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Pharmacy, Dept of Q.A.CONTENTS: CONTENTS Intro of Immunoassay Structure and preparation of antibodies Four categories of immunoassay methodology ( competitive and noncompetitive , and homogeneous and heterogeneous ) immunoassay-types Radioimmunoassay procedureSlide 3: Introduction to Immunoassays“Immuno”& “assay” : “Immuno”& “assay” “Immuno” refers to an immune response that causes the body to generate antibodies, and “assay” refers to a test. Thus, an immunoassay is a test that utilizes immunocomplexing when antibodies and antigens are brought togetherDEFINITION: DEFINITION 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 known as an immuno-complex.Immunoassay: Antibodies, Antigens and Analytes Defined: 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: An Antigen 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.An analyte: An analyte An analyte is anything measured by a laboratory test. In immunoassay testing, the analyte may be either an antibody , or an antigen .Antibodies: Antibodies Antibodies possess high a) specificity and b) affinity for a specific antigen . It is the specific binding of an antibody to an antigen that allows the detection of analytes by a variety of immunoassay methods.Structure of Antibodies: Structure of Antibodies Antibodies (Ab) are a type of protein called immunoglobulins . The most common one is immunoglobulin G (IgG). IgG is a protein composed of two main structural and functional regionsPreparation of Polyclonal and Monoclonal Antibodies: Preparation of Polyclonal and Monoclonal Antibodies Antibody reagents are developed from either polyclonal or monoclonal antibodies. Polyclonal antiserum (serum from blood containing the desired antibodies) is generated in animals, most commonly sheep , rabbits , or goats.Monoclonal antibodies: Monoclonal antibodies Monoclonal antibodies differ from polyclonal antibodies in that they are highly specific for a single epitope on a multivalent antigen (see Figure 1-3). They are produced from a single cell line using hybridoma technology and mouse myeloma cell lines . Hybridomas are antibody-producing tumor cells that produce many copies of the same antibody and grow easily in laboratory cell culture.An advantage: An advantage An advantage of monoclonal antibodies is that the hybridoma cell line that produces them is potentially “immortal” and can produce the same antibodies consistently and indefinitely . A polyclonal antisera produced by immunization of animals can vary from animal to animal, and a useful antiserum may no longer be available if the single animal that produces it dies.Categories of Immunoassay Methodologies: Categories of Immunoassay Methodologies The immunoassay methodologies are: noncompetitive and competitive immunoassays , and homogeneous and heterogeneous immunoassayslabelled material: labelled material 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.Competitive and Noncompetitive Immunoassays: Competitive and Noncompetitive Immunoassays The measurement of analyte in an immunoassay is achieved by using either a competitive or a noncompetitive format.Competitive Format : 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.Noncompetitive (Sandwich) Method: 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 (Figure 1-10).Homogeneous and Heterogeneous Immunoassay Methods: 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. Types of Immunoassays: Radioimmunoassays (RIAs) utilize a radioactive label (usually 125I, 3H or 14C), which emits radiation that can be measured with a beta or gamma counter or scintillation counter. Types of Immunoassays TYPES OF IMMUNOASSAY CONT’D: In the Enzyme Multiplied Immunoassay (EMIT), the drug in the sample and the drug labeled with G6PD compete for antibody binding sites. Binding inhibits enzyme activity, while free enzyme remains active to interact with. Enzyme activity/absorbance is directly proportional to drug concentration. TYPES OF IMMUNOASSAY CONT’D TYPES OF IMMUNOASSAY CONT’D: Reaction components are absorbed or bound to the surface of a solid phase, commonly a well of a microtiter plate Absorbance is measured using a micro-plate reader Sample absorbance is inversely proportional to drug concentration TYPES OF IMMUNOASSAY CONT’D Enzyme linked immunosorbant assay (ELISA): competitive, heterogeneous EIATYPES OF IMMUNO ASSAY CONT’D: Reaction mixture is excited by planepolarized light. As the tracer returns to a lower energy state, it emits light; polarization is measured. The polarization value of the sample is inversely proportional to analyte concentration. TYPES OF IMMUNO ASSAY CONT’D In the Fluorescent Polarized Immunoassay, the drug in the sample competes with fluorescein-labeled drug for antibody binding sites. DEFINITION: Radioimmunoassay is a highly sensitive and specific assay method that uses the competition between radiolabelled and unlabelled substances in an antigen-antibody reaction to determine the concentration of the unlabeled substance, which may be an antibody or a substance against which specific antibodies can be produced. DEFINITIONSlide 31: In the year 1959, Drs. Rosalyn Yalow & Soloman Berson invented the radioimmunoassay, which applied the use of radioisotopes in the measurement of insulin. The RIA is the predecessor of modern immunoassays. Dr. Rosalyn Yalow became the first female to win a Nobel Prize with her work on the radioimmunoassay.Slide 32: Radio immunoassays are classified based on the media used for immobilisation of antigen. They are of two types Solid Phase(sandwich method)- for both antigen and antibody. Soluble Phase(classical RIA method)Solid Phase RIA: : It includes addition of an antiserum to excess of labelled antigen. Due to affinity of antibodies complex are formed and precipitation occurs. Measurement of the label gives the estimation of antigen. Ag* + Ab Ag* + Ag*-Ab Ag*-Ab (excess (antiserum) Free soluble precipitated labelled antigen complex complex antigen) Solid Phase RIA:Slide 35: It includes binding of labelled antigen to antibody. This binding is inhibited by unlabelled antigen. The extent of inhibition is the measurement of unlabelled material Standard curves : Soluble Phase RIA:Slide 37: RADIO IMMUNOSORBENT ASSAY:Slide 38: RADIOALLERGOSORBENT TEST:Applications of Radioimmunoassay: 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 Applications of RadioimmunoassaySlide 40: REFERENCES: Wild, David (Ed.). (2005). The Immunoassay Handbook . Kidlington, Oxford: Elsevier. Evans, Susan (2004, June 15). Retrieved January 19, 2008, from SACB Online Web site: http://sacb.org.sg/ Lee, Tim (2007, May 27). Antigens. Retrieved January 19, 2008, from Immunology Bookcase Web site: http://pim.medicine.dal.ca/atg.htm Monoclonal Antibody Production. Retrieved January 19, 2008, from Lecture Notes Web site: http://www.college.ucla.edu/webproject/micro7/lecturenotes/finished/monoclonal.html Masseyeff , RF (1991).Standardization of immunoassays.. Ann Ist Super Sanita . 27 , 427-436. Moody, David E. (2006).Immunoassay in Forensic Toxicology. Encyclopedia of Analytical Chemistry . http://www.troopers.state.ny.us/Forensic_Science/Lab_Sections/Toxicology/ http://www.abbottdiagnostics.com Elements of immunology by S.A.RASTOGI-Pgno-257 You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.