Immunoassays Group 1: Kateryna Dyakiv, Emina Hodzic, Chris Mecinski, Nicholas Anaya, and Andrew Strmic BACKGROUND • Immunoassays - laboratory technique using the binding between an antigen and its homologous antibody to identify and quantify the specific antigen or antibody in a sample • Immunoassays are based on the reaction of an analyte or antigen (Ag) with a selective antibody (Ab) to give a product (Ag–Ab) that can be measured. The reactants are in a state of equilibrium that is characterized by the law of mass action. • Immunoassay is clearly not the best analytical method for all analytes in all situations. • However, immunoassay technology is important for the analyst because it complements the classical methods, thus providing a conﬁrmatory method for many compounds and the only reasonable analytical choice for others USES TYPES • ELISA – Enzyme Linked ImmunoSorbent • Primary screen of biological samples for drugs • Forensic Urine Drug Testing • Human Performance Testing • RIA – Radio-ImmunoAssay • Postmortem Investigation • EMIT – Enzyme Multiplied Immunoassay • Used for high throughput analyses • Uses less volume of sample Assay Technique • FRET – Fluorescent Resonance Energy Transfer • FPIA – Fluorescent Polarized ImmunoAssay • KIMS – Kinetic Interaction of Microparticles in Solution • CEDIA – Cloned Enzyme Donor ImmunoAssay • Lateral Flow Immunoassays HOW IT WORKS • An immunoassay is a test that uses antibody and antigen complexes to generate a measurable result, or in other words a test that utilizes immunocomplexing when antibodies and antigen are brought together. [*Note: an antibody (antigen complex) is also called an immuno-complex)] • Immuno is an immune response that causes the body to generate antibodies. • Immunoassays utilize one or more select antibodies to detect analytes of interests, which can be those that are naturally present in the body (thyroid hormone0, those that the body produces but not typically present (cancer), or those that do no naturally occur in the body (abused drug). • When a specific binding of an antibody to an antigen occurs, you are able to detect the analytes present. ADDITIONAL INFO • Especially used for detection of drugs in blood and urine. • Antibodies that react with drugs are produced in animals such as rabbits by combining the drug with a protein and injecting this combination into an animal. • Drug protein complex acts as an antigen producing antibodies within the animal. • Recovered blood serum contains antibodies specific or nearly specific to the drug. • A particular serum is added to a urine or blood sample designed to interact with a specific drug such as opiates, cannabanoids, amphetamines, Etc. STRENGTHS • Very useful in detecting a variety of diseases (all detections are done in a lab) such as: • Infections (infectious diseases) • Autoimmune diseases • Cardiovascular diseases • Very useful in detecting the presence of drugs • Very useful for handling a large amount of specimens that have to be quickly tested daily because of the required urinary test needed from people for certain jobs or careers they want to get into • Testing labs have access to many prepared sera from the animals that are injected with any of the different types of drugs • The serum that is added to the urine specimen is unique and made to interact with cocaine, opiates, amphetamines, methadone, etc. WEAKNESSES • Less sensitive than other tests • In some cases, negative results do not exclude the presence of a drug • Are not great for individual testing, more practical for testing a large group of people • Some types of Immunoassays such as ELISA (Enzyme Linked ImmunoSorbent Assay) are expensive and can give a false positive EXAMPLES • Can be used for pregnancy tests • The pregnancy test detects the human chorionic gonadotropin, a hormone produced by the fertilized egg. • Can detect drugs from athletes’ blood • The test will contain the specific antigen for detection. • Detect heart disease by measuring levels of CK-MB. • Rheumatoid Factor - tests for the presence of autoimmune antibodies. (For patients with rheumatoid arthritis.) • West Nile Virus - tests for antibodies that a person made in response to an infection with that virus • Antibodies made in response to a vaccination to assure that the vaccination were successful. REVIEW 1) What is an immunoassay? 2) How does it work? 3) What is it’s ability to detect disease a strength or weakness? 4) What is the fact that it is less sensitive a strength or weakness? 5) A baseball player is suspected of using steroids. How can we use the test to determine if this assumption is true or false? ANSWERS 1) It is a laboratory technique using the binding between an antigen and its homologous antibody to identify and quantify the specific antigen or antibody in a sample. 2) Immunoassays utilize one or more select antibodies to detect analytes of interests by observing whether a specific binding of an antibody to an antigen occurs. 3) STRENGTH 4) WEAKNESS 5) To verify whether the baseball player is using or not, you can test his urine. If the antibodies react with the antigens of the drug than you can claim that he is using. SOURCES • Immunoassays (2011). In BIO Ventures for Global Health. Retrieved April 2, 2013 • Immunoassay tests . (n.d.). Encyclopedia of surgery: a guide for patients and caregivers. Retrieved April 3, 2013, from http://www.surgeryencyclopedia.com/FiLa/Immunoassay-Tests.html • Laboratory methods. (n.d.). Lab tests online. Retrieved April 2, 2013, from https://labtestsonline.org/understanding/features/methods/start/1 • Saferstein, R. (2011). Immunoassay Techniques. In Forensic Science (Second ed., pp. 289-290). Saddle River, NJ: Pearson Education, Inc. • Yeakel, J. (Director) (2013, April 2). Immunoassay techniques. Powerpoint. Lecture conducted from The center for forensic science research education.