANTAGES: The main draw backs of RIA are the expense and hazards in preparing and handling the radioactive antigen. Both 131 I or 125 I emit gamma radiation that requires special counting equipment. The body concentrates Iodine atoms- radioactive or not – in the thyroid gland where they are incorporated I thyroxine (T 4 ). Labs require special license to handle radioactive material. Requires special arrangements for - storage of radioactive material - radioactive waste disposal.

20. GENERAL USES:- In Endocrinology - Insulin, HCG, Vasopressin - Detects Endocrine disorders - Physiology of Endocrine function. In Pharmacology - Morphine - Detects drug abuse or drug poisoning. Oncology: - Early cancer detection and diagnosis Diagnosis and treatment of peptic ulcers.

21. Applications: Screening donated blood for evidence of viral contamination by - HIV 1 and HIV 2 ( prescence of anti- HIV antibodies). - Hepatitis C (prescence of antibodies) - Hepatitis B (Testing for both antibodies and a viral antigen). Measuring hormone levels - HCG ( as a test for pregnancy) - LH ( Determine the time of ovulation) - TSH, T 3 and T 4 ( for thyroid functions)

22. Detecting allergens in food and house dust RAST :- “Radioallergosorbent test“ to detect specific Ig E antibodies to suspected or known allergens. Ig e is the antibody with type I allergic response: pollen. The amount of radioactivity is proportional to the serum Ig E for the allergen. Detecting illicit drugs eg:- cocaine, opiates, Δ -9-tetra hydrocannabinol, the active ingredient inn marijuana. Detecting infections - Sexually transmitted agents like HIV, Syphilis, and Chlamydia.