1. DON XAVIER N.D GUEST LECTURER ST.ALBERT’S COLLEGE

2. RADIO IMMUNOASSAY(RIA) It is a competitive binding assasy Immunoassay is a method for utilizing immunological properties, when radioisotopes is used then called as Radioimmunoassay was first described in a paper by Rosalyn Sussman Yalow and Solomon Berson (1960) for serum insulin assay. RADIO IMMUNO ASSAY

3.  Technique is based on the competition between unlabelled antigen(Ag) and radioactively labeled antigen(Ag*)  In RIA when there is an excess of antigen molecule (labeled & unlabelled) for a known antibody (Ab). So a competition occurs for same sites of antibody Ag + Ab Ag.Ab Ag* Ag*Ab

4. The increase in the Ag leads to the decrease in the Ag*Ab complex. So we can manipulate the total amount of unlabelled antigen(Ag) bound to the antibody can be calculated from the amount of Ag*Ab complex. In RIA the selection of radiolabel is determined by the structural and sensitivity requirements of immuneresponse system. I131 or I125 is generally employed as radiolabel by attaching to the tyrosine residues of proteins.

6. PROCEDURE OF RIA Mix sample containing drug with fixed quantity of labeled drug and antibody Allow to equilibrate - incubate Separate drug bound to antibody from unbound drug –Charcoal adsorption of antibody (and bound drug) –Antibody - antibody binding precipitates bound drug –Antibody bonded to container Measure radioactivity associated with bound labeled drug –low drug concentration means more bound radioactivity and higher measurement –high drug concentration means less bound radioactivity and lower measurement Determine standard curve –Non-linear plot of radioactivity versus concentration –Logit-log concentration plot is linear

8. Total [Drug]Bound [Drug]Free [Drug] 060 342 633 1224

9. BENEFITS OF RIA ADVANTAGES  Used in assay of any compound that is immunogenic in nature.  It has high sensitivity  Precision comparable to other physico- chemical techniques  It can be automated so minimum handling is required  Low cost

10. DISADVANTAGES  Half life of the reagents are very low  Radiological hazards are involved  Requires use a good technician

12. INTRODUCTION TO ELISA ELISA or enzyme-linked immunosorbent assay, is an immunoassay technique involving the reaction of antigen and antibody in vitro. ELISA is a sensitive and specific assay for the detection and quantitation of antigens or antibodies. ELISA tests are usually performed in microwell plates. The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity.

13. A 96-WELL MICROTITER PLATE USED FOR ELISA

14. COMPONENTS OF AN ELISA Antibody: IgG fraction of serum purified by affinity chromatography Enzyme: Horse Radish Peroxidase (HRP) MW 44, 000, glycoprotein with 4 lysine residues Substrate: TMB (3,3',5,5', tetramethylbenzidine) The enzyme acts as a catalyst to oxidize substrate in the presence of Hydrogen peroxide to produce a blue color. Reaction stopped with dilute acid to cause complex to turn yellow.

15. PRINCIPLE OF ELISA Antibody is immobilized on micro-plate wells Competition between in sample and labeled enzyme for antibody binding sites The unbound material is washed out Chromogenic substrate added to develop color Resulting color is read in a spectrophotometer

17. TYPES OF ELISA DIRECT ELISA INDIRECT ELISA SANDWICH ELISA COMPETETIVE ELISA

18. Variations in the enzyme-linked immunosorbent assay (ELISA) technique, similar to RIA except using an Enzyme (alkaline horseradish Peroxidase, & galactosidase) : safer & less costly. to detect Ab (HIV, HCV) to detect Ag

19. DIRECT ELISA  The direct ELISA uses the method of directly labeling the antibody itself.  Microwell plates are coated with a sample containing the target antigen  The binding of labeled antibody is quantitated by a colorimetric, chemiluminescent, or fluorescent end- point.

20. INDIRECT ELISA The indirect ELISA utilizes an unlabeled primary antibody in conjunction with a labeled secondary antibody. Since the labeled secondary antibody is directed against all antibodies of a given species (e.g. anti-mouse), it can be used with a wide variety of primary antibodies (e.g. all mouse monoclonal antibodies).

21. SANDWICH ELISA 1. Plate is coated with a capture antibody 2. Sample is added, and any antigen present binds to capture antibody 3. Detecting antibody is added, and binds to antigen 4. Enzyme-linked secondary antibody is added, and binds to detecting antibody 5. Substrate is added, and is converted by enzyme to detectable form.

22. COMPETITIVE ELISA In this Unlabeled antibody is incubated in the presence of its antigen. These bound antibody/antigen complexes are then added to an antigen coated well. The plate is washed unbound antibody is removed. The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal. For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.

23. ELISA Reverse method & device (ELISA-R m&d) A newer technique uses an solid phase made up of an immunosorbent polystyrene rod with 4-12 protruding ogives. The entire device is immersed in a test tube containing the collected sample and the following steps (washing, incubation in conjugate and incubation in chromogenous) are carried out by dipping the ogives in microwells of standard microplates pre-filled with reagents.

25. PRECAUTIONS Negative control with strong signal 1. The excessive background signal can be caused by inadequate rinsing of plates. 2. Reagents not sufficiently diluted 3. Inadequate blocking of plates or non-specific binding of enzyme conjugate. 4. The appearance of color in negative control wells may also indicate cross-reactivity of secondary antibody with components in the antigen sample.

26. Positive control with no signal 1. Microwell plates not coated properly. 2. Reagents applied in wrong order or step omitted. 3. Secondary antibody not matched to the species of primary antibody. 4. Enzyme conjugate defective or inhibited by contaminant. 5. Detector antibody not compatible with capture antibody (for sandwich assays).

27. ELISA with weak signal 1. Wash buffer not adequately drained after every wash step. 2. Inadequate incubation times. 3. Detection reagents too dilute. Perform checkerboard titrations. 4. Enzyme conjugate defective or inhibited by contaminant. 5. Substrate defective or contaminated. 6. Microwell plates poorly coated. 7. Loss of capture antibody during blocking/washing. Decrease or eliminate use of Tween-20.

28. APPLICATIONS Screening donated blood for evidence of viral contamination by HIV-1 and HIV-2 (presence of anti-HIV antibodies) hepatitis C (presence of antibodies) hepatitis B (testing for both antibodies and a viral antigen) Measuring hormone levels HCG (as a test for pregnancy) LH (determining the time of ovulation) TSH, T3 and T4 (for thyroid function)

29. Detecting infections sexually-transmitted agents like HIV, syphilis and chlamydia hepatitis B and C Toxoplasma gondii Detecting allergens in food and house dust Measuring "rheumatoid factors" and other autoantibody in autoimmune diseases like lupus erythematosus Measuring toxins in contaminated food Detecting illicit drugs, e.g., cocaine opiates