RIA

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PRESENTED BY :- PATEL MITUL A. M.PHARM, SEM- 1 ROLL NO: 4 DEPT. OF QUALITY ASSURANCE GUIDED BY :- MR.DHARMENDRASINH BARIA ASSISTANT PROFESSOR DEPT. OF PHARMACEUTICAL CHEMISTRY DHARMAJ DEGREE PHARMACY COLLAGE , DHARMAJ A SEMINAR ON RADIOIMMUNOASSAY

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Introduction Principle of RIA Theory Requirements Methods Merits & De-Merits Applications Related Techniques References CONTENT:-

STORY OF RIA: 

STORY OF RIA Radioimmunoassay: An assay based on the reversible and non- covalent binding of an antigen (hapten) by a specific antibody employing radioactivity labelled antigen (hapten) to measure the fraction of the antigen (hapten) bound to a substoichiometric amount of antibody. In fact, RIA is a heterogeneous, limited reagent and competitive assay. Dr. Rosalyn Yalow became the first female to win a Nobel Prize with her work on the radioimmunoassay.

INTRODUCTION: 

INTRODUCTION An immunoassay is a test that uses antibody and antigen complexes as a means of generating a measurable result. An antibody : antigen complex is also know as an immune – complex. Immunoassays are different from other types of laboratory tests, such as colorimetric tests, because they use antibody: antigen complexes to generate a signal that can be measured. In contrast, most routine clinical chemistry tests utilize chemical reactions between the reagent (a solution of chemicals or other agents) and patient sample to generate a test result.

General terms :-: 

General terms :- ANTIBODY :- is a protein that is produced by in response to an “invading” (foreign) substances. Some immunoassay test for the presence of antibodies to cancer molecules. ANTIGEN :- is the substance that the body is trying to “fight off” (eliminate or reduce) by mounting an immune response. Some immunoassay test for antigens directly , rather than looking for the antibodies. AN IMMUNOGEN :- is a substance that elicits immune response. E.g. drug – protein conjugate . ANALYTE :- Is anything measured by a laboratory test.

IMMUNO ASSAY :-: 

IMMUNO ASSAY :- An Immunoassay methods can be highly sensitive and specific. In this assay determine WHAT ? Immunoassay is used to determine antigen concentration by means of competitive binding of labelled (Ag*) unlabelled (Ag) antigen to the antibody (Ab) specific for that antigen. The competition can be represent. Ab + Ag* Ab .Ag* + Ag Ab . Ag.

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If the antibody cannot distinguish between Ag* and Ag, then the proportion of Ab.Ag* to Ab.Ag at equilibrium will be equal to the original proportion of Ag* to Ag . After equilibrium , the bound and free fractions of antigen are separated and analyzed for labelled antigen. So , the greater the concentration of unlabelled antigen in the sample , the more labelled antigen that will displaced from the bound form. Hence a standard curve is a prepared with a series of solutions each containing the same total amounts of Ab and Ag* , but with increasing amounts of Ag. The standard curve is a plot of percent labelled antigen bound against added antigen concentration.

Why label is required ?: 

Why label is required ? All immunoassays require the use of labeled material in order to measure the amount of antigen or antibody present. A label is a molecule that will react as part of the assay, so a change in signal can be measured in the blood:reagent solution. Examples of a label include- a radioactive compound, an enzyme that causes a change of color in a solution, a substance that produces light.

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Labeled antibodies allow detection of antigen/antibody complexes in immunoassays Labeled antigen also allows detection of antigen/antibody complexes in immunoassays

CLASSIFICATION OF IMMUNOASSAY :-: 

CLASSIFICATION OF IMMUNOASSAY :- Competitive & Non-competitive immunoassays. 2.Homogeneous & Heterogeneous immunoassays. 3.Limited reagent assays & Excess reagent assays.

COMPETITIVE IMMUNOASSAY :-: 

COMPETITIVE IMMUNOASSAY :- One step competitive immunoassay Two step competitive immunoassay

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Competitive format Amount of antigen is indirectly related to the amount of label (signal) in competitive formats

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14 Noncompetitive (Sandwich) Method Noncompetitive sandwich method of immunoassay

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15 Amount of antigen is directly related to the amount of label (signal) in competitive formats

HOMOGENEOUS AND HETEROGENEOUS IMMUNOASSAY METHIDS :-: 

HOMOGENEOUS AND HETEROGENEOUS IMMUNOASSAY METHIDS :-

LIMITED REAGENT ASSAY :-: 

LIMITED REAGENT ASSAY : - Many conventional RIAs follow limited reagent assay protocols. The following scheme depicts the AgAb reaction: Ag AgAb + Ab Ag* Ag*Ab With limited amount of Ab, the unlabeled antigen (analyte) compete with the labeled antigen Ag* for limited binding sites. Bound fraction [AgAb] is separated from free [Ab], and the signal [Ag*Ab] complex i.e. the Ab fraction not occupied by the analyte is measured. The amount of analyte is inversely proportional to the bound [Ag*Ab] complex in a hyperbolic function.

EXCESS – REAGENT ASSAY :-: 

EXCESS – REAGENT ASSAY :- This protocol is utilized by- 1. Immunoradiometric Assays (IRMA). 2. Two-site or sandwich Assays. Here the excess Ab is labeled.(In case of sandwich assay an excess amount of first Ab used to capture analyte from sample matrix, and a labeled second Ab provides the signal for quantitation.) IRMA : Ag + Ab* AgAb* Sandwich assay: Ag + Ab1 Ag-Ab1 + Ab2* Ab1-Ag-Ab2* Bound fraction is separated from free; the signal [AgAb*] or [Ag-Ab1-Ab2*] complex is measured. The amount of analyte is proportional to the bound complex in a hyperbolic function.

Following are the list of immunochemical method :-: 

Following are the list of immunochemical method :- radioimmunoassay (RIA) Immunoradiometric assay (IRMA) fluorescence immunoassay (FIA) Enzyme immunoassay (EIA) - Enzyme multiplied immunoassay technique(EMIT) - Enzyme linked immunosorbent assay (ELISA) - Micro particle enzyme immunoassay (MEIA) Chemiluminescence assay Mass spectrometric immunoassay

RADIOIMMUNOASSAY :- : 

RADIOIMMUNOASSAY :- PRINCIPLE :- The principal of RIA involves competitive binding of radio labeled antigen and unlabeled antigen to a high affinity antibody. The labeled antigen is mixed with antibody at a concentrations that saturates the antigen – binding sites of the antibody. Then the test samples of unlabeled antigen of unknown concentration are added in progressively larger amounts. The antibody does not distinguish labeled from unlabeled antigen, so the two kinds of antigen compete for available binding sites on the antibody.

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As the concentration of unlabeled antigen increases, more labeled antigen will be displaced from the binding sites. The decrease in the amount of radio labeled 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 test sample. RIA measures the effect of varying concentration of a compound in a biological fluid on an in-vitro system containing radioactive standards of the compound and a specific antibody. In fact, RIA is a HETEROGENEOUS , LIMITED REAGENT ,and COMPETITIVE ASSAY.

CALCULATION OF A HYPOTHETICAL IMMUNOASSAY SYSTEM: 

CALCULATION OF A HYPOTHETICAL IMMUNOASSAY SYSTEM UNITES TAKEN AFTER EQUILIBRATION* % BOUND Y BOUND FREE D D* A D D* D D* D* 0 200 100 0 100 0 100 50.0 1.00 50 200 100 20 80 30 120 40.0 0.80 100 200 100 33 67 67 133 33.5 0.67 200 200 100 50 50 150 150 25.0 0.50 300 200 100 60 40 240 160 20.0 0.40 400 200 100 67 33 333 167 16.5 0.33 500 200 100 71 29 499 171 14.5 0.29 600 200 100 75 25 525 175 12.5 0.25 700 200 100 78 22 622 178 11.0 0.22 800 200 100 80 20 720 180 10.0 0.20 900 200 100 82 18 818 182 9.0 0.18 1000 200 100 83 17 917 183 8.5 0.17

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Where, D = Unlabeled antigen ; GRAPH : Y vs. LOG D D*= Labeled antigen ; A = Antibody ; Y = (Bound D*)/(Bound D*) 0

Requirements can be specified for the ideal immunoassay :-: 

Requirements can be specified for the ideal immunoassay :- The antigen –antibody reaction is equilibrium-controlled. Only a single class of binding site exists on the antibody. The antibody is completely specific for the antigen. The antibody can not distinguish between labeled and unlabeled antigen. The separation of bound from free antigen is complete.

General procedure for RIA :-: 

General procedure for RIA :- 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.

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The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured. 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 patient's serum to be derived. Determine standard curve :- 1. Non-linear plot of radioactivity versus concentration. 2. Legit-log concentration plot is linear.

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Radioimmunoassay Procedure

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TOTAL (DRUG) BOUND (DRUG) FREE (DRUG) 0 6 0 3 4 2 6 3 3 12 2 4

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29 Plot of Bound versus Total Drug Concentration Logit versus Log Total C Plot

COMPONENTS USED IN RIA :-: 

COMPONENTS USED IN RIA :- A tracer i.e. labelled ligand. A binder (antibody) which is the specific antiserum. A separation system to separate the ‘bound’ and ‘free’ phases. A standard i.e. the ligand (analyte ) in highly pure form. A ligand (analyte )- free human antiserum.

1) Tracer :-: 

1) Tracer :- The radioisotopes used are – a) Beta (POSITRON) emitters - 3 H and 14 C b) Gamma emitters – 125 I polypeptide or proteins antigens usually contain the aromatic amino acid tyrosine, which can be iodinated with the gamma emitter 125 I. The isotope is available as the salt Na 125 I; this is oxidized , and allowed to react with the antigen.

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Gamma Counter

Specific antiserum (binder ,antibody) :-: 

Specific antiserum (binder ,antibody) :- It is prepared by injecting (repeatedly) the antigen together with the Freund’s adjuvant (mix. Of neutral detergent , paraffin oil , and killed mycobacterium ) into a suitable animal such as guinea pig ,rabbit or goat. Molecules like thyroid hormones , steroids , drugs are not immunogenic. So they are conjugated to carrier proteins and polymers to make them immunogenic.

Carrier molecules :-: 

Carrier molecules :- Bovine serum albumine Egg albumin Gamma globulin Haemocynin Fibrinogen Active group of protein carriers for conjugation to heptanes :- Amino group of lysine -SH group of Cysteine. Imidazole ring of histidine -COOH group of aspartic and glutamic acid

3) Separation system :-: 

3) Separation system :- RIA requires a separation procedure because the bound fraction does not precipitate spontaneously at the low conc. Employed. Variety of procedures are available:- Physical method 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 :

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Second antibody method for separation:

4) A Standard i.e. The ligand (analyte) in highly pure form:: 

4) A Standard i.e. The ligand ( analyte ) in highly pure form: Drugs, protein, hormones etc. must be in pure form, so they can be diluted and aliquoted in precisely measured quantities. Standards 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 analyzed.

5) Ligand free human serum: 

5) Ligand free human serum It is prepared by treating human serum with charcoal that absorbs the smaller antigen. 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.

APPLICATION: 

APPLICATION Highly specific Highly sensitivity Diagnosis of hypo and hyperthyroidism by estimating thyroid hormones like T3 & T4 Management of diabetic patient by estimation of insulin, insulin antibodies. Wide application both invivo and invitro measurement of compounds of interest like insulin, gastrin , glucagon, hormones etc. Determining drugs in biological fluid like blood and urine.

1-For Endocrinology Insulin, vasopressin Detects Endocrine Disorders Physiology of Endocrine function 2-For Pharmacology Morphine Detect Drug abuse or Drug Poisoning Study Drug kinetic 3-Epidermiology Hepatitis B 4-Clinical Immunology Antibodies for Inhalant allergens Allergy Diagnosis 5-Oncology Carcinoembryonic antigen Early Cancer Detection and Diagnosis.: 

1-For Endocrinology Insulin, vasopressin Detects Endocrine Disorders Physiology of Endocrine function 2-For Pharmacology Morphine Detect Drug abuse or Drug Poisoning Study Drug kinetic 3-Epidermiology Hepatitis B 4-Clinical Immunology Antibodies for Inhalant allergens Allergy Diagnosis 5-Oncology Carcinoembryonic antigen Early Cancer Detection and Diagnosis.

Following are lists of compounds for which immunoassay were developed :-: 

Following are lists of compounds for which immunoassay were developed :- DRUGS STEROIDS POLYPEPTIDE HORMONE AMPHETAMINE ANABOLICS ACTH BARBITURATES ANDROGENS FSH DIAZEPAM CORTICOSTEROIDS GH DIGOXIN ESTROGENS GLUCAGON GENTAMICIN PROGESTERONE INSULIN METHADON STEROIDS LH MORPHIN GLUCOCORTICOIDS PROLACTIN NARCOTIN PENICILLINS PROSTAGLANDIN

LIMITATION: 

LIMITATION Radiation hazards Requires specially trained persons Laboratory require special license to handle radioactive material Requires special arrangements for ; Requisition, storage of radioactive material Radioactive waste disposal.

REFERENCES: 

REFERENCES Analytical Biochemistry: David J, Holme and Hazel peck. 3 rd edition; Page no. 228-37 Biochemistry : S. satyanarayan . 4 th Edition; Page no. 228 Text book of bio technology by vyas and dixit 2 nd edition; Page no. 312-21 Biochemistry by Agrawal ; 2 nd edition; Page no. 128-31 A Textbook of PHARMACEUTICAL ANALYSIS by Kenneth A. Connors; A Witey - interscience Publication; 10 th edition Page no. 553-65