Radio immuno assay


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Radioimmunoassay By- Nidhi Lathia M.pharm (Sem -I) Pharmaceutics Atmiya Institute of Pharmacy Rajkot.

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Immunoassay: Antibodies, Antigens and Analytes Defined An antibody is a protein that is produced by the body in response to an “invading” (foreign) substance.* Antibodies are produced as part of the body’s immune response to protect itself. For instance, some immunoassays test for the presence of antibodies to cancer molecules. Thus, if the antibodies are present, it means invading cancer cells are, too. An antigen is the substance that the body is trying to “fight off” (eliminate or reduce) by mounting an immune response. Some immunoassays test for antigens directly, rather than looking for the antibodies. In a test to measure the concentration of a therapeutic drug, for example, the drug is the antigen that binds to the antibody. 2 Radioimmunoassay

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Immunogen : An Immunogen is a substance that elicits immune response i.e. drug-protein conjugate. Analyte: An analyte is anything measured by a laboratory test. In immunoassay testing, the analyte may be either an antibody, or an antigen. * AN EXCEPTION IS THE CASE OF AUTOIMMUNE DISEASES , WHERE THE BODY PRODUCES ANTIBODIES TO NATURALLY OCCURRING PROTEINS RATHER THAN FOREIGN SUBSTANCES. 3 Radioimmunoassay

What is ‘LABEL’?:

What is ‘LABEL’ ? 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 a part of the assay, so change in signal can be measured in the solution. The label can be applied during the manufacture of the reagent to either Ab or Ag. Example of label includes- A radioactive compound An enzyme that causes a change of colour in solution A substance that produces a light 4 Radioimmunoassay

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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. The technique of radioimmunoassay has revolutionized research and clinical practice in many areas, e.g., blood banking diagnosis of allergies endocrinology Dr. Rosalyn Yalow became the first female to win a Nobel Prize with her work on the radioimmunoassay. 5 Radioimmunoassay

History and Background:

History and Background 1972 First Hepatitis RIA 1972 EMIT Assay 1975 Bead-based Assays 1978 Coated-tube assays 1985 HIV Immunoassay 1993 TroponinAssays 1997 Automated Cytokine Assays (Evans) 6 Radioimmunoassay

Immunoassays at a Glance:

Immunoassays at a Glance Immunoassays are a group of sensitive analytical tests that utilize very specific antibody/antigen complexes to produce a signal that can be measured and related to the concentration of a compound in solution . Immunoassays also produce qualitative data in terms of the presence (+) or absence (-) of a compound in the body. They are used in a lot of laboratories, including hospitals labs, and have been widely used in the special area of Forensic Toxicology to screen for drugs and other chemicals in the body. 7 Radioimmunoassay


Principle It is based on the competition between unlabelled antigen & a fixed amount of the corresponding radio labelled antigen for limited number of antibody binding sites in fixed anount of antiserum. At equilibrium, the unbound antigen (F)+ Antigen-antibody complex(Ag-Ab) are separated & 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). 8 Radioimmunoassay


Principle 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. k1 Ag* + Ab k1 Ag*-ab and , k2 Ag + Ab k1 Ag-Ab k2 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 9 Radioimmunoassay

Principle :

Principle Fig 3.7.1 RIA before and after Incubation - Blank and Three Standard Samples A Blank and Three Standard Samples 10 Radioimmunoassay

Production of Antibodies:

Production of Antibodies The production of antibodies is an important process in the use of immunoassays because it is the antibody-antigen complexes that form that the device uses for its results. Antibodies can be called monoclonal or polyclonal, depending upon the technique used to produce it. Monoclonal antibodies involve these basic steps and result in very specific antibodies that bind only to one antigen epitope , which in turn reduces the occurrence of false positives in the immunoassay: A mouse (or rabbit) is immunized by being injected with an antigen; the antigen generates an antibody response in the animal. The mouse is sacrificed and the antibody forming cells are isolated from the mouse's spleen. Monoclonal antibodies are produced by fusing single antibody-forming cells with myeloma cells. The resulting cell is called a hybridoma . This hybridization makes the cells “immortal.” The hybridomas contain large amounts of antibodies, and can easily be cultured into populations of cells that contain identical antibodies. These antibodies are called " monoclonal antibodies " because they are produced by the identical offspring of a single, cloned antibody producing cell. (UCLA) 11 Radioimmunoassay

Production of Antibodies Cont’d:

Production of Antibodies Cont’d A diagram depicting the steps involved in monoclonal antibody production. 12 Radioimmunoassay

Production of Antibodies Cont’d:

Production of Antibodies Cont’d Polyconal antibodies are more likely to produce a false positive because they are less specific to antigen epitopes and have varying binding affinities; they may bind of molecules similar to their antigens. Production includes these steps: A mouse (or rabbit) is immunized by being injected with an antigen; the antigen generates an antibody response in the animal. The animal is bled and the antibodies are collected; the blood contains a heterogenous mixture of antibodies of varying binding affinities and specificities. 13 Radioimmunoassay

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Radioimmunoassay 14 Multiple antigen specificities of polyclonal antibodies Uniform specificities of monoclonal antibodies

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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 15 Radioimmunoassay

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Classification of immunoassays: Competitive & Non-competitive immunoassays. 2.Homogeneous & Heterogeneous immunoassays. 3.Limited reagent assays & Excess reagent assays. 16 Radioimmunoassay

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Competitive format In competitive formats, unlabelled analyte (usually antigen) in the test sample is measured by its ability to compete with labeled antigen in the immunoassay. The unlabeled antigen blocks the ability of the labeled antigen to bind because that binding site on the antibody is already occupied. Thus, in a competitive immunoassay, less label measured in the assay means more of the unlabeled (test sample) antigen is present. The amount of antigen in the test sample is inversely related to the amount of label measured in the competitive format . Amount of antigen is indirectly related to the amount of label (signal) in competitive formats Radioimmunoassay 17

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One step competitive immunoassay Two step competitive immunoassay 18 Radioimmunoassay

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Noncompetitive (Sandwich) Method Noncompetitive assay formats generally provide the highest level of assay sensitivity and specificity and are applied to the measurement of critical analytes such as cardiac and hepatitis markers. This format is referred to as a “sandwich” assay because analyte is bound (sandwiched) between two highly specific antibody reagents Noncompetitive sandwich method of immunoassay 19 Radioimmunoassay

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Figure -11 Amount of antigen is directly related to the amount of label (signal) in competitive formats 20 Radioimmunoassay

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Homogeneous and Heterogeneous Immunoassay Methods Immunoassay methods that require separation of bound Ab- Ag* complex are referred to as heterogeneous immunoassays. Those that do not require separation are referred to as homogeneous immunoassays . Homogeneous methods have been generally applied to the measurement of small analytes such as abused and therapeutic drugs. Since bound complex releases a differentiated signal from the unbound reactants, homogeneous methods do not require the separation of the bound Ab-Ag* from the free Ag* and thus they are generally much easier and faster to perform. 21 Radioimmunoassay

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Figure -12 Homogeneous and heterogeneous immunoassays 22 Radioimmunoassay

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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) competes 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. 23 Radioimmunoassay

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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. 24 Radioimmunoassay

The Technique :

The Technique A mixture is prepared of radioactive antigen Because of the ease with which iodine atoms can be introduced into tyrosine residues in a protein, the radioactive isotopes 125 I or 131 I are often used. antibodies against that antigen. Known amounts of unlabeled ("cold") antigen are added to samples of the mixture. These compete for the binding sites of the antibodies. 25 Radioimmunoassay

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At increasing concentrations of unlabeled antigen, an increasing amount of radioactive antigen is displaced from the antibody molecules. The antibody-bound antigen is separated from the free antigen in the supernatant fluid, and the radioactivity of each is measured. 26 Radioimmunoassay

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27 Radioimmunoassay

Reagents used in RIA:

Reagents used in RIA A tracer i.e. a labelled ligand. A binder (Antibody) which is the specific antiserum. A separation system to separate the ‘bound’ and ‘free’ phase. A standard i.e. the ligand (analyte) in highly pure form. A ligand (analyte) - free human antiserum. 28 Radioimmunoassay


Tracers The radioisotopes used are – Beta (positron) emitters- 3 H , 14 C, 11 C, 13 N, 15 O, 18 F Gamma emitters- 125 I, 111 In, 123 I, 131 I, 75 Se Positron vs. Gamma Isotopes: Positron emitters are more appropriate for the direct labeling of drug molecules since the γ-emitting radionuclides are rarely an integral part of the drug molecule structure. The positron (β-emitting) radionuclides are mainly restricted for invitro experiments. The γ-emitting radionuclides are useful for in-vivo imaging. 29 Radioimmunoassay

Radiolabelling Techniques:

Radiolabelling Techniques Isotope Exchange Reactions Introduction of a Foreign Label Labeling with Bifunctional Chelating Agents Biosynthesis 30 Radioimmunoassay

Specific antiserum (Binder, Ab):

Specific antiserum (Binder, Ab ) It is prepared by injecting (repeatedly) the antigen together with the Freund’s adjuvant into a suitable animal such as guinea pig, rabbit or goat. Freund’s adjuvant: It is a mixture of neutral detergent , paraffin oil and killed mycobacterium. It increases the antibody response of the injected antigen Molecules like thyroid hormones, steroids, drugs are not immunogenic. So they are conjugated to carrier proteins and polymers to make them immunogenic. Carrier Molecules: Bovine Serum Albumin (BSA). Egg Albumin. Gamma globulin. Haemocyanin. Fibrinogen. Thyroglobulin. Synthetic Polypeptides. E.g. Poly Lysine, Poly Glutamic acid. Synthetic polymers. E.g. PVP. 31 Radioimmunoassay

Specific antiserum (Binder, Ab):

Specific antiserum (Binder, Ab ) Active group of Protrin carriers for conjugation to heptanes: Amino group of Lysine. –SH group of Cysteine. Imidazole ring of Histidine. –COOH group of Aspartic and Glutamic acid. Phenolic group of Tyrosine. –OH group of Serine. Terminal -NH 2 and –COOH groups. Active positions on rings of Tyrosine, Tryptophan and Histidine can be attached to diazepam salt. 32 Radioimmunoassay

Specific antiserum (Binder, Ab):

Specific antiserum (Binder, Ab ) After 2-3 weeks of immunization, serum of the animal is collected, tested for antibody and suitably diluted before storage. Good quality antiserum is obtained after 3 – 4 booster doses. Antibodies produced are polyclonal i.e. mixture of antibodies specific to different epitopes on the antigen. They have different specificity and avidity and it may vary from animal to animal and batch to batch quality. Monoclonal antibodies are produced by ‘Hybridoma Technique’ propagated from a single cell, uniform in their binding characteristics within batch and also from batch to batch. 33 Radioimmunoassay

Characterization of Antiserum:

Characterization of Antiserum Titre – It is the degree to which the antiserum can be diluted so that it is usable in the assay. The dilution of the antiserum that corresponds to 30% - 50% of binding is taken as the titre of that antiserum. It depends upon – The amount of tracer used, The incubation volume and temperature The % binding with the tracer adopted in the optimized assay. Avidity – It is the energy of binding to the specific antigen. It is expressed in terms of equilibrium constant K. Higher the ‘ avidity ’ greater the sensitivity of RIA. 34 Radioimmunoassay

Characterization of Antiserum:

Characterization of Antiserum 3. Sensitivity – It is the lowest of ligand which can be distinguished from the absence of ligand in that assay. 35 Radioimmunoassay

Characterization of Antiserum:

Characterization of Antiserum 4. Specificity – It is the ability of the antibody to react with specific antigen and no other antigen. It is not always absolute, and may react with chemically resembling analyte. 36 Radioimmunoassay

Separation system:

Separation system 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. 37 Radioimmunoassay

Separation system:

Separation system Basis of method Material/technique used 1. Differential migration of the bound & free fraction by- (a) Differences in charge. Paper chromatography Electrophoresis. (b) Differences in relative molecular mass Gel filtration , Gel equilibration. 2. Adsorption of the free fraction Coated charcoal Ion exchange Resin 3. Precipitation of the bound fraction by- (a) Organic solvent Ethanol, Dioxane , PEGs (b) Salt Sodium or Ammonium sulphate (c) Second antibody 4. Solid phase Methods Coated on tubes or discs (a) First antibody Covalently linked to sephadex paper discs (b) Second antibody Covalently linked to solid matrix. Techniques employed for the separation of bound and free antigen . 38 Radioimmunoassay


Standard A standard i.e. the ligand (analyte) in highly pure form: Drugs, proteins, 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. 39 Radioimmunoassay

Ligand free human serum:

Ligand free human serum It is prepared by treating human serum with charcoal that absorbs the smaller antigens. 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. 40 Radioimmunoassay

Assumptions for RIA validity:

Assumptions for RIA validity The standard is structurally identical with the analyte. However, the labelled antigen (Ag*) need not be structurally identical with the analyte , provided that it closely mimics their behaviour of the analyte with the antibody. The equilibrium constant K between the antibody (Ab) and antigen (Ag) and that between labelled antigen (Ag*) and antibody (Ab) is of the same order of magnitude. The separation of bound and free phases is perfect which is not always the same case. One molecule of antibody (Ab) binds to only one molecule of antigen (Ag) or labelled antigen (Ag). All the reactions between the antibody (Ab) and antigen (Ag) and that between labelled antigen (Ag*) and antibody (Ab) are taken to equilibrium, although many assays are done under non-equilibrium conditions. 41 Radioimmunoassay

Advantages & Disadvantages of RIA:

Advantages & Disadvantages of RIA Advantages:- Highly specific: Immune reactions are specific High sensitivity : Immune reactions are sensitive Indirect method of analysis Saturation analysis Disadvantages:- Radiation hazards: Uses radiolabelled reagents Requires specially trained persons Labs require special license to handle radioactive material Costly Requires special arrangements for Requisition, storage of radioactive material radioactive waste disposal. 42 Radioimmunoassay


Applications 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 Endocrinology Insulin, HCG, Vasopressin Detects Endocrine Disorders Physiology of Endocrine Function Pharmacology Morphine Detect Drug Abuse or Drug Poisoning Study Drug Kinetics Epidemiology Hepatitis B Clinical Immunology Antibodies for Inhalant Allergens Allergy Diagnosis Oncology Carcinoembryonic Antigen Early Cancer Detection and Diagnosis 43 Radioimmunoassay

RIA for Insulin:

RIA for Insulin Reagents used :- Each kit is sufficient to run 250 tubes and contain following reagents: Asaay buffer: 0.05M Phosphosaline buffer, pH 7.4, containing 0.025m EDTA, 0.08% Sodium azide & 1% RIA grade BSA. Antiserum: Guinea pig anti rat insulin serum in assay buffer. 125 I -Insulin: 125 I –Insulin label , HPLC purified. Label Hydrating Buffer: Assay buffer containing normal guinea pig IgG as carrier. Standards: Purifies rat Insulin standard buffer at different concentration. Quality controls 1 & 2: Purified rat Insulin in assay buffer. Precipitating reagent: Goat anti-guinea pig IgG serum, 3% PEG & 0.05% triton X-100 in 0.05M Phosphosaline, 0.025M EDTA , 0.08% sodium azide. Radioimmunoassay 44

Specimen collection & storage:

Specimen collection & storage Radioimmunoassay 45

Assay procedure [Day 1]:

Assay procedure [Day 1] Radioimmunoassay 46

Assay procedure [Day 2]:

Assay procedure [Day 2] Radioimmunoassay 47

Assay procedure [Day 2]:

Assay procedure [Day 2] Radioimmunoassay 48


GUIDELINES TO SET UP A RIA LABORATORY Radioimmunoassay 49 Handling facility and Staff requirement for starting RIA laboratory


GUIDELINES TO SET UP A RIA LABORATORY Radioimmunoassay 50 Other facilities required :-

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Radioimmunoassay 51

Enzyme Immunoassay:

Enzyme Immunoassay In EIAs, enzyme labels are used in place of radioactive labels. Typical enzyme labels are alkaline phosphatase, horseradish peroxidase, B-galactosidase, glucose oxidase, p-nitrophenyl phthalate and o-nitro phenyl phthalate galactosidase. Whereas RIAs use radioactivity to measure the concentration of analyte, EIAs typically use a change in color, emission of light, or other signal. Specific equipment is required to quantitate the amount of enzyme present by measuring the specific change that occurred. Radioimmunoassay 52

Enzyme Immunoassay:

Enzyme Immunoassay The two types of Enzyme immunoassay are- Enzyme mediated (multiplied) Immunoassay tevhnique(EMIT) Enzyme linked immunosorbent assay (ELISA). Enzyme immunoassay 53


EMIT Enzyme mediated (multiplied) Immunoassay technique It has been widely used for rapidly assaying micro amount of drug and substance in biological fluid. Key elements are- Compound to be measured (heptan). Enzyme labelled molecules of compound. Specific antibody that binds the compound. Enzyme chromogenic substrate i.e. initially colourless but gives colour with enzyme It is homogeneous method of analysis means there is no need to wash excess of enzyme labelled antigen. Method does not involve solid phase system but enzymes are active in free form but inactive in bound form. Enzyme immunoassay 54


EMIT Two basic test principles are involved in EMIT- The enzyme must retain enzymatic activity even after the heptan or antigen conjugation takes place. 55 Enzyme immunoassay


EMIT 2. The enzyme activity of the heptan-enzyme conjugate is reduced or inhibited when the heptan reacts with specific antibody. EMIT drug assay begins by adding to a patient specimen, an excess of specific antibodies that will bind to the drug being measured. If drug molecules are present, they immediately bind to antibody sites. The enzyme labelled drug is then added to the mixture. 56 Enzyme immunoassay


EMIT Antibody binding sites not occupied by molecules of the drug in the specimen and are immediately filled with molecules of added enzyme labelled drug. Enzyme activity is then reduced because only free enzyme-labelled drug conjugate can act on the substrate. The amount of substrate converted from a colourless to a coloured product in a given period of time is determined by the amount of free enzyme left in the mixture. 57 Enzyme immunoassay


EMIT This result in colour change i.e. measured by spectrophotometrically. The specimen drug concentration is measured by comparing the absorbances of test and standard. The absorbance obtained is directly proportional to the drug concentration in sample. As it is homogeneous assay there is no need for separation of bound from unbound compound as it is necessary with other Immunoassay like ELISA. Sensitivity of EMIT can be increased by using enzyme reaction followed by fluorescent substrate but there may be a problem of quenching. Application: Rapid detection of virus, hormones, drugs, insecticides etc. 58 Enzyme immunoassay


ELISA A 96-well microtiter plate being used for ELISA 59 Enzyme immunoassay


Equipments 60 Enzyme immunoassay

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61 Enzyme immunoassay


ELISA Kit 62 Enzyme immunoassay


ELISA Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology , as well as a quality control check in various industries. 63 Enzyme immunoassay

Types of ELISA:

Types of ELISA Indirect ELISA Sandwich ELISA Competitive ELISA Reverse ELISA 64 Enzyme immunoassay

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Variations in the enzyme-linked immunosorbent assay (ELISA) technique, similar to RIA except using an Enzyme (alkaline ⓟ, horseradish peroxidase, & β-galactosidase) : safer & less costly. to detect Ab (HIV, HCV) to detect Ag to detect Ag 65 Enzyme immunoassay


Application Detecting infections Sexually transmitted agents like HIV, syphilis & Chlamydia Hepatitis B and C Toxoplasma gondii Detecting allergens in food and house dust Measuring ‘rheumatoid factors’ and other autoantibody in autoimmune disease like Lupus erythematosus. Measuring toxins in contaminated food. 66 Enzyme immunoassay


Application Screening donated blood for evidence of viral contamination by:- HIV-1 & HIV-2 (presence of anti-HIV antibodies). Hepatitis C (presence of antibodies) Hepatitis B (testing for both antibodies & a viral antigen) Measuring hormone levels HCG (as a test for pregnancy) LH (determining the time of ovulation) TSH, 3 AND T4 (for thyroid function) 67 Enzyme immunoassay


Application ELISA is used to detect many bacterial and viral antigens including HIV, malaria, cholera, measles and mumps. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. 68 Enzyme immunoassay

ELISA for Insulin :

ELISA for Insulin 69 Enzyme immunoassay

ELISA for Insulin :

ELISA for Insulin 70 Enzyme immunoassay

Fluorescence immunoassay:

Fluorescence immunoassay

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72 Enzyme immunoassay

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