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Ria 112070804007

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Ria 112070804007

  2. 2.  An assay based on the reversible and non-covalent binding of an antigen by a specific antibody employing radioactivity labeled antigen to measure the fraction of the antigen bound to a substoichiometric amount of antibody. The technique of radioimmunoassay has revolutionized research and clinical practice in many areas, e.g.,  blood banking  diagnosis of allergies  endocrinology
  3. 3.  It is based on the competition between unlabelled antigen and a fixed amount of the corresponding radio labelled antigen for limited number of antibody binding sites in fixed amount of antiserum. At equilibrium, the unbound antigen (F)+Antigen-antibody complex(Ag-Ab) are separated and quantified. Ag(larger) + Ab → Ag-Ab Ag*(smaller) + Ab → Ag*-Ab In std. Condition, amount of labeled antigen bound to the antibody decreases as the amount of unlabelled antigen increases in sample. The reaction between Ab and Ag* is quantified by counting either the bound (B) or the free labeled compound (F).
  4. 4.  The amount of labeled compound bound to antibody is dependent on  The amount of ligand (Ag +Ag *) present  The amount of antibody (Ab) present  The equilibrium constant/ affinity constants k1’ and k2’ where, k1’ = k1/k2 and k2’=k3/k4. Ag* + Ab Ag*-ab and , Ag + Ab Ag-Ab In assay ag* and Ab are constant as binding of Ag* solely depends on amount of Ag present. % F = [F/ (F+B)]*100. % B = [B/ (F+B)]*100
  5. 5.  A known quantity of an antigen is made radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine attached to tyrosine. 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. 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 radio labeled antigen for antibody binding sites. As the concentration of "cold" antigen is increased, more of it binds to the antibody, displacing the radio labeled variant, and reducing the ratio of antibody-bound radio labeled antigen to free radio labeled antigen. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured.
  6. 6.  With this technique, separating bound from unbound antigen is crucial. Initially, the method of separation employed was the use of a second "anti- antibody" directed against the first for precipitation and centrifugation. Using known standards, a binding curve can then be generated which allows the amount of antigen in the patients serum to be derived. Determine standard curve  Non-linear plot of radioactivity versus concentration.
  7. 7.  Radioimmunoassay (RIA) is a very sensitive technique used to measure concentrations of antigens (for example, hormone levels in the blood) without the need to use a bioassay. It can measure one trillionth of a gram of material per milliliter of blood. Because of the small sample required for measurement, RIA quickly became a standard laboratory tool. It is structurally specific as Antigen: Antibody reaction are highly specific. Therefore, RIA includes specificity of antigen antibody reaction and sensitivity of radioactivity measurement. It is an indirect method of analysis since neither the radioactive standard nor the antibody is present in the original sample. It is a saturation analysis as active reagent (antibody) is added in smaller quantity than that of analyte (antigen).
  8. 8.  Prolonged reaction time (in days) as a consequence highly diluted reagent is used. Radioactive Iodine used in is not a cheap reagent. Possible health hazards due to handling of radioisotopes. All the reagents must be added precisely. Limited assay range. Lack of direct linear relationship between analyte concentration and signal response. Difficulty of automation. Lengthy counting time. 
  9. 9. 1)A tracer i.e. a labeled ligand. Carbon-14 Tritium-311 Iodine-125 13’I 13N 15O2) A binder (Antibody) which is the specific antiserum. 3)A separation system to separate to separate the ‘bound’ and ‘free’ phases. 4)A standard i.e. the ligand (analyte) in highly pure form. 5)A ligand (analyte) - free human antiserum.
  10. 10.  RIA requires a separation procedure because the bound fraction does not precipitate spontaneously at the low concentration employed. Variety of procedures are available that exploit physicochemical or immunological differences between the two fractions. They are: Physical methods include: filtration, chromatography, electrophoresis, charcoal-dextran adsorption, adsorption on ion-exchange resin. Chemical methods include: organic solvents, such as ethanol, dioxane and PEG, or salts, such as sodium, zinc and ammonium sulphate, to precipitate antibody bound heptan. Solid phase system. No one is ideal in establishing a new assay as two or more principles employed for separation.
  11. 11.  RIA has many uses, Narcotics (drug) detection, Blood bank screening for the hepatitis (a highly contagious condition) virus, Early cancer detection, Measurement of growth hormone levels, Tracking of the leukemia virus, Diagnosis and treatment of peptic ulcers, and Research with brain chemicals called neurotransmitters
  12. 12.  Endocrinology  Insulin, HCG, Vasopressin  Detects Endocrine Disorders  Physiology of Endocrine Function Pharmacology  Morphine  Detect Drug Abuse or Drug Poisoning  Study Drug Kinetics
  13. 13.  Epidemiology  Hepatitis B Clinical Immunology  Antibodies for Inhalant Allergens  Allergy Diagnosis Oncology  Carcinoembryonic Antigen  Early Cancer Detection and Diagnosis
  14. 14.  INTRODUCTION: ELISA is an important immunological technique for detecting and measuring the amount of antigen or antibody in the sample using various enzymes. PRINCIPLE:  It is based on the measurements of the enzyme labeled antibody. In this antigen or antibody are linked to the enzymes and forms the complex with enzymes which is measured by colorless substances to coloured compound.
  15. 15.  It is useful technique for determining serum antibody concentration. It has also found application in the food industry in detecting potential food allergens such as milk, peanuts, walnuts, almonds, and eggs. It is used to assay of hepatitis B-antigen. It is used for detection of anti-HIV-1 and anti-HIV-2 anti body in the patient serum. An out off point may be determined by comparing it with a known standard.
  16. 16. ELISA RIA Specificity + + Sensetivity + + Cost + - Reagent shelf life + - Fully automated - + Safety + - Small diagnostic laboratories + -
  17. 17.  Principle: competition between endogenous insulin in body fluids i.e. blood plasma and exogenous radioactive insulin for the binding sites on anti insulin antibody.Formerly developed techniques:1) A Laborious techniques developed by yallow et all in which paper electrophoresis was used to separate antibody bound insulin from free insulin.2) Morgan et all utilized the double antibody techniques wherein antiguinea pig gamma-globulin is used to precipitate the soluble complex formed between insulin and guinea pig anti insulin.
  18. 18.  Components used in the assay: Radio labeled tracer: insulin Analyte: Immunoreactive insulin Buffer: phosphate buffer ph 7.4 Adsorbent: Zirconyl phosphate Separation system: by centrifugation
  19. 19.  Here we are going to describe RIA of digitoxin-3-hemisuccinyl-leucine using antiserum elicited by digoxin 3-hemisuccinate – BSA conjugate and then measuring concentration of digoxin in digitalized patients. REQUIREMENTS: Specific antiserum: Immunization of rabbit with digoxin 3-hemisuccinate-BSA conjugate. Tracer: digoxin 3 hemisuccinyl-[3H] leucine Analyte: serum digoxin in digitalized patient Buffer: phosphate saline buffer(ph-7.4) Adsorbent: dextran coated charcoal suspension Separation system: centrifugation
  20. 20.  Digoxin free serum + digoxin 3-hemisuccinyl [3H] leucine+ assay buffer +diluted antiserum Add 0.1ml non labeled digoxin in test tubes. All tubes are shaken in a vortex mixture and incubated at 4c. A dextran coated charcoal suspension is added to each test tube. which was then vortexed, incubated for 10min in an ice bath and centrifuged at 1700*g for 10 min at 4°c. The supernatant is then transferred to a counting vial, scintillation solution is added and the radioactivity is counted in liquid scintillation counter. The radioactivity of the bound antibody is calculated after correction for the blank value. The dose-response curve is constructed using duplicate samples.
  21. 21. (1)James w. Munson ”Pharmaceutical Analysis modern method” , part A, page no:412-422(2) Dr. Chandrakant R.kokare “Pharmaceutical Microbiology principle & application”, 6th edition,Nirali publication, page no:23.16-23.17