GROUPE I PRESENTATION L3

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CHEMICAL PATHOLOGY ASSIGNMENT   :

CHEMICAL PATHOLOGY ASSIGNMENT

GROUP I MEMBERS:

GROUP I MEMBERS AHSL3/05/1001 AHSL3/05/1013 AHSL3/05/1018 AHSL3/05/1004 AHSL3/05/1037 AHSL3/05/1075 AHSL3/05/1005 AHSL3/05/1061

RADIO IMMUNOASSAY:

RADIO IMMUNOASSAY Is an immunologic technique that uses radioisotopes to detect antigen or antibodies in biological fluids. Purpose is to determine the concentration of an antigen in solution. Is a competitive binding assay.

PRINCIPLE:

PRINCIPLE To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine attached to tyrosine. This radiolabeled 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 radiolabeled antigen ("hot") for antibody binding sites.

CON,T:

CON,T 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 antigen to free radiolabeled antigen. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured using a gamma counter. Using known standards, a binding curve can then be generated which allows the amount of antigen in the patient's serum to be derived.

FIGURE:

FIGURE

PROCEDURE :

PROCEDURE REAGENTS USED. 1 The antibody reagents used in RIAs are frequently attached to solid supports such as plastic tubes or polystyrene beads . The antibodies specifically recognize an antigen or hapten , i.e , a drug or a low molecular mass steroid hormone of interest . The antibody reagents can be characterized as either: polyclonal (a mixture of purified antibodies recognizing different antigenic determinants of an antigen or the same portion of the antigen with differing affinities). or monoclonal (a solution containing identical antibody molecules recognizing a single antigenic determinant).

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2. The radiolabelled antigen or hapten is a purified molecule covalently labeled with a radioisotope . The radioisotope most frequently used is sodium iodine ( 125 I ). 3 . Buffers are used in commercial RIA kits as diluents and generally contain EDTA , bovin serum albumin to reduce non specific bindings and preservative . 4 . Standards and controls consist of human source material , sodium azide and a stated concentration of antigen , hapten, or antigenic antibody of interest . 5. Patient sample.

ASSAY PROCEDURE :

ASSAY PROCEDURE Add known amounts of the test sample + labelled antigen into the microtitre wells Incubate  allow the reaction to reach completion Decant & wash contents of the well  removes all unbound antigens Radioactivity remaining in the Microtitre wells measured by a Counter [GM counter , Scintillation counter etc ] Intensity of radioactivity is inversely correlated with the concentration of antigens in the test sample. Sensitive to very low concentration of antigens

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Antibody

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Labeled Antigen Labeled Antigen + Sample

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Separate bound from free: Antibody labeled tubes can be simply decanted Liquid-phase antibodies need to be precipitated Use a second antibody PEG Centrifugation

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Competing reactions that form basis of radioimmunoassay; * indicates the labeled antigen, and † “in known standard solutions or unknown samples.”

SENSITIVITY &DETECTORS :

SENSITIVITY &DETECTORS This assay is very sensitive because it uses a high gamma emitting radioisotope label, 125 I, which detects a lower antigen concentration at the level of picomolar . The detector used in RIAs is GAMMA COUNTER or SCINTILLATION COUNTER;It measures gamma rays or electromagnetic radiation emitted by very high energy radioisotopes( 125 I ).

CLINICAL UTILITY:

CLINICAL UTILITY Radioimmunoassay (RIA) is a very sensitive in vitro assay technique used to measure concentrations of antigens (for example, hormone levels in the blood ) by use of antibodies. As such, it can be seen as the inverse of a radiobinding assay, which quantifies an antibody by use of corresponding antigens.

APPLICATIONS OF IMMUNOASSAYS :

APPLICATIONS OF IMMUNOASSAYS Analysis of hormones, vitamins, metabolites, diagnostic markers Eg . ACTH, FSH, T3, T4, Glucagon, Insulin, Testosterone, vitamin B12, prostaglandins, glucocorticoids , Therapeutic drug monitoring: Barbiturates, morphine, digoxin , Diagnostic procedures for detecting infection ( HIV, Hepatitis A, B etc)

GAMMA COUNTER:

GAMMA COUNTER

ADVANTAGE AND DISADVANTAGES :

ADVANTAGE AND DISADVANTAGES ADVANTAGES: The use of a high gamma emitting radioisotope label, 125 I ,which creates an assay that is sensitive enough to detect antigen at the picomolar concentration or lower. Radioimmunoassay is easly automated and practical if large batches are to be tested. Highly specific: Immune reactions are specific High sensitivity : Immune reactions are sensitive Sensitive: nanogram levels or lower Minimal reagents Qualitative & Quantitative Qualitative  Eg HIV testing quantitative assays  Eg Therapetic Drug Monitoring Greater scope : Wells can be coated with Antigens OR Antibodies Suitable for automation  high speed

DISADVANTAGES :

DISADVANTAGES Radioisotope labels have a short shelf life of one to two months, which cause inefficiency, high cost and waste . These substances(iodine)can also be inconvenient and expensive to dispose of and store . Radiation hazards: Uses radiolabeled reagents which can be harmful to human . Requires specially trained persons Labs require special license to handle radioactive material Requires special arrangements for : Requisition, storage of radioactive material radioactive waste disposal.

REFERENCES :

REFERENCES Clinical chemistry concepts&application,2003,Shauna C. Anderson&Susan Cockayne www.uludag.edu.tr/~sahinas/radioimmunassay.ppt www.cdc.gov/nchs/data/nhanes/nhanes.../l10am_c_met_cpeptide.pdf www.ncbi.nlm.nih.gov/pubmed/3381240 www.sciencedirect.com/science/article/pii/0022175986903388 Yalow RS, Berson SA (1960). "IMMUNOASSAY OF ENDOGENOUS PLASMA INSULIN IN MAN" . The Journal of clinical investigation 39 (7): 1157–75. Acebedo G, Hayek A, Klegerman M, Crolla L, Bermes E, Brooks M (1975). "A rapid ultramicro radioimmunoassay for human thyrotropin ". Biochem . Biophys . Res. Commun . 65 (2): 449–56.

CON,T:

CON,T THANK YOU FOR YOUR ATTENDANCE

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